Plasmodium knowlesi is now the main cause of human malaria within Malaysian Borneo. While data is increasingly available on symptomatic cases, little is known about community-level patterns of exposure and infection. Understanding the true burden of disease and associated risk factors within endemic communities is critical for informing evidence-based control measures.Primarily impacting poor, rural populations, the zoonotic malaria P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2–8.2%) of the population, compared with 16.1% (14.6–17.7%) and 12.6% (11.2–14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work  and higher levels of forest cover  and clearing  around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.We conducted comprehensive surveys in three areas where P. knowlesi and associated risk factors within endemic communities. Results indicate community–level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.This is the first study to describe serological exposure to  Plasmodium knowlesi is a species of malaria parasite found in wild macaque populations which is now the main cause of human malaria in Malaysian Borneo. Spread from macaques to people through infected mosquitoes, human P. knowlesi malaria cases have primarily been reported in adult men working in forests or plantations. However, little data is available on the extent of asymptomatic infections or people exposed to P. knowlesi not reporting to clinics. We conducted comprehensive surveys of three case study communities in Malaysian Borneo and Palawan, the Philippines with varying numbers of P. knowlesi cases reported. In addition to testing for infection, we measured species-specific antibody responses to P. knowlesi and other malaria species to identify exposed individuals. Few asymptomatic infections were detected and varying levels of P. knowlesi exposure was detected between sites. P. knowlesi exposure was identified in both men and women and associated with farm work and forest and clearing around the house. Spatial patterns and risk factors for P. knowlesi differed from other malaria species, highlighting the need for knowlesi specific disease control measures. Results suggest more people are exposed to P. knowlesi than are identified at clinics and exposure to P. knowlesi may occur in different demographic groups and geographic areas than previously reported. Plasmodium knowlesi in 2004 and advent of routine diagnosis of malaria cases by molecular methods, increasing numbers of human P. knowlesi cases have been reported in Southeast Asia and P. knowlesi is now the most common cause of human malaria in Malaysian Borneo [P. falciparum and vivax, the emergence of P. knowlesi presents a challenge to malaria elimination programmes. Despite increasing amounts of data available for symptomatic malaria cases presenting at hospital facilities, little is known about patterns of P. knowlesi exposure and infection at a community level [After the initial recognition of a large number of human cases of the zoonotic malaria n Borneo –3. AlthoP. knowlesi requires a detailed understanding of environmental and social risk factors. Carried by long and pig-tailed macaques (Macaca fasicularus and M. nemestrina), environmental changes affecting contact between people, mosquito vectors and simian hosts are believed to contribute to this apparent emergence of P. knowlesi in people [Anopheles balabacensis, the main knowlesi vector, has been associated with forest environments but is also found in peridomestic and agricultural areas [P. knowlesi infections, including in women and children, demographic groups comprising a minority of cases reported to facilities [Effectively targeting resources to identify and control n people , 6. Anopal areas , 8. Assoal areas . Additiocilities –14.P. falciparum and P. vivax e.g. [P.knowlesi have only recently been described[Patterns of community-level exposure can be assessed by the prevalence of specific antibodies against malaria parasites; these antibodies reflect exposure to previous infection and can be used to characterise the level of transmission and identify areas or groups with higher transmission . These svax e.g. , 20, 21bdescribed.P. knowlesi and other malaria species in three case study communities where P. knowlesi transmission has been reported; a largely deforested and highly fragmented site at Matunggong, Kudat, an area with large patches of secondary forest bordering large scale clearing for an oil palm plantation in Limbuak, Pulau Banggi in Sabah, Malaysia and an area with intact secondary forest and some remaining primary forest in Bacungan, Palawan, The Philippines . P. knowlesi is the main cause of reported human malaria in both the Matunggong and Limbuak sites while only few sporadic P. knowlesi cases have been reported from Bacungan [P. knowlesi in 2014 was 12 for Matunggong, 2 for Limbuak and 0 for Bacungan.This study aimed to characterise these community level patterns of serological exposure to and prevalence of asymptomatic parasitemia of lippines . These aBacungan –25. BaseThis study was approved by the Medical Research Sub-Committee of the Malaysian Ministry of Health (NMRR-14-713-21117), the Institutional Review Board of the Research Institute for Tropical Medicine, Philippines and the Research Ethics Committee of the London School of Hygiene and Tropical Medicine (8340). Written informed consent was obtained from all participants or parents or guardians and assent obtained from children under 18 in this study and all methods were performed in accordance with relevant guidelines and regulations.P. knowlesi cases and all households within these communities were enumerated and geo-located. All individuals were asked to participate in the study and consenting individuals were interviewed on demographic characteristics, movement patterns, malaria prevention methods and land use practices. Individuals were excluded if they were less than 3 months old, had not primarily resided in the area for the past month or could not be reached after three attempts to contact them, including during evenings and weekends. Finger-prick blood samples were collected to test for malaria infection and exposure; these included blood smears to detect malaria parasites by microscopy and approximately 200μl whole-blood specimens collected in a tube containing EDTA  and three 20μl spots stored on filter paper . Filter paper was dried and stored with desiccant at 4°C.This study involved comprehensive sampling of all individuals residing within the study areas. Study sites were selected based on the locations of previously reported clinical Plasmodium small subunit ribosomal RNA (ssRNA) was used to identify malaria infected individuals, as described by [Plasmodium genus were then screened using species specific primers targeting the ssRNA region; for P. knowlesi these included PkF1140 (5’-GATTCATCTATTAAAAATTTGCTTC-3’) and PkR1150 (5' GAGTTCTAATCTCCGGAGAGAAAAGA 3') for 35 cycles at 50°C, 72°C and 94°C. All products were visualised on a 2% agarose gel. PCR for malaria infection was performed at laboratories at the Universiti Sabah Malaysia in Malaysia and Research Institute for Tropical Medicine in the Philippines, with PCR validation of a subset of samples at the London School of Hygiene and Tropical Medicine in the UK.All blood smears were examined by trained malaria microscopists. DNA was extracted from filter paper or 10 μl blood pellets using the Chelex-100 boiling method and a nested polymerase chain reaction (PCR) method targeting the ribed by . This asP. vivax , P. falciparum  and PfMSP-119 (PMID: 8078519) and P.knowlesi SERA3 antigen 2[Pk serine repeat antigen (SERA) 3 antigen 2  is a novel recombinant protein, N-terminally located between positions 826–998 aa, inclusive. SERA3 (1079 aa) belongs to a multigene family whose members encode a papain-like cysteine protease domain (ref: PMID: 21423628). In P falciparum, the N-terminal domain of SERA 5 is showing promise as a potential vaccine candidate . The recombinant protein was expressed in Escherichia coli and affinity purified by a GST tag. Knowlesi -exposed hospital clinical case control samples showed antigen specific reactivity to the SERA3 antigen 2 recombinant when compared to responses from European malaria naïve and Ethiopian vivax-exposed serum samples  Eluates were tested in duplicate at a final concentration of 1:1000 for all antigens except 1:2000 for PfAMA-1. In addition, blank wells and a dilution series of the appropriate positive plasma pool were added per plate. Positive controls based on a hyper-immune endemic adult Tanzanian pool (PMID: 15792998), a lyophilised anti-malaria patient sample  and pooled Pk-exposed hospital serum samples were used to assay for P. falciparum, P. vivax and P. knowlesi antigens, respectively. Polyclonal rabbit anti-human IgG-HRP  was used at 1/15,000 dilution and plates were developed using TMB . Optical density (OD) values were measured at 450 nm with a microplate reader. Values in excess of 1.5 CV between duplicates were considered fails and re-ran. OD values were corrected by subtracting the background of the blank well per plate. For P. falciparum and P. vivax OD readings, values were normalised between plates using a standardised control. Normalisation was not done for P. knowlesi results due to the lack of standard control. All serological analysis was performed at the Universiti Malaysia Sabah and the London School of Hygiene and Tropical Medicine.Enzyme-linked immunosorbent assays (ELISA) were performed as previously described . BrieflyAll households and roads within these areas were geo-located using a hand-held GPS . Land cover maps were derived from LANDSAT 8 30m resolution satellite images  and supehttp://www.R-project.org).These classified land cover maps were used to calculate distance from the household to the forest edge. The proportions of different land types surrounding all households were evaluated for 100m, 500m and 1000m buffer radii. Additionally, the level of forest fragmentation was assessed within 500m and 1000m of each household; this was represented as the ratio of forest perimeter to forest area as described by . All geoP. falciparum and P. vivax as described by [P.knowlesi antigen a more parsimonious cutoff value was defined as the mean OD plus 5 standard deviations due to a lack of prior data. Because the assays were run in different laboratories, cut off values were defined separately for each antigen, malaria species and location . For P. falciparum and P. vivax, individuals were considered positive if they were positive for either MSP-1 and/or AMA-1. Reversible catalytic models were fit to age sero-prevalence data using maximum likelihood methods; these models were then used to generate age sero-prevalence curves and estimate the seroconversion rate (SCR) [Questionnaire data was collected electronically using Pendragon Forms VI  and analysed using R statistical software. To define sero-positive individuals, mixture models were fit for normalised optical densities (ODs), with the distribution of ODs modelled as two Gaussian distributions. Cut off values to define sero-prevalence for each antigen were defined as the mean OD of the sero-negative population plus 3 standard deviations for ribed by . For thete (SCR) . Evidencte (SCR) . Models P. knowlesi sero-positivity were evaluated using multivariate logistic regression, with household included as a random effect to account for correlation between individuals from the same household. An additional model was developed to compare individuals sero-positive for P. knowlesi with