Plasmodium vivax is the most widespread cause of malaria with a high burden of disease. Progress in reducing the global malaria burden has stalled with no vaccines available partly due to a limited knowledge of targets and mechanisms of protective immunity. We developed a platform to quantify antibody functions to multiple P. vivax antigens and dissect immunity in a longitudinal cohort of children from Papua New Guinea at risk of P. vivax malaria. We identified antigens targeted by multiple functional antibodies, including interactions with Fcγ receptors, which mediate different cellular effector functions, and complement fixation, advancing our understanding of P. vivax immunity. We identified specific antigens targeted by antibodies associated with protection from P. vivax malaria. Evaluating thousands of possible combinations, we identified subsets of antigens in the most protective combinations providing leads for developing highly protective multi-antigen P. vivax vaccines eliciting multi-functional antibody responses to achieve and sustain elimination.

[This corrects the article DOI: 10.1371/journal.pntd.0004639.].

BACKGROUND:Mycoplasma genitalium infection in pregnancy is increasingly reported at similar frequencies to other sexually transmitted infections (STIs). Knowledge on its contribution to adverse pregnancy outcomes is very limited, especially relative to other STIs or bacterial vaginosis (BV). Whether M. genitalium influences birthweight remains unanswered. METHODS:Associations between birthweight and M. genitalium and other STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis) and BV in pregnancy were examined in 416 maternal-newborn pairs from a prospective cohort study in Papua New Guinea. FINDINGS:Compared to uninfected women, M. genitalium (-166.9 g, 95% confidence interval [CI]: -324.2 to -9.7 g, p = 0.038) and N. gonorrhoeae (-274.7 g, 95% CI: -561.9 to 12.5 g, p = 0.061) infections were associated with lower birthweight in an adjusted analysis. The association for C. trachomatis was less clear, and T. vaginalis and BV were not associated with lower birthweight. STI prevalence was high for M. genitalium (13.9%), N. gonorrhoeae (5.0%), and C. trachomatis (20.0%); co-infections were frequent. Larger effect sizes on birthweight occurred with co-infections of M. genitalium, N. gonorrhoeae, and/or C. trachomatis. CONCLUSION:M. genitalium is a potential contributor to lower birthweight, and co-infections appear to have a greater negative impact on birthweight. Trials examining the impact of early diagnosis and treatment of M. genitalium and other STIs in pregnancy and preconception are urgently needed. FUNDING:Funding was received from philanthropic grants, the National Health and Medical Research Council, and the Burnet Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Populations of the Eastern Highlands of Papua New Guinea (EHPNG, area 11,157 km2) lived in relative isolation from the rest of the world until the mid-20th century, and the region contains a wealth of linguistic and cultural diversity. Notably, several populations of EHPNG were devastated by an epidemic prion disease, kuru, which at its peak in the mid-twentieth century led to some villages being almost depleted of adult women. Until now, population genetic analyses to learn about genetic diversity, migration, admixture, and the impact of the kuru epidemic have been restricted to a small number of variants or samples. Here, we present a population genetic analysis of the region based on genome-wide genotype data of 943 individuals from 21 linguistic groups and 68 villages in EHPNG, including 34 villages in the South Fore linguistic group, the group most affected by kuru. We find a striking degree of genetic population structure in the relatively small region (average FST between linguistic groups 0.024). The genetic population structure correlates well with linguistic grouping, with some noticeable exceptions that reflect the clan system of community organization that has historically existed in EHPNG. We also detect the presence of migrant individuals within the EHPNG region and observe a significant excess of females among migrants compared to among non-migrants in areas of high kuru exposure (p = 0.0145, chi-squared test). This likely reflects the continued practice of patrilocality despite documented fears and strains placed on communities as a result of kuru and its associated skew in female incidence.

