The World Health Organization has yet to endorse deployment of topical repellents for malaria prevention as part of public health campaigns. We aimed to quantify the effectiveness of repellent distributed by the village health volunteer (VHV) network in the Greater Mekong Subregion (GMS) in reducing malaria in order to advance regional malaria elimination.Plasmodium spp. infection. The median age of participants was 18 years, approximately half were female, and the majority were either village residents (46%) or forest dwellers (40%). No adverse events were reported during the study. Generalised linear mixed modelling estimated the effect of repellent on infection detected by rapid diagnostic test (RDT) (primary outcome) and polymerase chain reaction (PCR) (secondary outcome). Overall Plasmodium infection detected by RDT was low , but infection detected by PCR was higher . There was no significant protection against RDT-detectable infection . In Plasmodium-species-specific analyses, repellent protected against PCR-detectable P. falciparum , but not P. vivax infection . Repellent effects were similar when delayed effects were modelled, across risk groups, and regardless of village-level and temporal heterogeneity in malaria prevalence. The incremental cost-effectiveness ratio was US$256 per PCR-detectable infection averted. Study limitations were a lower than expected Plasmodium spp. infection rate and potential geographic dilution of the intervention.Between April 2015 and June 2016, a 15-month stepped-wedge cluster randomised trial was conducted in 116 villages in Myanmar (stepped monthly in blocks) to test the effectiveness of 12% N,N-diethylbenzamide w/w cream distributed by VHVs, on In this study, we observed apparent protection against new infections associated with the large-scale distribution of repellent by VHVs. Incorporation of repellent into national strategies, particularly in areas where bed nets are less effective, may contribute to the interruption of malaria transmission. Further studies are warranted across different transmission settings and populations, from the GMS and beyond, to inform WHO public health policy on the deployment of topical repellents for malaria prevention.ACTRN12616001434482).Australian and New Zealand Clinical Trials Registry , where malaria vectors bite outdoors and are not confined to biting at night .We wanted to know whether distributing repellent through established village health volunteer (VHV) networks in the GMS would prevent malaria infection.We randomised 116 villages in Myanmar to receive repellent through VHV networks using a stepped-wedge design and followed the villages up for 15 months to detect malaria by rapid diagnostic test (RDT) and highly sensitive polymerase chain reaction (PCR).We found that distribution of repellent through the VHV network did not reduce RDT-detectable infection, but appeared to reduce the odds of PCR-detectable infection.The apparent protective effect of repellent was consistent across risk groups  and villages, suggesting that repellent distributed by VHVs may be an effective intervention across a range of transmission settings and populations.Distribution of topical repellent by the VHV network may be an effective supplementary intervention for reducing malaria in the GMS.P. falciparum and 2030 for all malaria species, particularly in the context of limited effectiveness of traditional vector control interventions and spreading drug resistance.Incorporation of repellent into national malaria control programme national strategic plans for malaria elimination may advance achieving GMS malaria elimination targets of 2025 for Further studies are warranted across different transmission settings and populations, from the GMS and beyond, to inform World Health Organization public health policy on the deployment of topical repellents for malaria prevention. While there have been major gains in reducing the global burden of malaria since the turn of the millennium, the progress in reducing malaria has recently stalled . Vector Although there is strong evidence for topical repellents’ efficacy against mosquito biting , and somIn the Greater Mekong Subregion (GMS), the emergence and spread of artemisinin-resistant malaria has accelerated the malaria elimination agenda, with GMS countries and WHO committing to eliminating malaria in the region by 2030. The overall approach includes providing universal access to malaria testing and treatment, and universal coverage of all at-risk populations with long-lasting insecticidal nets (LLINs) or IRS . In GMS Plasmodium falciparum and P. vivax infection detected by rapid diagnostic test (RDT)—the routine method of malaria diagnosis in VHV networks in the GMS—as well as by polymerase chain reaction (PCR) (which exhibits higher sensitivity), and explored the moderating effect of intervention adherence and risk group on these associations. Among other reasons (outlined below), the stepped-wedge trial design was chosen to permit seminal analysis of the presence of delayed effects of repellent distribution as well as the impact of heterogeneity in malaria prevalence, at both a village and a temporal level, on the effectiveness of repellent in reducing malaria. Finally, a cost-effectiveness analysis of repellent distribution in the context of a large-scale malaria control and elimination programme was conducted in order to inform policy.In order to inform the rollout of repellent as part of a national strategic plan for malaria elimination, we conducted a stepped-wedge cluster randomised controlled trial to determine the effectiveness of the addition of repellent into the malaria services package delivered by the VHV network on reducing malaria infection in Myanmar. We examined the impact of repellent distribution on This trial is registered in the