those sero-positive for non-zoonotic malaria species. Explanatory variables included age, gender, site, individual and household level farming activities, residence in the area, elevation and distance to forest. Additionally, the proportions and configuration of different land types were extracted for each household at 100m, 500m and 1000m radii and categorised as greater or less than 30% coverage within a specific radius in the final model. Univariate analysis was conducted for all explanatory variables and variables with p < 0.2 were included in multivariate analyses. For highly correlated variables (such as land cover proportions at different radii), single variables were selected based on marginal increases in Akaike Information Criterion (AIC). The final adjusted models were developed by retaining all variables significant at a 0.05 level and variables were added in a forward stepwise fashion to check for interactions. Potential residual spatial autocorrelation of exposure to P. knowlesi was assessed separately for all sites using Moran’s I.Risk factors associated with P. knowlesi and nonzoonotic malaria species was explored through correlograms, plots of spatial autocorrelation with lag distances. First, ODs were log-transformed and adjusted for age by linear regression as described by [P.knowlesi and each other antigen were plotted. Correlation ranges were determined by significance values (p < 0.05) of individual bins of lag distances of 500m. Pairwise correlation between antibody responses was determined using a simple Mantel test to test the significance of associations [Correlation between spatial patterns of exposure to ribed by . For eacciations , 36.P. knowlesi cases were reported from the Bacungan study site while one P. knowlesi case was reported in the Limbuak site and two cases were reported in Matunggong site. The median age of participants was 24 years (age range 3 months– 99 years) and similar proportions of men and women were sampled in all study sites. While only 22% (538/2503) of individuals reported their primary occupation as farming or plantation work, the majority of individuals  reported their household engaged in some agricultural activities  and Matunggong (n = 1162) sites in Sabah and in September 2014 in Bacungan, Palawan (n = 546). During this time, no clinical tivities . The protivities . The MatP. knowlesi mono-infections by PCR. All PCR infections were re-confirmed at the laboratory in the U.K. Both of these individuals were male plantation workers (ages 21 and 25) residing in the same household. An additional two individuals in Matunggong were microscopy negative but identified as P. knowlesi infected by molecular methods; these included a three year old girl and 33 year old woman residing in different villages within the study site. Only one out of these four infected individuals identified self-reported history of fever. None of the survey participants in either the Limbuak or Bacungan sites were positive by microscopy or PCR and no infections with any other malaria species were identified in any participants.Two microscopy positive individuals were identified from the Matunggong, Kudat site; these were both subsequently identified as P. knowlesi  followed by 6.8% (79/1162) in Matunggong Kudat. Bacungan, Palawan had the lowest sero-prevalence . Similar reactivity to P. knowlesi was observed in men : med: 0.035, IQR: 0.006–0.094) and women  and gender was not significantly associated with P. knowlesi sero-positivity .Overall, 7.1% (178/2503) of the population surveyed was seropositive to knowlesi . ExposurP. falciparum and P. vivax were higher in all sites, with 16.1% (364/2266) of individuals sero-positive to one or both P. falciparum antigens and 12.6% (270/2141) positive for one or more P. vivax antigens. Sero-prevalence to P. falciparum was 16.9% (196/1162) in Matunggong, 13.5% (107/795) in Limbuak and 10.4% (61/587) in Bacungan. In contrast, reactivity to P. vivax was highest in Limbuak  with sero-prevalences of 6.9% (80/1162) and 9.7% (57/587) in Matunggong and Bacungan respectively. Due to insufficient samples and non-systematic errors in labelling, results for all antigens were not available for all individuals. Out of individuals with complete test results for all antigens, 25.7% (499/1941) of participants were sero-positive to at least one species of malaria and 7.9% (54/1941) were sero-positive for two or more malaria species. Of individuals exposed to P. knowlesi, 29.7% (53/ 178) were also positive for P. falciparum or P. vivax antigens. There was no evidence of correlation between P. knowlesi and other antigens tested  had antibodies to P. falciparum and 2.9% (23/792) to P. vivax. Changes in age sero-prevalence were more pronounced for P. falciparum and P. vivax, with 32.9% (78/237) and 28.1% (64/228) reactivity to P. falciparum and P. vivax in individuals over the age of 60 years. In contrast, antibodies for P. knowlesi were detected in 9.4% (25/265) of individuals over 60 years old and the highest sero-prevalence was detected in adults from 45–60 years old . As reactivity to P. knowlesi was low and not evenly distributed through the population, seroconversion rates (SCR) for P. knowlesi could not be calculated. Historical changes in falciparum transmission intensity were apparent in all sites and SCR models fitted with two forces of infection suggest substantial reductions in P. falciparum transmission occurred 18–30 years ago (p < 0.05)  . Strong P. knowlesi sero-positivity . There was no significant spatial correlation detected between age-adjusted antibody responses for P. knowlesi and other malaria species for either Matunggong or Limbuak . Comparisons between P. knowlesi and other malaria species could not be evaluated for Bacungan due to the low prevalence of P. knowlesi sero-positivity.Based on Moran’s I, there was no evidence of residual spatial autocorrelation for P. knowlesi through antigen specific antibody responses and associated risk factors and is one of few studies to assess P. knowlesi carriage prevalence at a community level. Results indicate spatial and temporal patterns of P. knowlesi transmission differ markedly from other non-zoonotic malaria species within the region. Although P. knowlesi sero-positivity was associated with some landscape attributes within these communities, extensive cross sectional surveys are needed to identify ecological risk factors across a broader geographic scale.This is the first study to describe exposure to P. knowlesi transmission intensity between sites. Although formal comparisons between P. knowlesi infection and exposure could not be undertaken due to the low prevalence of parasite carriage, these geographical differences in transmission mirror hospital-based reporting rates in the study sites at Kudat, Pulau Banggi and Palawan [P. knowlesi and P. vivax indicate the possibility of cross reactivity between antigens, relatively low numbers of individuals were identified as sero-positive for both knowlesi and vivax  and individuals could have been plausibly exposed to both species due to the co-endemicity of these species within this region. Additional work has been done to characterise response to P. knowlesi in vivax-exposed individuals and validate these antigens for population-based studies[Sero-prevalence data indicate distinct heterogeneities in  Palawan –25. Thesd studies.P. falciparum and P. vivax cases within the past 20 years following the scale up of malaria control and elimination programmes [P. falciparum[P. knowlesi exposure was identified in children under 5 in all sites, suggesting recent or on-going transmission albeit at a low level. Further work is needed to refine P. knowlesi serological analysis to allow for antigenic variation, identify further antigenic targets and assess the differential responsiveness of individuals and longevity of antibody responses [Changes in seroconversion rates can also reflect temporal changes in malaria transmission. In Sabah, state-wide malaria notification records describe dramatic decreases in clinical alciparum. These cesponses .P. knowlesi, levels of exposure between different demographic groups varied markedly from clinical case reports. While clinical P. knowlesi has been commonly reported in adult men, men and women had similar antibody reactivity to P. knowlesi antigens in all sites [P. knowlesi infection, more men could develop clinical infections due to higher exposure or number of bites; however, this requires further research to assess [P. knowlesi affects groups which may be underrepresented by current passive surveillance systems.Despite these similarities between existing case data and community-level exposure to ll sites , 39. Witll sites . Asymptoll sites , 12. As o assess . Larger P. knowlesi exposure was also associated with landscape factors. Both the proportion of forest cover and cleared areas around the household were positively associated with knowlesi sero-positivity, potentially reflecting the higher vectorial capacity and sporozoite rates reported in secondary forest within these study sites [P. knowlesi and forest activities, data on movement into different environments was not available for all survey participants [dy sites . Althougicipants , 41. Insicipants , 42, 43.P. knowlesi were more likely to reside in areas with higher proportions of forest cover; this may reflect differences in disease dynamics between species or temporal changes in transmission. Because of the longevity of antibody responses and the rapid rates of land use change within these areas, seroreactivity to non-zoonotic species is probably more likely to be associated with past rather than current environmental factors. The main mosquito vector species of P. knowlesi, Anopheles balabacensis, was historically incriminated as the main vector of other human malaria species within these same areas [These spatial patterns differed markedly from exposure to other non-zoonotic malaria species. Individuals with antibodies to me areas , 45. Whime areas , 42, 46.me areas .P. knowlesi risks across wider populations. As this study surveyed three relatively homogenous populations, there was minimal variation in environment, ethnicity, socioeconomic status and access to healthcare within each site. Identifying environmental and population-level risk factors will require randomised sampling across a wider ecological gradient; community level data on presence and absence of exposure and infection are required to understand spatial heterogeneity of disease transmission and develop and refine predictions of disease risk [The main limitations of this study are the non-randomised population sampling approach and limited geographical scale. While this study describes fine scale patterns of malaria exposure and infection within these three case study communities, these results cannot be generalised to extrapolate ase risk . AdditioP. knowlesi infection and exposure within these communities and illustrates how serologic markers can be used to describe differences in transmission intensity between malaria species in low transmission settings. Results from these surveys indicate patterns of P. knowlesi exposure and infection within the community may be substantially different from cases detected by passive surveillance systems. Cross sectional surveys across a broader geographical scale are needed to describe spatial variation in transmission intensity and identify associated environmental and population-based risk factors. Integration of serology into these surveys would provide vital information on rare infections for control programmes [Despite these limitations, this study describes ogrammes .S1 Checklist(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigP. falciparum AMA-1 antibody density; b. P. falciparum MSP-1 antibody density.a. (TIF)Click here for additional data file.S4 FigP. vivax AMA-1 antibody density; b. P. vivax MSP-1 antibody density.a. (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