Background Globally, 2.5 million babies die in the first 28 days of life each year with most of these deaths occurring in low- and middle-income countries. Early recognition of newborn danger signs is important in prompting timely care seeking behaviour. Little is known about women’s knowledge of newborn danger signs in Papua New Guinea. This study aims to assess this knowledge gap among a cohort of women in East New Britain Province. Methods This study assessed knowledge of newborn danger signs (as defined by the World Health Organization) at three time points from a prospective cohort study of women in East New Britain Province, factors associated with knowledge of danger signs after childbirth were assessed using logistic regression. This study includes quantitative and qualitative interview data from 699 pregnant women enrolled at their first antenatal clinic visit, followed up after childbirth ( n = 638) and again at one-month post-partum ( n = 599). Results Knowledge of newborn danger signs was very low. Among the 638 women, only 9.4% knew three newborn danger signs after childbirth and only one knew all four essential danger signs defined by Johns Hopkins University ‘Birth Preparedness and Complication Readiness’ Index. Higher knowledge scores were associated with higher gravidity, income level, partner involvement in antenatal care, and education. Conclusion Low levels of knowledge of newborn danger signs among pregnant women are a potential obstacle to timely care-seeking in rural Papua New Guinea. Antenatal and postnatal education, and policies that support enhanced education and decision-making powers for women and their families, are urgently needed.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

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.

Pregnant women in resource-limited settings are highly susceptible to anemia and iron deficiency, but the etiology of postpartum anemia remains poorly defined. To inform the optimal timing for anemia interventions, changes in iron deficiency-attributable anemia through pregnancy and postpartum need to be understood. In 699 pregnant Papua New Guinean women attending their first antenatal care appointment and following up at birth and 6 and 12 months postpartum, we undertake logistic mixed-effects modeling to determine the effect of iron deficiency on anemia and population attributable fractions, calculated from odds ratios, to quantify the contribution of iron deficiency to anemia. Anemia is highly prevalent during pregnancy and 12 months post-partum, with iron deficiency increasing the odds of anemia during pregnancy and, to a lesser extent, postpartum. Iron deficiency accounts for R72% of anemia during pregnancy and 20%-37% postpartum. Early iron supplementation during and between pregnancies could break the cycle of chronic anemia in women of reproductive age.

Seasonal malaria chemoprevention (SMC) is recommended by the World Health Organization for the sub-Sahel region in sub-Saharan Africa for preventing malaria in children between 3 months and 5 years old. Since 2016, the Malian National Malaria Control Program has deployed SMC countrywide during its high malaria transmission season at a rate of four monthly cycles annually. The standard SMC regimen includes sulfadoxine-pyrimethamine (SP) plus amodiaquine (AQ). Resistance against SP is suspected to be rising across West Africa, and thus assessing the effectiveness of an alternative antimalarial drug for SMC is needed to provide a second-line regimen when it is ultimately needed. It is not well-understood whether SMC is effective in preventing malaria in children five years of age or older. The primary goal of the study is to determine compare two SMC regimens (SP-AQ and dihydroartemisinin-piperaquine [DHA-PQ]) in preventing uncomplicated P. falciparum malaria in children between 3 months and 9 years old. The study design is a three-arm cluster-randomized design comparing SP-AQ and DHA-PQ arms in two age groups (under 5 years of age and 5 to 9 years of age), and a control group for children aged 5 to 9 years. The study was performed in Mali’s Koulikouro District, a rural area situated in southwest Mali with historically high rates of malaria transmission. The primary outcome for the study is P. falciparum incidence for two SMC regimens in children under 10 years of age. Should DHA-PQ provide an acceptable alternative to SP-AQ, a plausible alternative to conventional SMC would be available as a second line prevention option in the event of SP resistance or drug supply shortages. A significant byproduct of this effort included bolstering district health information systems for rapid identification of severe malaria cases. The study began on July 1, 2019. Through November 2022, a total of 4,556 children between 3 months and 5 years old were enrolled. Routine evaluation of antimalarial drugs is needed to establish appropriate SMC age targets. The study goals here may impact public health policy and provide alternative therapies in the event of drug shortages or resistance. ClinicalTrials.gov NCT04149106, https://clinicaltrials.gov/ct2/show/NCT04149106