Australian New Zealand Clinical Trials Registry  and was approved by the Ethics Review Committee on Medical Research involving Human Subjects, Department of Medical Research, Ministry of Health and Sports, Myanmar Government , and the Alfred Hospital, Melbourne, Australia 95/15). The ethics review committee of the Department of Medical Research, Ministry of Health and Sports, requested that there be no commercial advantage for the product during the trial. Consequently, the repellent was provided in plain unbranded tubes, and the investigators registered the trial after completion of fieldwork  to minimise public disclosure. The study protocol has been published previously [5/15. TheBetween April 2015 and June 2016, a 15-month stepped-wedge cluster randomised trial was conducted in Myanmar with a target sample of 116 villages (clusters). Originally, in order to maximise power, we sought to confine data collection to the ‘rainy’ seasonal period only. However, due to logistical constraints in integrating research into implementation activities and in order to model temporal effects of repellent distribution, we conducted the study continuously through the ‘cool’, ‘hot’, and ‘rainy’ seasonal periods. Villages from 8 townships identified as having malaria services gaps according to National Malaria Control Programme services data were recruited, in Bago East (16 villages), Kayin (61 villages), and Kayah (39 villages) states , a WHO-recommended positive control for testing the effectiveness of mosquito repellents ,12. FielRepellent was distributed in 2 stages. In the first stage, repellent was introduced and distributed to all villagers  at village-level community meetings by VHVs and Karuna Mission Social Solidarity (KMSS) malaria officers . Any village members absent from community meetings were identified by VHVs, and repellent was distributed to them individually, external to the meeting.In the second stage, in order to establish and maintain consistent distribution, villagers were asked to return any empty repellent tubes to the VHV for replacement . The nature of the study and proper application of repellent was explained verbally . On a monthly basis, malaria officers communicated with VHVs regarding stock levels, with stock typically kept at 20%–30% of the initial procurement. Repellent was delivered to villages between 1 and 2 months prior to the onset of the randomly allocated distribution start month and stored in an appropriate secure location prior to commencement of the distribution process.Household-level mapping was considered as a primary mode of repellent distribution; however, it was determined that village-level distribution through community meetings, with targeted follow-up of non-attenders would be the most pragmatic and cost-effective method of distribution.P. falciparum or P. vivax  infection determined by SD Bioline P.f/P.v combo RDT, measured monthly by VHVs. This RDT is routinely used by VHVs in the field for active and passive case detection. Passive case detection refers to villagers presenting to VHVs for testing, and active case detection refers to VHVs seeking out infection in their village  and performing a minimum number of tests set by the programme.The primary outcome was Plasmodium spp. infections; molecular markers of artemisinin resistance; and antimalarial antibody levels in individuals as determined by enzyme-linked immunosorbent assay [P. falciparum and P. vivax infection in dried blood spots  . Given tP. falciparum or P. vivax  infection (determined by RDT and PCR). Repellent was included as a binary variable that indicated a village’s repellent distribution status as being either without  or with repellent (intervention state). This repellent variable was modelled as a time-varying (monotonic) variable whereby the variable changed in value from control to intervention for each village at a specific stage across the 15 months of the study. Models were generalised through use of a logit link function and binomial distribution , generalised structural equation modelling (GSEM) was used to apply crossed random effects multinomial logit regression analyses. In this modelling, random effects for village and month were constrained to be equal across Plasmodium spp..To explore the extent to which any association between repellent distribution and PCR-detectable infections varied across Given its direct impact on efficacy, we also performed an a priori specified additional analysis of the frequency of repellent application at the village level as the principal indicator of intervention implementation quality. At the end of the study, VHVs were asked to assess village frequency of repellent application  as occurring ‘never’, ‘monthly’, ‘weekly’, or ‘daily’ on average, and this variable was modelled in per protocol analysis. In addition, we explored the extent to which risk group membership  might moderate repellent effectiveness using a group by intervention fixed-effect interaction term in modelling.ICCs were estimated for both village and temporal heterogeneity using estimated model variance components. Given the correct specification of the GLMM, the effect estimates for repellent distribution were unbiased in light of village attrition, assuming missing monthly RDT and PCR data were missing at random (MAR) .Stata version 15.1 was used for all statistical analyses, randomisation, and power estimation [We calculated the additional costs associated with adding repellent distribution to malaria services already programmatically funded and implemented by VHVs, excluding study-related costs. All costs are reported in 2015 US dollars and are given in Plasmodium spp. infection detected by RDT was lower than expected, with 50 infections  detected over the duration of the trial  were excluded due to security concerns, with 2 villages