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.The Total IgG and IgG subclasses 1, 2, and 3 that recognize four alleles of PvDBPII , 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  were also tested in a subset of children. The association of presence of IgG with age, cumulative exposure  and prospective risk of clinical malaria were evaluated.P. vivax episodes . High levels of total IgG and IgG1 to PvEBPII correlated strongly with protection against clinical vivax malaria compared with IgGs against all PvDBPII variants . Antibodies to PvDBPII AH and PvEBPII showed evidence of an additive effect, with a joint protective association of 70%.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 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.Antibodies to the key parasite invasion ligands PvDBPII and PvEBPII are good correlates of protection against  Plasmodium vivax is responsible for most malaria infections outside Africa, with 13.8 million vivax malaria cases reported annually worldwide. Antibodies are a key component of the host response to P. vivax infection, and their study can assist in identifying suitable vaccine candidates and serological biomarkers for malaria surveillance. The binding of P. vivax Duffy binding protein region II (PvDBPII) to the Duffy Antigen Receptor for Chemokines (DARC) is critical for P. vivax invasion of reticulocytes. Although the binding residues for DARC are highly conserved across PvDBPII, the parasite displays high sequence diversity in non-binding residues of PvDBPII. Other regions such as PvDBPIII-V are relatively conserved. Recently, sequencing of P. vivax field isolates, identified a homologous erythrocyte-binding protein (PvEBP), which harbors a domain, region II (PvEBPII), that is homologous to PvDBPII. To date, there has been limited investigation into the naturally acquired immunity to both PvDBPIII-V and PvEBPII in human populations. Using a longitudinal cohort study, we have characterized the serological response to PvDBPII, PvDBPIII-V, and PvEBPII among 1–3 years old PNG children and investigated associations with protection against clinical malaria. This study shows that both total IgG and IgG3 to the predominant PvDBPII AH allele in PNG, and total IgG and IgG1 to PvEBPII were associated with protection from P. vivax malaria. Plasmodium vivax, which is the most widely distributed plasmodium species that infects humans [P. vivax to relapse from dormant stages in the liver [P. vivax are urgently needed [s humans , is conshe liver . These lhe liver . Thus, ay needed . Vacciney needed , it willP. vivax preferentially invades young red blood cells called reticulocytes. Invasion into these cells relies on the interaction between parasite proteins and reticulocyte receptors. A well-characterized ligand-receptor pair involved in invasion is the interaction of P. vivax Duffy binding protein (PvDBP) with the Duffy Antigen Receptor for Chemokines (DARC) [P. vivax malaria in West Africa, where populations are generally DARC negative, highlights the central role of this pathway in P. vivax infection [s (DARC) . The virnfection . PvDBP infection , 9. It hnfection . No funcnfection , the potP. vivax controls parasite densities leading to reduced risk of clinical disease in the second and third year of life [P. vivax invasion of reticulocytes provide the rationale of PvDBP as promising vaccine candidate [P. vivax endemicity, the most predominant allele of PvDBPII is AH with a proportion of 26% in circulating strains [P. vivax strains circulating in PNG [P. vivax infection and reduced parasite densities [P. vivax malaria in young children in PNG of 1–3 years age.In highly endemic areas of PNG, naturally acquired immunity against  of life . Immune  of life . Strong  of life . In addiandidate . Howeverandidate , 17. In  strains . In parag in PNG . When ang in PNG . The preensities , 19. Altensities . The reaPlasmodium erythrocyte-binding protein, including a N-terminal signal peptide, a Duffy-binding like domain , a C-terminal cysteine-rich domain, and a transmembrane domain [P. vivax antigens in plasma from naturally exposed children in PNG, antibodies to both PvDBPII and PvEBPII were frequently identified among five-antigen combinations with the strongest protective effects against clinical malaria [Whole genome sequencing of Cambodian field isolates identified a second putative erythrocyte binding protein (PvEBP) with all the features of a e domain . Althouge domain . The gene domain . In a re malaria . A more P. falciparum antigens [Plasmodium antigens, a switch from a predominant IgG1 response in young children to an increase or even predominance of IgG3 response in older individuals is a characteristic feature of natural acquisition of clinical immunity to malaria [The IgG isotype determines antibody function, and in humans, cytophilic IgG1 and IgG3 are important mediators of pathogen clearance. Numerous studies have reported that IgG subclass profiles differ among antibodies targeting different antigens –27. The antigens . In addi malaria –30. It rPlasmodium infection is considered to be one of the key driving forces of the evolution of the human genome. Polymorphisms in RBC proteins are particularly common in malaria endemic regions [GYPCΔex3) [Plasmodium infection [P. falciparum infection among the population with this phenotype [P. vivax and its relationship with the acquisition of immunity remains unknown. regions –33. GerbYPCΔex3) . GerbichYPCΔex3) . HoweverYPCΔex3) . Few stunfection –39, withhenotype . Its potP. vivax infection, clinical episodes, and Gerbich negativity was explored.In this study, parameters of naturally acquired immunity to four variants of PvDBP , as well as PvEBPII were characterized in PNG children of 1–3 years of age from a 16-month longitudinal cohort study. In addition, levels of total IgG to PvDBPII, IgG subclass, and presence of anti-PvDBPII BIAbs were measured and their association with P. vivax infections were genotyped, allowing for the calculation of the incidence of genetically distinct blood-stage infections acquired during follow-up  [GYPCΔex3 deletion associated with the Gerbich blood group was done by PCR, as previously described [GYPCΔex3 deletion were considered to be Gerbich negative. Plasma samples collected at the start and at the end of the study from 224 children were used in the present study.Plasma samples used in the current study were collected as part of a longitudinal cohort study of young PNG children (1 to 3 years old) previously described . In brie molFOB) . Host geescribed . ChildreE. coli. Proteins were solubilized from inclusion bodies, purified by affinity chromatography, followed by refolding and ion exchange chromatography as per methods described earlier [E. coli. The protein was purified by metal affinity chromatography. Recombinant PvEBPII (aa 161–641) with a C-terminal 6-His tag was expressed as a soluble protein in E. coli SHuffle cells. Following cell lysis, the recombinant PvEBPII was purified from cell lysate as a soluble protein by metal affinity chromatography using standard procedures.The four PvDBP variants used in this study were binding domain II from strains AH, O, P and Sal1 . The rec earlier . An 11766 beads, we used 0.300 μg/mL of PvDBPII AH, 0.125 μg/mL of PvDBPII O, 0.094 μg/ mL of PvDBPII P, 0.225 μg/mL of PvDBPII Sal, 0.031 μg/mL of PvDBPIII-V and 0.250 μg/mL of PvEBPII. The Luminex multiplex bead-based antibody detection assay was performed as described elsewhere with the following modifications [Recombinant PvDBPII, PvDBPIII-V, and PvEBPII fragments were conjugated to Luminex Microplex microspheres as previously described . To conjications , 43. Plaications . NegativAn ELISA plate-based semi-quantitative binding assay was used to test binding of PvDBPII with DARC and estimate the binding inhibitory activity of serum as described earlier . Briefly−5 or 1/51200 to 0.02 or 1/50) using a five parametric logistic regression model as described previously [P. vivax episodes (defined as axillary temperature > 37.5°C or history of fever in the preceding 48 hours with a concurrent parasitemia > 500 P. vivax /μl) and prevalence of infection diagnosed by PCR and light microscopy over the 16 months of follow-up [GYPCΔex3 genotypes were analyzed using GEE.Standard curves from each Luminex assay plate were used for transformation of MFIs into relative antibody units . These scores were then added up for each different combination. The scores of any combination were equally divided into three groups and used in our GEE model.https://doi.org/10.5061/dryad.n14p52b [All datasets were available in the Dryad repository: .n14p52b .Ethics clearance was obtained from the PNG Medical Research and Advisory Committee (MRAC 05.19) and the Walter and Eliza Hall Institute (HREC 07/07) for the use of field samples. All parents/guardians of the participants signed a consent form prior to enrollment. The Melbourne control sample was obtained under ethics approval HREC 13/07.The pooled serum from immune PNG adults was assumed to represent the equilibrium antibody levels to all antigens achieved following repeated natural exposure. Here, we determined at enrollment the number of children who had already acquired IgG levels equivalent to >50%, >25%, >10%, >5%, or >1% of the IgG levels in adults . Plasma Total IgG levels were strongly correlated between all proteins measured at the beginning of the cohort study , with the strongest correlation found between PvDBPII AH and PvDBPII O variants. Total IgG to PvEBPII shows weak to moderate correlation with different PvDBP antigens (Spearman’s rho = 0.22–0.63) . SimilarP. vivax infection was 55.4% (124/224) among young PNG children at enrolment, as determined by PCR (P. vivax infection by PCR had significantly higher IgG levels (P<0.015) to all variants of PvDBPII, PvDBPIII-V and PvEBPII  had IgG1 levels exceeding 25% of adult levels . In contPCR-positive children had increased IgG1 for PvDBPII AH, PvDBPII O, and PvEBPII . ConsistP. vivax clinical episodes, respectively, compared to children with low antibody levels ; high versus low antibody levels   . IgG to  <0.001) . Therefo). Combinations of 3 antigens did not show an additional increase in protection  . In a multivariate model incorporating IgG1 and IgG3 for all antigens, only IgG3 to PvDBPII AH and IgG1 to PvEBPII remained associated with clinical protection   . For IgG≤ 0.006) .Plasma obtained at first (n = 8) and last visit (n = 8) exhibited substantial binding inhibitory antibodies against diverse PvDBPII alleles . BindingP. vivax infections showed moderate-high blocking activity . Although median blocking activity did not vary with age (P>0.19), five of the six children with high levels of inhibitory, strain-transcending antibodies were older than 21 months of age (P = 0.115).Twelve children (7.14% of 168 tested) had ≥60% blocking activity for at least one variant of PvDBPII. Eight children (4.76%) had high levels (≥80% blocking activity) of inhibition to one variant, six of which showed high blocking activity against all three variants. Children with concurrent P. vivax infections were no longer associated with an increase in blocking activity. Only three children (1.9%) had high blocking antibodies (≥80%) against all three variants at both time points. Among the three children with constant high blocking activity, two (66.7%) were homozygous for the Gerbich blood group (i.e. GYPCΔex3) compared to 14 (10.5%) in those with lower or no blocking activity (P = 0.036).Blocking activity in plasma samples collected at the end of follow-up was very similar to the start of the study . After 1P. vivax malaria, children with high blocking ability against AH , O , Sal1 , or all three alleles combined  at enrolment showed a tendency for a reduced incidence of P. vivax episodes of any density , except for PvEBPII (P = 0.501) . These rP. vivax. Antibody responses against PvDBPII were higher in those Gerbich-negative, a common red blood cell polymorphism within the East Sepik region of PNG. We also observed a strong association with protection for total IgG and IgG1 antibodies to PvEBPII, but no difference by Gerbich phenotype.This study confirmed that total IgG and infrequently detected BIAbs against PvDBPII were associated with an overall lower incidence rate of clinical vivax malaria in young children who were developing clinical immunity to P. vivax exposure indicates that these functional blocking antibodies are difficult to acquire under conditions of natural exposure. Nevertheless, since the target epitope of BIAbs to PvDBPII was conserved [In this study, only 4.8% (8/168) of the young children aged 1 to 3 years had acquired high levels of BIAbs (> 80% binding inhibition) against at least one PvDBPII allele, with six children exhibiting binding-inhibitory antibodies against diverse strains, while none of them had obtained levels higher than of 90% of BIAbs against any PvDBPII allele. An earlier study among school-age children (5–9 years) in PNG identified 9% of children with BIAbs >90% to PvDBPII  and in tonserved , once PvIn contrast to the DARC conserved binding residues, polymorphisms thought to be associated with immune evasion  are commP. falciparum showed that antibodies to regions III-V of PfEBA175 and PfEBA140 could inhibit P. falciparum invasion [Regions II and VI of PvDBP are cysteine-rich domains and under immune selection, while regions III to V do not have a known function . Neverthinvasion . Our stuP. vivax ligand with a stronger binding preference for Duffy-positive than Duffy-negative reticulocytes in vitro [Among all antigens tested, the strongest association of protection from vivax malaria was identified for PvEBPII. In one previous study, PvEBPII was characterized as a functionally and antigenically distinct in vitro , suggestSome polarization of IgG1 towards IgG3 was only identified for PvDBPII AH, as similar switch towards a balanced IgG1/IgG3 response was observed in the narrow age range of our study population. The possible reason for this might be related to the higher circulation of AH strain in this setting, thus young children might be exposed to the AH strain enabling them to acquire higher IgG3 to AH. Apart from the possible higher exposure, by comparing with other alleles included in this manuscript, AH was shown to have two special mutations K371E and K386Q, both of which were mapped to SD2. As most of the PvDBPII polymorphism are in this subdomain, our results suggest that these two mutations were important for immune reactivity to PvDBPII AH. Whereas, after a longer period of immune exposure to other strains, including O, P and Sal1, significantly higher levels of IgG3 to them were also observed in the adults. Consistent with previous reports , 28, theP. falciparum and/or P. vivax infection [P. vivax malaria among young children aged 1 to 3 years with limited clinical immunity. However, Gerbich negativity was not associated with significant protection against blood-stage infection in school-aged children (5–14 years) from another PNG longitudinal cohort study [SLC4A1Δ27 deletion in the human Band 3) were 3.3 times more likely than non-SAO children to have high levels of PvDBPII-specific BIAbs [In addition to antibody-mediated protection against vivax malaria, it is believed that specific red blood cell polymorphisms can induce resistance to clinical malaria . There infection . Our lonrt study . It is art study . In thisic BIAbs . The mecP. vivax malaria episodes in PNG children and further strengthens the rationale for PvDBPII as a potential vaccine antigen. Interestingly, both naturally acquired immunity to PvDBPII and Gerbich homozygosity showed significant protection against P. vivax. Antibodies to PvEBPII were more strongly association with clinical protection against P. vivax malaria in these young children. Further studies will be needed to clarify the mechanism of protection afforded by Gerbich and the importance of PvEBPII in P. vivax reticulocyte invasion and immune protection.In summary, our study highlights the association of total antibody and BIAbs to PvDBPII variants with lower risk of clinical S1 Checklist(DOC)Click here for additional data file.S1 FigThe heat map colors correspond to the Spearman’s correlation coefficient, ranging from 0  to 1 . P<0.001–0.152.(TIF)Click here for additional data file.S2 Fig(A) The comparison of antibody levels between enrolment and end of the cohort study. Red represents the first visit and blue represents the last visit. P values were calculated using Wilcoxon signed-rank sum method. P values <0.05 were considered significant. (B) Association of antibody levels from the last visit of the study and the molecular force of blood stage infection (molFOB). The blue lines show the association between antibody responses and molFOB predicted by linear regression models. The shaded regions depict the variation in the data . X-axis: molFOB, y-axis: total IgG antibody responses for each antigen. P values are from general linear model. P values and were deemed significant if <0.05.(TIF)Click here for additional data file.S3 FigAntibody levels of crude mean fluorescence intensity (MFI) were log10 transformed. Solid lines represent antibody levels among adults in a two-fold serial dilution starting from 1/50. Only the median antibody levels among children for each subclass  were presented by dashed lines.(TIF)Click here for additional data file.S4 FigAntibody levels of crude mean fluorescence intensity (MFI) were shown in X axis and count of each level were represented in Y axis. Antibody levels of total IgG, IgG1, IgG2 and IgG3 were depicted in pink, yellow, blue, and light slate blue respectively.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Plasmodium spp. detection in blood units must have a high detection threshold, and the prevention of TTM relies entirely on the exclusion of potentially infected donors. However, in Brazilian blood banks, the screening test relies on blood thick smears examination.Malaria can be transmitted by blood transfusion through donations collected from asymptomatic or parasitic donors. The parasites are released into the bloodstream during its life cycle and will therefore be present in donated blood by infected individuals. All cases of transfusion-transmitted malaria (TTM) notified since 2005 in Brazil were fatal. A good screening tool for Plasmodium falciparum, Plasmodium vivax, and standardized for use in Plasmodium malariae. The analytic sensitivity of this mt-qPCR methodology was performed using a sample of P. vivax.The molecular diagnostic based on mitochondrial DNA (mtDNA) using real time PCR (mt-qPCR) was improved to detect P. vivax was determined (0.000006 parasites/µL). This method was tested to detect P. vivax and P. falciparum in individuals from two malaria-endemic areas in Brazil, Amazon region (Pará and Rondônia states), the samples were collected in 10 reference units of two blood banks (Pará/nine cities and Rondônia/Porto Velho), and parasites mtDNA were detected in 10 of 2224 potential blood donors (0.45%). In all 10 positive samples, only P. vivax was detected.The mt-qPCR was highly efficient, and the analytic sensitivity for Molecular diagnostic using mt-qPCR was effective in revealing infected potential donors with good perspectives to be applied as screening routine of asymptomatic carriers for preventing transfusion-transmitted malaria in blood banks. Plasmodium genus. In addition to being primarily transmitted by bites of female mosquitoes of Anopheles species, malaria is readily transmitted by blood transfusion through donations collected from asymptomatic or parasitic donors, especially those individuals with low parasite densities [Malaria is an infectious disease caused by parasites of the ensities –4. The pensities –7.The erythrocyte concentrate is the main blood component involved in transfusion-transmitted malaria (TTM), but there are reports of transmission by fresh frozen plasma, cryoprecipitate and platelets concentrate. When preserved in blood units, the parasite remains viable for a long period of time, even after storage. After transfusion, the disease begins in a matter of days or weeks, with severe and often fatal evolution, especially in immunodepressed individuals, young children, pregnant women, and people who have never been exposed to malaria parasites , 7.Plasmodium genus that infect humans, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi [Haemovigilance is required to identify and prevent the occurrence or recurrence of unwanted transfusion-related transmissions and to increase the safety, efficacy and efficiency of blood transfusions . This viknowlesi , 9.Plasmodium infections through blood transfusion [Malaria transmitted by blood transfusions has been described since the early 1900s. The first report on malaria as a consequence of an artery-to-vein blood transfusion was described in 1910 by Woolsey , 10. Hownsfusion . HoweverPlasmodium infections is essential for malaria control or eradication [Plasmodium, but this test has limited sensitivity to detect asymptomatic infections with low parasitaemia [Plasmodium species in the case of individuals with low parasitaemia [An accurate diagnosis of dication , 9, 12. sitaemia . The lacsitaemia .P. falciparum and P. vivax [In this context, for the first time, an innovative strategy was developed to detect the malaria parasites based on mitochondrial DNA (mtDNA) of malaria parasites, P. vivax . EffortsP. vivax . After dP. vivax –21 or loP. vivax , 23, theP. vivax .P. falciparum, and even its aggressive implementation has few lasting impacts upon endemic P. vivax [Historically, the conventional malaria control has mostly taken aim at P. vivax , 12. In P. vivax , 25. In P. vivax .P. vivax is the main species. In the past two decades, the numbers of malaria cases caused by this parasite have varied between 100,000 and 500,000 per year, representing approximately 85% of all reported cases [P. vivax is able to develop latency stages (hypnozoites) in the liver, which leads to relapses, and its life cycle occurs inside the reticulocytes, these factors may contribute to an increase in the number of individuals with low parasite load, which are not usually detected by healthcare facilities [Plasmodium infection has been reported [In Brazil, malaria is endemic in the Amazon region, where ed cases , 27. Beccilities , 25. Morreported . This isP. falciparum, P. vivax and P. malariae. In addition, for the P. vivax parasite, the prevalence of positive samples was determined as units for TTM occurrence upon the transfusion of blood or its components from infected donors living in malaria-endemic urban areas in Brazil.In this study, the performance of molecular approach was improved for detection of malaria parasites via mitochondrial DNA as a