Pathogen genomic surveillance demands rapid, low-cost genotyping solutions for tracking infections. Here we use single nucleotide polymorphism (SNP) barcodes to generate practical information for malaria surveillance and control. Using 91 Plasmodium falciparum genomes from three provinces of Papua New Guinea (PNG), we assessed SNP panels with different allele frequency characteristics. A 191 ‘local’ SNP barcode captured similar patterns of population structure evident with 5786 ‘whole genome’ SNPs. Geographically informative SNPs (iSNPs, F ST>0.05) show increased population clustering whilst randomly selected SNPs (rSNPs) and SNPs with similar allele frequencies ( F ST<0.05) amongst different countries (universal, uSNPs) or local PNG populations (balanced, bSNPs) indicated little clustering. SNP panels must be validated in local settings to ensure they capture the diversity and population structure of the target population. Applied to 727 P. falciparum isolates from 16 provinces of PNG, the full barcode identified variable transmission dynamics, and eight major sub-populations, as well as the source of a malaria outbreak in a low transmission setting.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementSample collections, DNA extractions and molecular diagnosis were funded by the Global Fund to end AIDS Tuberculosis and Malaria (GFATM). Genetic and genomic data collection for this was funded through a National Health and Medical Research Council (NHMRC) of Australia Project Grant Number GNT1027108. IM and MB are supported by NHMRC Research Fellowships (GNT1155075, GNT1102971). The authors acknowledge the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support Scheme (IRIISS).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics and Informed Consent. All samples were collected as part of ongoing studies in PNG investigating the impact of intensified malaria control efforts since 2004. All samples were collected with written informed consent from individuals or if children, consent was obtained from their parents, and guardians. Ethical approval for the study was obtained from the Papua New Guinea Institute of Medical Research Institutional Review Board (IRB 11/21, 12/29), the PNG Medical Research Advisory Council (MRAC 12/03, 13/08), the Walter and Eliza Hall Institute Human Research Ethics Committee (HREC 12/06, 13/14) and Deakin Human Research Ethics Committee (2020-282, 2020-283).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll raw whole genome sequence data is available at the European Nucleotide Archive using the accession numbers indicated in Table S1. The final SNP barcode dataset for 727 isolates and for 32 outbreak samples will be made available in a public repository upon publication, and in the interim will be available by contacting the corresponding author.

AbstractAs progress towards malaria elimination continues, the challenge posed by the parasite speciesPlasmodium vivaxhas become more evident. In many regions co-endemic forP. vivaxandPlasmodium falciparum, as transmission has declined the proportion of cases due toP. vivaxhas increased. Novel tools that directly targetP. vivaxare thus warranted for accelerated elimination. There is currently no advanced vaccine forP. vivaxand only a limited number of potential candidates in the pipeline. In this study we aimed to identify promisingP. vivaxproteins that could be used as part of a subunit vaccination approach. We screened 342P. vivaxprotein 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 theseP. vivaxproteins in further studies to confirm their potential and identify functional mechanisms of protection against clinical disease are warranted.