careful screening test of blood samples from donors, and compared the more sensitive molecular technique with those used in haemovigilance as the best strategies for detecting P. vivax  and P. malariae (400 parasites/µL) were obtained from patients with malaria. The positive sample of the 3D7 strain of P. falciparum was provided by the Evandro Chagas Institute/Ministry of health, Brazil. Samples of potential donors were collected in two blood bank centres. Among them, 1324 were obtained from nine reference units of the Fundação Centro de Hemoterapia e Hematologia do Estado do Pará (HEMOPA) and 900 were collected from blood donors in Porto Velho, the capital of the state of Rondônia. In samples collected in the state of Pará, the frequency of malaria parasites in potential donors was analysed according to age and annual parasite index (API), the gender ratio was approximately 4 males to 1 female.Positive samples of DNA samples were extracted from 150 µL blood by phenol/chloroform followed by ethanol precipitation method, with modifications . Then, DA qPCR was performed to determine the presence and absence of the malaria parasite mtDNA, based on primer extension and posterior hybridization with TaqMan probes on ABI Prism 7.500 sequence detection system  version 2.0.6 software, according to the manufacturer’s instructions.Plasmodium evaluated in this study. The probes identified the species P. falciparum (COX III—access n. GI8346992 and M76611), P. vivax (COX I—access n. GI63022502). The qPCR assay amplified mtDNA which was a part of the sequence generated by the primers as published by Cunha et al. [P. malariae (COX I—access n. KC175322.1), 3 different qPCR assays were performed.The probes were designed as described by Batista-dos-Santos et al. , targetia et al. , and P. As negative control, ultra-pure water previously tested for mitochondrial qPCR (mt-qPCR) was employed and a molecule of template DNA was used as endogenous positive control.P. vivax previously evaluated and quantified. For this, a serial dilution was performed in ultra-pure water (DF 10) until the concentration of 1:100,000, in triplicate, as previously described by Gama et al. [Evaluation of analytic sensitivity of the mt-qPCR methodology was performed using a sample of a et al. , using sa et al. , 32.3. After extraction, the pellet was precipitated in a volume of 50 µL (~ 600 parasites/µL). In addition, the solution was diluted to 1:1000 in ultra-pure water, which corresponded to approximately ~ 0.6 parasites/µL.The aliquot of 150 µL of blood was withdrawn from the sample containing 200 parasites/mmP. malariae, and the detection threshold was determined in comparison with P. falciparum and P. vivax using this molecular diagnosis. The amount of amplicon obtained by the amplification of mtDNA from malaria parasites showed variability according to the parasitaemia density of each Plasmodium species tested. This assay showed good reproducibility when tested for all three dilutions of each sample . The serial dilutions were established as follows: (i) the first solution contained the equivalent of 0.6 parasites/μL; (ii) the second solution contained 0.06 parasites/μL; (iii) the third solution had 0.006 parasites/μL; (iv) the fourth solution had 0.0006 parasites/μL; (v) the fifth solution had 0.00006 parasites/μL; and, (vi) the sixth solution contained 0.000006 parasites/μL . The detection thresholds for the P. falciparum and P. malariae species were not determined.The technique of amplification by mitochondrial qPCR was able to detect P. vivax mtDNA was detected. The distribution of blood donors by city and the prevalence of detected malaria parasites is shown in Table Plasmodium was observed in only 3 blood banks, and the numbers of positive samples were 5, 3 and 2 for those collected in Abaetetuba (Pará), Santarém (Pará) and Porto Velho (Rondônia), respectively. Regarding samples collected in Porto Velho, P. vivax was detected using both molecular diagnostic methods (mtDNA and 18S rRNA). The percentage of positive samples was 3.0, 1.5 and 0.2% for the Abaetetuba, Santarém and Porto Velho donor groups, respectively. All tested samples were negative for P. falciparum.A total of 2224 samples collected from blood donors were tested by molecular diagnosis using mt-qPCR. All participants were permanent residents of two different states in the Brazilian Amazon region. Of these, 1324 were collected from different blood banks of the state of Pará, and 900 were from blood donors from Porto Velho, the capital of the state of Rondônia. Malaria parasites were detected in 10 of 2224 blood donors (0.45%). In all 10 positive samples, only TTM is recognized in many countries where malaria transmission has been eliminated . AlthougConversely, the risk of transmitting malaria by blood transfusion in countries where parasite transmission is endemic can be high, and the healthcare systems clearly need alternative measures to prevented TTM, including tools for detecting the parasite or its components in the blood of potentially infected donors , 7, 11.Although the exact numbers are unknown, the frequency of TTM has been estimated as 1 case per 4 million units donated in non-endemic areas , 37. MorMalaria screening is mandatory in endemic regions and the directives of the Brazilian National Health Surveillance Agency (ANVISA) and the Brazilian Ministry of Health require high sensitivity tests. In spite of this, malaria screening in Brazilian blood banks relies on questionnaire data followed by microscopic examination of thick smears, which has several limitations, such as low counts of parasitaemia in asymptomatic patients which may lead to liability to human error .P. falciparum and P. vivax, respectively. The conventional PCR methodology was changed to a more sensitive and semi-automated technique using one-step qPCR and the same mtDNA sequences. Additionally, mt-qPCR specific to P. malariae was standardized. The conventional PCR identified small amplicons of 273 pb  and 290 pb (P. vivax) [In this study there was an improvement in the methodology developed by Cunha et al., which originally used a strategy based on mtDNA to amplify specific sequences of the Cytochrome C Oxidase genes, such as COXIII and COXI for . vivax)  allowing. vivax) .Plasmodium species  more frequently found as the infecting agents among the 5 species that cause human malaria. The results confirmed that potentially infected volunteers can be among the donors. Although the frequency was low, it represents an even more complicated issue considering that each donation can be distributed over erythrocyte concentrates, platelet concentrates, or serum or plasma/platelets concentrates. Plasmodium falciparum and Plasmodium vivax tests were included, but only samples infected with P. vivax were detected. This can be explained by the fact that this is an area of higher incidence of P. vivax. It is well-characterized as an endemic area where P. vivax causes approximately 85% of malaria episodes registered by the Brazilian Health System [This new methodology was applied for the detection of 3 h System , 27.P. malariae. This innovative approach, although only preliminarily tested one positive sample, proved to be a suitable screening tool for identification of infected patients. The detection threshold curve varied depending on the infecting species. This suggests that P. malariae, which frequently occur in low parasitaemia, can also be detected by mt-qPCR. Future studies may be conducted to validate this assay in a cross-sectional study. In Brazil, it is necessary because there are certain issues with the transmission of P. malariae that need to be addressed, which include a high prevalence of P. malariae infections in the Brazilian Amazon endemic area [Another contribution was the qPCR designed specifically for detecting mtDNA of mic area , as wellmic area . In bothThe major advantage of using mitochondrial DNA (mtDNA) in malaria diagnosis as originally reported  is the lP. vivax-infected patients were tested, and the estimated detection threshold was very low (0.000006 parasites/µL) when compared to the detection threshold of the nested PCR (0.004 parasites/µL) [Another important point to be considered is the volume of blood needed to obtain the DNA, since the amount of processed specimen determines the limit of detection . Some quites/µL) .P. vivax proved that it was possible to reach the hypothetical detection threshold to detect parasitaemias as low as 2.2 × 10−6 parasites/µL, as described by Hänscheid et al. [P. vivax at 2.4 × 10−5 parasites/4 µL (6.0 × 10−6 parasites/µL) density by analysing DNA from a suitable blood volume, thus achieving analytical sensitivity very close to the value estimated almost two decades ago [Two research groups discussed this issue and proposed a hypothetical detection threshold, using calculations that offered the hypothetical value as the threshold , 36. Thud et al.  in a comades ago , that isP. falciparum infections has illustrated how these infections have been studied for decades, and based on the data from these studies, it is possible to suggest that for blood transfusion-transmitted malaria, the inoculum should contain approximately 1000 parasites in a blood bag or bottle of approximately 500 mL. Thus, the transmission density approached an average of 2 parasites/mL [Malaria induced by blood transfusions from asymptomatic carriers is a relevant problem of haemovigilance. A review of the duration of sites/mL . It mustP. vivax) analysing only 150 µL out of a total of approximately 450 mL of blood.Finally, this analysis proved that it was possible to considerably improve the assay to detect the parasite mtDNA, compared to conventional PCR, where the detection threshold was 0.01 parasites/µL . Using mPrevention of TTM relies entirely on the exclusion of potentially infected donors. However, it can be very difficult since in many cases, the donor screening process is not efficient and fails to obtain accurate information on previous exposure to malaria and identification of the infected donors. It represents a situation that can particularly occur for donors in blood banks of endemic-malaria areas , 11, 39.Plasmodium species among donors that live in the Amazon region, which varies from 1 to 3%. Both studies included samples collected from donors of the blood banks of the Belém city, and the prevalence observed was similar, 1.34% [P. vivax [Analysing the frequency of positive donor detection by molecular diagnostic tools for samples from blood banks from the malaria-endemic area in Brazil, it was identified only two studies up until the present that determined the prevalence of positive samples of r, 1.34%  and 1% [r, 1.34% . It shouP. vivax , while oP. vivax . It can P. vivax . This moP. vivax –49.Plasmodium species in the blood banks of endemic areas or in micro-regions should be evaluated in addition to the possibility of reducing the risks of TTM. Malaria diagnostic research provides a basis for applied advances in malaria surveillance [A suitable screening tool for malaria detection in blood units must have high sensitivity and specificity; it must be fast and reduce the risk of TTM , 4. Conseillance , 13–15. In conclusion, qPCR is efficient for malaria molecular diagnostics, presenting promising results with good analytic sensitivity. It is a fast and easy molecular methodology to detect mtDNA of the three most frequent malaria parasites, presenting a potential for large-scale use in the prevention of transfusion-transmitted malaria by screening for potential donors as part of malaria haemovigilance in blood banks.