Much about the range of pathogens, frequency of coinfection, and clinical effects of reproductive tract infections (RTIs) among pregnant women remains unknown. We report on RTIs (Mycoplasma genitalium, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum subspecies pallidum, bacterial vaginosis, and vulvovaginal candidiasis) and other reproductive health indicators in 699 pregnant women in Papua New Guinea during 2015-2017. We found M. genitalium, an emerging pathogen in Papua New Guinea, in 12.5% of participants. These infections showed no evidence of macrolide resistance. In total, 74.1% of pregnant women had >= 1 RTI; most of these infections were treatable. We detected sexually transmitted infections (excluding syphilis) in 37.7% of women. Our findings showed that syndromic management of infections is greatly inadequate. In total, 98.4% of women had never used barrier contraception. These findings will inform efforts to improve reproductive healthcare in Papua New Guinea.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Introduction Left untreated, sexually transmitted and genital infections (henceforth STIs) in pregnancy can lead to serious adverse outcomes for mother and child. Papua New Guinea (PNG) has among the highest prevalence of curable STIs including syphilis, chlamydia, gonorrhoea, trichomoniasis and bacterial vaginosis, and high neonatal mortality rates. Diagnosis and treatment of these STIs in PNG rely on syndromic management. Advances in STI diagnostics through point-of-care (PoC) testing using GeneXpert technology hold promise for resource-constrained countries such as PNG. This paper describes the planned economic evaluation of a cluster-randomised cross-over trial comparing antenatal PoC testing and immediate treatment of curable STIs with standard antenatal care in two provinces in PNG. Methods and analysis Cost-effectiveness of the PoC intervention compared with standard antenatal care will be assessed prospectively over the trial period (2017–2021) from societal and provider perspectives. Incremental cost-effectiveness ratios will be calculated for the primary health outcome, a composite measure of the proportion of either preterm birth and/or low birth weight; for life years saved; for disability-adjusted life years averted; and for non-health benefits (financial risk protection and improved health equity). Scenario analyses will be conducted to identify scale-up options, and budget impact analysis will be undertaken to understand short-term financial impacts of intervention adoption on the national budget. Deterministic and probabilistic sensitivity analysis will be conducted to account for uncertainty in key model inputs. Ethics and dissemination This study has ethical approval from the Institutional Review Board of the PNG Institute of Medical Research; the Medical Research Advisory Committee of the PNG National Department of Health; the Human Research Ethics Committee of the University of New South Wales; and the Research Ethics Committee of the London School of Hygiene and Tropical Medicine. Findings will be disseminated through national stakeholder meetings, conferences, peer-reviewed publications and policy briefs. Trial registration number ISRCTN37134032.

Abstract Background Children in Papua New Guinea (PNG) experience high rates of malnutrition and poor growth - nearly half of children under 5 are stunted and 16% wasted. Methods We investigated predictors of infant growth over the first year of life using longitudinal data from mothers and infants in PNG. Between 2015 and 2018, 699 pregnant women were enrolled. At delivery, one, 6- and 12-months post-partum blood samples and anthropometric measurements were taken from mothers and infants. Using structural equation modelling with full information maximum likelihood, multivariate latent growth curve (LGC) modelling for infant weight and length (i.e. simultaneous estimation) was undertaken, and maternal factors that influenced growth investigated. Results A quadratic function for growth (weight and height) was estimated. Boys were larger at birth (49cm, 3.2kg vs. 48cm, 3.0kg; Wald χ2(2) =15.3, p<0.001) and gained more weight and length monthly (Wald χ2(4) =68.4, p<0.001). Maternal height, MUAC and number of antenatal healthcare visits were associated with birth weight and length, but not growth. Maternal nutrition and infections, breastfeeding and complementary feeding were not associated with birth size or growth. Conclusions Maternal height and MUAC and antenatal healthcare were associated with birth size and no maternal factors were associated with growth. Prenatal interventions to improve postnatal infant growth may be challenging in this environment Key messages Compared to conventional LGC analysis, multivariate LGC modelling using SEM provides less biased estimates of infant growth and factors associated with growth, particularly in the presence of missing data and infant-specific weight and height heterogeneity.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Unintended pregnancy is a major driver of poor maternal and child health in resource-limited settings. Data on pregnancy intention and use of family planning (FP) is scarce in Papua New Guinea (PNG), but are needed to inform public health strategies to improve FP accessibility and uptake. Data from a facility-based cross-sectional sample of 699 pregnant women assessed prevalence and predictors of unintended pregnancy and modern FP use among pregnant women in East New Britain Province, PNG. More than half (55%) the women reported their pregnancy as unintended. Few (18%) reported ever having used a modern FP method, and knowledge of different methods was low. Being single, separated or divorced (AOR 9.66; 95% CI 3.27–28.54), educated to a tertiary or vocational level (AOR 1.78 CI 1.15–2.73), and gravidity > 1 (AOR 1.43 for each additional pregnancy CI 1.29–1.59) were associated with unintended pregnancy; being accompanied by a male partner to ANC was associated with a reduced unintended pregnancy (0.46 CI 0.30–0.73). Factors associated with modern FP use included male partner involvement (AOR 2.26 CI 1.39–3.67) and gravidity > 1 (AOR 1.54 for each additional pregnancy CI 1.36–1.74). FP use also varied by the facility women attended. Findings highlight an urgent need for targeted interventions to improve FP knowledge, uptake and access, and male partner involvement, to reduce unintended pregnancies and their complications.

Plasmodium vivax , the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi , another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

ABSTRACT There is a pressing need for detailed knowledge of the range of pathogens, extent of co-infection and clinical impact of reproductive tract infections (RTIs) among pregnant women. Here, we report on RTIs ( Mycoplasma genitalium, Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , Treponema pallidum subspecies pallidum, bacterial vaginosis and vulvovaginal candidiasis) and other sexual and reproductive health indicators among 699 pregnant women in Papua New Guinea (PNG). We found widespread M. genitalium infection (12.5% of women), the first time this pathogen has been reported in PNG, with no evidence of macrolide resistance. Most pregnant women (76.2%) had at least one RTI, most of which are treatable. Excluding syphilis, sexually-transmitted infections were detected in 37.8% women. Syndromic management of infections is greatly inadequate and there was remarkably little use of contraception; 98.4% report never having used barrier contraception. This work has implications for improving maternal and child health in PNG. ARTICLE SUMMARY LINE This first report of Mycoplasma genitalium in Papua New Guinea finds a high burden (12.5%) among 699 pregnant women. Additionally, more than one in two women were positive for a treatable reproductive tract infection associated with poor health outcomes.

Monitoring the genetic structure of malaria parasite populations has been proposed as a novel and sensitive approach to quantify the impact of malaria control and elimination efforts. Here we describe the first population genetic analysis of sympatric Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) populations following nationwide distribution of long-lasting insecticide treated nets (LLIN) in Papua New Guinea (PNG). Parasite isolates from serial cross-sectional studies pre-(2005-6) and post-LLIN (2010-2014) were genotyped using microsatellite markers. Despite parasite prevalence declining substantially in these communities (East Sepik: Pf =54.9-8.5%, Pv =35.7-5.6%, Madang: Pf =38.0-9.0%, Pv : 31.8-19.7%), genetically diverse and intermixing parasite populations remained. P. falciparum diversity declined modestly post-LLIN relative to pre-LLIN (East Sepik: R s = 7.1-6.4, H e = 0.77-0.71; Madang: R s= 8.2-6.1, H e = 0.79-0.71). Unexpectedly, population structure present in pre-LLIN populations was lost post-LLIN, suggesting that more frequent human movement between provinces may have contributed to higher gene flow between provinces. P. vivax prevalence initially declined but increased again in one province, yet diversity remained high throughout the study period (East Sepik: R s=11.4-9.3, H e=0.83-0.80; Madang: R s=12.2-14.5, H e=0.85-0.88). Although genetic differentiation values increased between provinces over time, no significant population structure was observed at any time point. For both species, the emergence of clonal transmission and significant multilocus linkage disequilibrium (mLD) due to increased focal inbreeding post-LLIN was a strong indicator of impact on the parasite population using these markers. After eight years of intensive malaria control in PNG and substantial prevalence decline the impact on parasite population diversity and structure detectable by microsatellite genotyping was limited.