Plasmodium falciparum  parasites is a major hurdle to the control of malaria. This study monitored changes in the genetic diversity and the multiplicity of P. falciparum parasite infection in asymptomatic children living in southern Ghana at 3 month intervals between April 2015 and January 2016.Genetic diversity in merozoite surface protein 1 (msp 1) and merozoite surface protein 2 (msp 2) genes.Filter paper blood spots (DBS) were collected quarterly from children living in Obom, a community with perennial malaria transmission and Abura, a community with seasonal malaria transmission. Genomic DNA was extracted from the DBS and used in polymerase chain reaction (PCR)-based genotyping of the P. falciparum. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The geometric mean MOI in Abura ranged between 1.17 (95% CI: 1.08–1.28) and 1.48 (95% CI: 1.36–1.60) and was significantly lower  than that determined in Obom, where the geometric mean MOI ranged between 1.82 (95% CI: 1.58–2.08) and 2.50 (95% CI: 2.33–2.678) over the study period. Whilst the msp 1 R033:MAD20:KI allelic family ratio was dynamic, the msp 2 3D7:FC27 allelic family ratio remained relatively stable across the changing seasons in both sites.Out of a total of 787 samples that were collected from the two study sites, 59.2% (466/787) tested positive for msp 1 rather than msp 2 due to the constantly changing relative intra allelic frequencies observed in msp 1 and the fact that the dominance of any msp 2 allele was dependent on the transmission setting but not on the season as opposed to the dominance of any msp 1 allele, which was dependent on both the season and the transmission setting.This study shows that seasonal variations in parasite diversity in these communities can be better estimated by The online version of this article (10.1186/s12879-018-3350-z) contains supplementary material, which is available to authorized users. P. falciparum infections for informed interventions to be instituted [P. falciparum in high transmission areas with some infected people harboring multiple clones of the parasites. On the contrary, people living in low transmission areas tend to have low genetic diversity with a high proportion being monoclonal infections [The high prevalence of malaria in Ghana has resulted in a majority of adults and children older than 5 years attaining a semi-immune status due to past and continuous exposure to different parasite clones . Repeatestituted , 13. Thefections –17. Tranfections , 19. Thufections .merozoite surface protein 1 , merozoite surface protein 2  and the glutamate-rich protein  [msp 1 and msp 2 genes helps to measure MOI and transmission in a population. Although only a limited number of loci can be examined at a time and some minor parasite populations missed by conventional PCR [msp genotyping followed by agarose gel electrophoresis as their parasite diversity analysis tool [The most common and widely used tool for the estimation of parasite diversity is PCR based genotyping of a gene with high diversity such as 1035300) –25. The onal PCR  comparedonal PCR . Many resis tool –31.P. falciparum circulating in the middle belt of Ghana has been extensively characterized [The genetic diversity of cterized , 33. Howcterized , 31 or icterized , 35. ThiPlasmodium falciparum parasite prevalence in Obom has been noted to be higher than in Abura [The study was conducted in community primary schools in two areas of southern Ghana; Obom, a semi-rural community in the Ga South Municipality of the Greater Accra Region with high and perennial malaria transmission and Abura, a semi-urban community in the Cape Coast Municipality of the Central Region with low and seasonal malaria transmission . In 2010in Abura , 39.Fig.P. falciparum parasites. The same children were followed up during subsequent sampling visits.After obtaining parental consent, 237 children between the ages of 6 and 12 years were recruited in a multiple cross sectional study; 83 from Obom and 154 from Abura. Filter paper (Whatman® 3 mm) blood spots (DBS) were prepared from ~ 50 μL of finger pricked blood that was collected in April, June and October 2015 as well as in January 2016. The filter paper blots were air-dried and stored desiccated at room temperature. None of the children sampled exhibited any signs or symptoms of clinical malaria at the time of blood draw and were classified as either healthy with no PCR detectable parasites or asymptomatic, harboring PCR detectable g for 1 min), the extracted DNA was transferred into a labelled 0.6 mL microcentrifuge tube and then stored at − 20 °C.Total genomic DNA (gDNA) was extracted from the dried blood spot (DBS) using the Chelex extraction procedure , 41. BriP. falciparum [2, 200 nM dNTP mix, 200 nM rPLU5 and rPLU6 primer set  gene was used to detect lciparum . The 15 8S rRNA gmsp 1 block 2 and msp 2 block 3 were amplified using a protocol adapted from the WHO protocol for malaria parasite genotyping [2, 200 nM each of forward and reverse primers for both msp 1 and msp 2  for msp 1 KI and msp 2 FC27 alleles, MRA-155G (HB3) for msp 1 MAD20/msp 2 FC27 alleles and MRA-200G (R033) for msp 1 RO33/msp 2 3D7 alleles) were included in each set of PCR reactions. PCR products were resolved for an average of 50 min at 120 V on a 2% agarose gel stained with 0.5 μg/mL ethidium bromide. After the electrophoresis, the gels were visualized under UV trans-illumination using a Toyobo FAS-III gel doc system and then analyzed.The polymorphic regions of notyping  and siminotyping , 44. Allmsp 1 or the two msp 2 allelic family PCR reactions. A sample containing a clonal infection produced a single product after the three msp 1 PCR reactions as well as a single product after the two msp 2 reactions. A simplified method of estimating multiplicity of infection (MOI), where MOI of a sample was taken as the highest number of PCR products (amplicons) obtained after the family specific msp 1 or msp 2 PCR amplifications was used. Multiplicity of infection was generalized and reported as the geometric mean MOI for infections in the group of children from one study site at each time point.A sample containing a multiclonal infection produced more than one amplified fragment after either the three msp 1 and msp 2 family alleles was calculated as the ratio of the number of PCR products obtained for each family to the total number of gene-specific PCR products identified. The MOI for msp 1 or msp 2 in a sample was estimated as the total number of distinct msp 1 or msp 2 PCR amplicons detected in that sample, the total MOI of a sample is the highest number of PCR fragments obtained after both msp 1 and msp 2 genotyping [msp 1 and msp 2 allele at the different time points. Statistical significance was defined as p value ≤ 0.05 unless otherwise stated.The geometric mean, column statistics, unpaired T test and One-way analysis of variance were obtained using GraphPad Prism v5.0. The distribution of the different allelic families was presented as proportions. The frequency of notyping , 45. A cp = 0.8, Unpaired T test) from the mean age of 9.10 years in Abura . Asymptomatic parasite carriage over the course of the study was 41% (197/481), ranging between 13.7% (13/95) and 64% (89/139) in Abura and 88%, ranging between 80% (64/80) and 96.4% (80/83) in Obom .At enrolment, there were 46.5 and 45.3% male children in Obom and Abura respectively. The mean (SEM) age of the children in Obom was 8.96 years, which was not significantly different (ra Table . Out of msp 1 or msp 2 gene amplifications was considered as the number of independent parasite clones co-existing within an infection and as the multiplicity of that infection. The msp 1 and msp 2 genes were successfully amplified from 73.8% (344/466) and 82.5% (385/466) of the P. falciparum positive samples respectively. The samples yielded amplicons compared to the 57.4% of asymptomatic samples that yielded products in Abura. The msp 2 amplification efficiencies were similar in both sites, 83% (223/269) in Obom and 82.2% (162/197) in Abura  in Obom and 82.2% (162/197) in Abura  than the lowest geometric mean MOI of 1.82 (95% CI 1.58–2.08) at the same site, which occurred in January 2016  .The highest number of fragments obtained after either the msp 1  and msp 2 (3D7 and FC27) allelic families. Each of the multiple msp 1/msp 2 PCR products was considered as a different parasite. The msp 1 and msp 2 alleles were characterized based on the size of the PCR products using 50 bp and 100 bp DNA ladders as size standards. Variations in the sizes of the largest and smallest PCR products within each family specific PCR was noted both msp 1 and msp 2 family members in the two study sites. The approximate size of the smallest and largest alleles of msp 1 was 120 bp and 350 bp respectively for parasites from Obom and 150 bp and 320 bp respectively for parasites from Abura. No obvious trends were observed in the diversity of the smallest and largest sized PCR fragments obtained after msp 1 genotyping in both study sites across the various time points. However, in Obom the largest sized msp 2 fragment, the 400 bp variant persisted from April through to January while in Abura, no trend in the diversity of the largest and smallest sized msp 2 PCR fragments were observed across the time points . The proportion of parasites belonging to the K1 family reduced from 38.8% in April 2015 to 24.3% in July 2015, increased again in October 2015 to 48.8% and reduced slightly to 43.7% in January 2016. The parasites belonging to MAD20 allelic family increased steadily from April (22.4%) to July 2015 (42.8%), reduced to 27.1% in October 2015 and 27.7% in January 2016  in both Obom  and Abura   Fig. . Thus thmsp 2 family ranged from 57% in July 2015 to 43.3% in January 2016, while the proportion of parasites belonging to the FC27 family ranged from 43% in July to 56.7% in January 2016 with slight variation between the seasons  or Abura   Fig. , d. ThusP. falciparum and the multiplicity of P. falciparum infections over changing malaria seasons in one community with perennial and high malaria transmission and another with seasonal and low malaria transmission. The most prevalent circulating parasite clones at different time points in different transmission settings were also identified.This study sought to characterize the genetic diversity of p > 0.05, Dunn’s Multiple comparison test). This is to be expected in perennial transmission settings, where mosquito vectors and malaria transmission only vary slightly between seasons [In Abura a lot children were lost to follow up at between July and October 2015  as well as the dominance of any msp1 allele across the seasons and between the two transmission settings Primers used for Additional file 2:Plasmodium falciparum parasite carriage in study participants. (DOCX 15 kb)


Plasmodium falciparum and Plasmodium vivax infections are important causes of adverse pregnancy outcomes in the Asia-Pacific region. We hypothesised that monthly intermittent preventive treatment (IPT) or intermittent screening and treatment (IST) with dihydroartemisinin–piperaquine is more effective in reducing malaria in pregnancy than the existing single screening and treatment (SST) strategy, which is used to screen women for malaria infections at the first antenatal visit followed by passive case detection, with management of febrile cases.transmission site), Indonesia. Eligible participants were 16–30 weeks pregnant. Clusters  were randomly assigned (1:1:1) via computer-generated lists to IPT, IST, or SST clusters. In IPT clusters, participants received the fixed-dose combination of dihydroartemisinin-piperaquine (4 and 18 mg/kg per day). In IST clusters, participants were screened with malaria rapid diagnostic tests once a month, whereas, in SST clusters, they were screened at enrolment only. In all groups, participants with fever were tested for malaria. Any participant who tested positive received dihydroartemisinin–piperaquine regardless of symptoms. The primary outcome was malaria infection in the mother at delivery. Laboratory staff were unaware of group allocation. Analyses included all randomly assigned participants contributing outcome data and were adjusted for clustering at the clinic level. This trial is complete and is registered with ISRCTN, number 34010937.We did an open-label, three-arm, cluster-randomised, superiority trial in Sumba  and Papua  were randomly assigned to SST, IPT, or IST clusters (26 clusters each). Of 3553 women screened for eligibility, 2279 were enrolled . At enrolment, malaria prevalence was lower in IST (5·7%) than in SST (12·6%) and IPT (10·6%) clusters. At delivery, malaria prevalence was 20·2% (128 of 633) in SST clusters, compared with 11·6% (61 of 528) in IPT clusters  and 11·8% (84 of 713) in IST clusters . Conditions related to the pregnancy, the puerperium, and the perinatal period were the most common serious adverse events for the mothers, and infections and infestations for the infants. There were no differences between groups in serious adverse events in the mothers or in their infants.IST was associated with a lower prevalence of malaria than SST at delivery, but the prevalence of malaria in this group was also lower at enrolment, making interpretation of the effect of IST challenging. Further studies with highly sensitive malaria rapid diagnostic tests should be considered. Monthly IPT with dihydroartemisinin–piperaquine is a promising alternative to SST in areas in the Asia-Pacific region with moderate or high transmission of malaria.Joint Global Health Trials Scheme of the Medical Research Council, Department for International-Development, and the Wellcome Trust. Plasmodium falciparum and Plasmodium vivax are associated with adverse pregnancy outcomes.Approximately 70% of 125·2 million pregnancies in malaria-endemic areas occur in the Asia-Pacific region annually,Evidence before this studyPlasmodium falciparum and Plasmodium vivax are co-endemic. The following search terms were used: “(intermittent OR IPT OR prophylaxis OR prevention) AND  AND (pregnan* OR trimester OR gestation)”. The names of each country in these regions were added as additional search terms. Two IPT trials were identified, one in the Solomon Islands and the other in Papua New Guinea, of which both had used sulfadoxine–pyrimethamine. No IST trials were identified. Our literature search confirmed that few intermittent screening trials or chemoprevention trials in pregnancy have been done outside of Africa. WHO does not have a prevention strategy for malaria in pregnancy in the Asia-Pacific region, where about 70% of the global number of pregnancies in malaria-endemic areas occur. Most countries in this region use a single screening and treatment (SST) strategy for malaria at the first antenatal visit.We searched the Malaria in Pregnancy Library and PubMed from their inception to Sept 20, 2018, without language restrictions, for relevant trials of chemoprevention with intermittent preventive treatment (IPT) or intermittent screening and treatment (IST) of malaria in pregnancy. We restricted the search to areas in the Asia-Pacific region, Central and South America, the horn of Africa, and Madagascar, where Added value of this studyTo our knowledge, this study is the first prevention trial to compare monthly IPT or IST with the antimalarial dihydroartemisinin-piperaquine against the existing SST strategy with dihydroartemisinin–piperaquine for the control of malaria in pregnancy in the Asia-Pacific region. The study was designed to support the Indonesian Ministry of Health and WHO in the development of strategies for the control of malaria in pregnancy in Indonesia and the wider Asia-Pacific region.Implications of all the available evidenceOur results do not support a role for IST with the existing standard malaria rapid diagnostic tests or for IPT with dihydroartemisinin–piperaquine in areas in the Asia-Pacific region with lower malaria transmission. However, our results confirm earlier findings from east Africa and show that IPT with dihydroartemisinin–piperaquine should be considered as a potential strategy to reduce the risk of malaria infection, and the associated adverse consequences in pregnancy in areas in the Asia-Pacific region with moderate to high levels of malaria transmission and high levels of resistance to sulfadoxine–pyrimethamine.Three completed trials in areas of high sulfadoxine–pyrimethamine resistance in Kenya and UgandaIn areas with predominantly low malaria transmission, alternative strategies involving regular screening and treatment approaches should also be considered. For example, in refugee camps on the Thai–Myanmar border, the introduction of weekly screening for malaria and treatment of pregnant women who tested positive reduced maternal mortality from malaria substantially.P falciparum infections that are predominantly sequestered in the placenta, or low-grade infections that are below the limit of detection by standard microscopy or malaria rapid diagnostic tests, which are particularly common with P vivax. Furthermore, because of the parasite's dormant forms in the liver, a single P vivax infection might cause multiple relapses during pregnancy, when radical cure with primaquine is contraindicated. The SST strategy has the additional limitation of potentially missing re-infections or asymptomatic parasitaemia in later stages of pregnancy.Limitations of these screening strategies include the failure to detect Here, we report the results of the first trial in the Asia-Pacific region designed to compare the safety and efficacy of monthly IST or IPT with dihydroartemisinin–piperaquine with the standard SST strategy for decreasing the risk of malaria infection in pregnancy.P falciparum and P vivax: Sumba Island,20We did an open-label, two-site, three-arm, cluster-randomised, superiority-trial in areas in eastern Indonesia that are co-endemic for Antenatal clinics were eligible for inclusion if they had at least ten new pregnancies per year and were located within 1·5 h drive from the study offices .Pregnant women of any gravity attending their first antenatal visit were eligible if they had a viable pregnancy between 16 and 30 weeks' gestation, were residents in the study catchment areas, were willing to complete the study schedule and deliver their baby at the study clinics or hospital, and had not yet been screened for malaria. Exclusion criteria comprised high-risk pregnancies due to pre-existing conditions likely to cause complication in the current pregnancy , severe malaria at presentation, treatment with antimalarials in the previous month, HIV positivity, a family history of sudden death or any known cardiac condition, current use of medication known to prolong the QTc interval, a history of allergy to dihydroartemisinin–piperaquine, and residence outside study area or plans to move within 6 months.Ethical approval was obtained from the Liverpool School of Tropical Medicine, the Eijkman Institute for Molecular Biology, and the National Institute of Health Research and Development (Litbangkes), Ministry of Health, Jakarta, Indonesia. Written informed consent was obtained from all participants. The trial protocol is provided in the The 78 antenatal clinic clusters were matched in triplicate on the basis of location, size, and malaria transmission intensity . Before At enrolment, demographic, socioeconomic, and educational information, and data on ownership and use of LLINs, were collected, and medical and obstetric histories were taken. Gestational age was assessed by fundal height, and fetal viability confirmed by doppler ultrasonography. The pregnant women's axillary temperature, blood pressure, weight, and mid-upper arm circumference were measured, and a blood sample was taken for malaria microscopy, molecular malaria diagnostics  visit only. At subsequent monthly visits, they were tested with malaria rapid diagnostic tests if they were febrile (axillary temperature ≥37·5°C) or had a history of fever in the previous 48 h. The procedures in the IST group were identical to those in the SST group, except that participants were screened with malaria rapid diagnostic tests at each scheduled monthly visit. Participants in the IPT group received 4 mg/kg per day dihydroartemisinin and 18 mg/kg per day piperaquine  at each monthly visit, at which they were not screened for malaria, unless they were febrile or had a history of fever in the past 48 h. The dose was the same throughout pregnancy and consisted of the standard 3-day course of two tablets for participants weighing less than 36 kg, three tablets for participants weighing 36–75 kg, or four tablets for participants weighing 75 kg or more at enrolment. The first dose was provided with a glass of water as directly observed therapy in the clinic. Participants were provided with the remaining two doses to be taken at home. All participants were contacted on day 2 and visited on day 3 to assess adherence and tolerance. In case of vomiting within 30 min, the full dose was repeated. Additionally, all participants who were positive for malaria on rapid diagnostic tests (positive HRP2 or pLDH bands) in all groups were treated with dihydroartemisinin–piperaquine (the same 3-day weight-based treatment as used for the IPT group). Participants with a history of dihydroartemisinin–piperaquine intake in the previous 4 weeks received quinine–clindamycin (10 mg/kg twice daily for 7 days).At delivery, a maternal blood sample was taken for the same malaria metrics, and placental and umbilical-cord blood samples for histology, malaria rapid diagnostic tests, microscopy, and LAMP and PCR . NewbornWe extracted DNA from dried blood spots using the Chelex method and tested for malaria using LAMP Pan-kits. LAMP-positive samples and 5% of negative samples were then tested by qPCR for verification and species identification, and discordant samples retested with nested-PCR .Electrocardiography was done in a subgroup of 33 participants in the IPT group (selected through convenience sampling) to establish whether previously documented transient QTc prolongation associated with piperaquine increased in magnitude with subsequent courses .The primary outcome was malaria (any species) at delivery, defined as a composite of maternal malaria  or placental malaria , detected by any method and by each method separately. Placental malaria infection detected by histology was classified as active acute, active chronic, active any, past, or any (active or past). In post-hoc analyses, malaria infection in the peripheral blood was stratified further by species. In addition, maternal anaemia  was assessed at delivery.Secondary outcomes during pregnancy comprised maternal malaria, detected with any method and by each method separately. This outcome was further stratified by patent infection  and sub-patent infection . Morbidity outcomes assessed during pregnancy comprised clinical malaria  and unscheduled clinic visits for any reason and for all reasons unrelated to malaria.Newborn secondary efficacy outcomes included congenital malaria, mean cord haemoglobin concentration, fetal anaemia (haemoglobin <12·5 g/dL), mean birthweight, low birthweight (<2500 g), mean gestational age, preterm delivery (<37 weeks' gestation), mean birthweight for gestational age (Z scores), small for gestational age,Safety outcomes included serious adverse events in the mother or infant, overall and by system organ class and preferred Medical Dictionary of Regulatory Affairs term; maternal deaths; congenital anomalies; and QTc prolongation.The trial was initially designed to detect a 50% reduction in malaria at delivery with IPT or with IST relative to SST across both sites pooled. Following recommendations from the ethics committee in Indonesia on June 27, 2014, to stop recruitment in Sumba because of the unexpected low malaria prevalence in the area, a blinded interim re-estimation of sample size was done with the aim to provide the study with 80% power across both sites pooled and 85% power in Papua alone to detect at least a 50% reduction in the primary outcome  population included all randomised participants with outcome data. We also assessed all efficacy outcomes in the per-protocol population, which included participants in the modified ITT population who attended every scheduled visit and took all study doses on each occasion. The safety-population included participants who received at least one dose of study drug in any of the study arms.Generalised estimating equation (GEE) models, with treatment group as a predictor and clinic as a cluster effect, were used. Log binomial GEE models were used to obtain risk ratios (RRs) for binary outcomes (including the cumulative risk), and linear GEE models to obtain mean differences for continuous outcomes. The unadjusted analysis, stratified by site, was considered the primary analysis. Because matching was ineffective and the number of triplets small in each site, unmatched analysis of the matched data was done to maximise power as soon as it became clear from the comparison of the baseline data that the matching was not successful.vs multigravidae), and use of insecticide-treated net during pregnancy. Simple imputation was used for missing covariates (<1%); no imputation was used for missing outcome variables. These same covariates, as well as a post-hoc covariate for species on enrolment, were used for subgroup analyses by adding them as interaction terms with treatment group. GEE Poisson regression, with time of follow-up as an offset, was used to obtain incidence rate-ratios.Secondary, covariate-adjusted analyses of the efficacy endpoints were done with seven prespecified, individual-level covariates and one post-hoc, cluster-level covariate . The individual-level covariates were study site , malaria status at enrolment (binary), season during pregnancy , socioeconomic status , 78 clusters (57 in Sumba and 21 in Papua) were randomly assigned to SST, IPT, or IST (26 clusters each). 3553 women were screened for inclusion, of whom 2279 (64·1%) were enrolled , with a median number of scheduled follow-up visits of three  in SST clusters compared with 11·6% (61 of 528) in IPT clusters  and 11·8% (84 of 713) in IST clusters . The incidence of clinical malaria with IST was lower than with SST, but this difference was not significant  or mean maternal haemoglobin levels  between The main adverse events associated with dihydroartemisinin–piperaquine were nausea, headache, and vomiting within 7 days after drug intake. In the IPT group, nausea occurred after 63 (3%) of 2058 courses, headache after 68 (3%) of 2050 courses, and vomiting after 87 (4%) of 2058 courses . There wIn total, 33 participants in the IPT group were enrolled in the nested cardiac monitoring. Dihydroartemisinin–piperaquine was associated with a mean QTcF prolongation of 20 ms (SD 19·6) and a mean QTcB prolongation of 14·8 ms (17·6). Neither the mean QTcF nor mean QTcB increased with the total number of dihydroartemisinin–piperaquine courses administered .P falciparum and P vivax in Indonesia, IPT with dihydroartemisinin–piperaquine compared with the predominantly passive detection afforded by the existing standard SST strategy resulted in a reduction of about 41% in the prevalence of malaria infection at delivery, a similar reduction in its incidence during pregnancy, and a 78% reduction in the incidence of clinical malaria during pregnancy. The effect was evident for both P falciparum and P vivax infections , had a greater potential for bias than trials based on randomisation of individuals. The lower prevalence of malaria at enrolment in the IST arm occurred by chance in both Sumba and Papua, despite our attempt to balance the randomisation by malaria transmission using locally available annual parasite incidence data from the government . The uneIn conclusion, the effect of IST relative to SST was difficult to ascertain as the 44% difference detected at delivery was already evident at baseline (55% difference) and very few participants tested positive by malaria rapid diagnostic test in this setting, where about 90% of infections were below the limit of detection. Further studies with highly sensitive malaria rapid diagnostic tests should be considered. By contrast, our results suggest that in areas in the Asia-Pacific region with moderate transmission and high-grade sulfadoxine–pyrimethamine and chloroquine resistance, the strategy of monthly IPT with dihydroartemisinin–piperaquine could be an effective alternative to the existing policy of single screening and treatment.feiko.terkuile@lstmed.ac.uk; to gain access, data requesters will need to sign a data access agreement, and the de-identified database will be transferred by email.All individual-participant data collected during this trial will be available to access, after de-identification. Data and documents, including the study protocol and statistical analysis plan will be available. Data access will be provided to researchers after a proposal has been approved by an independent review committee identified for this purpose. An agreement on how to collaborate will be reached based on any overlap between the proposal and any ongoing efforts. Data will be available beginning at 3 months after publication of this Article. Proposals should be directed to