substituted with alternate villages selected randomly. A total of 32,194 RDTs were performed across 114 villages during the 15-month study period . The meaPlasmodium spp. infection after repellent distribution by VHVs, independent of time and season   . The difference in the impact of repellent across the 2 species was statistically significant . Although there were some differences in the impact of repellent on PCR-detectable infection across village residents, forest dwellers, and migrants, these differences were not statistically significant  . Conside= 0.026) . HoweverPlasmodium spp. infection, there was marked heterogeneity  compared to control periods (ICC = 0.42) (see Plasmodium spp. infection detected by RDT (ICC = 0.003). However, we did observe higher temporal heterogeneity in the probability of Plasmodium spp. infection detected by PCR (ICC = 0.13)  and no village-specific differences in the association between repellent and infection  . For PCRnfection ; S6 TextPlasmodium spp. infection was time-dependent, we modelled the impact of repellent distribution on Plasmodium spp. infection delayed by 1 and 2 months after distribution  than when assuming that the impact of repellent distribution occurred instantaneously . The time dependence of the effect of repellent distribution was less consistent for species-specific analyses , there was a descriptive pattern of lower risk of Plasmodium spp. infection detected by RDT with higher levels of VHV-reported frequency of repellent use . A similar descriptive pattern was observed for malaria infection detected by PCR across levels of exposure, but again this was not statistically significant   reported that, on average, study participants applied repellent ‘daily’ and that the majority  also applied the repellent as per instructions. Six villages  experienced repellent stock-out periods. In these analyses, the intervention was measured as an ordinal time-varying exposure, where villages could be in either a control or an intervention state, which was marked by ‘monthly’, ‘weekly’, or ‘daily’ average use. Although these analyses  showed t= 0.195) .The total cost of the intervention during the study was US$76,138 . In total, repellent was provided for 237,701 person-months, or 19,808 person-years, resulting in a cost of approximately US$3.8 per person per year. The control provision  was provided for a total of 215,675 person-months, or 17,973 person-years.Cost-effectiveness analysis based on the overall infection prevalence observed during the study  found thPlasmodium spp. infections, apparent protection was observed against PCR-detected P. falciparum, but not P. vivax, infection. Importantly, for PCR-detectable infections, there was low heterogeneity of this protective effect between villages, and the effect was similar between village residents and migrants/forest workers, suggesting that repellent as an intervention to reduce malaria is likely to be applicable across a range of transmission settings and populations. Large-scale distribution of repellent delivered through VHVs, or similar providers, as part of malaria control and elimination programmes may be an effective public health strategy to target residual malaria transmission not covered by conventional vector control measures in Myanmar and the GMS more broadly.The results from this trial demonstrated that, although incorporation of repellent delivery into VHV-delivered malaria services did not reduce RDT-detected P. falciparum, but not P. vivax, infections, and this could be seen as providing discriminant validity for repellent effectiveness. The limited effect on P. vivax is most likely explained by the large proportion of P. vivax infections that are caused by relapses from dormant liver stages, rather than being new infections acquired from mosquito bites, whereas P. falciparum does not have a dormant liver stage. This strengthens the internal validity of our study because it suggests that repellent can specifically protect against new Plasmodium spp. infections. Whether the density of a new Plasmodium spp. infection reaches the detection limits of routine RDTs or PCR is dependent on the level of naturally acquired immunity, which controls parasitaemia. RDT-negative, PCR-detectable infections are important in malaria elimination settings because they often go undetected and untreated and can contribute to ongoing malaria transmission [P. falciparum. Studies in the GMS have shown that PCR-detectable infections can harbour transmissible gametocytes, and increases in PCR-detectable infections also increase the entomological inoculation rate [One of the barriers to including or recommending repellents for malaria prevention is the lack of evidence on implementation strategies that are effective. To our knowledge, this trial is the first to evaluate topical repellent distribution as part of a broader public health campaign, establishing evidence for the effectiveness of repellent distributed by the network of VHVs who deliver endorsed malaria interventions in Myanmar and the GMS more broadly. Our findings suggest that implementation using the VHV network model, or similar infrastructures, could be a mechanism for providing repellents for malaria prevention at the required scale. We found that the impact of repellent was species-specific; repellent distribution significantly reduced PCR-detectable smission . This ision rate –18. Furtion rate ,20. TherP. falciparum infections by 28% to 82%, with varying degrees of statistical significance [P. falciparum infections , and the researchers determined that their study was underpowered to detect the magnitude of effect observed, but confidence intervals did contain the magnitude of the apparent protective effect we observed  [Plasmodium spp. infection detected by RDT, the routine method of malaria diagnosis in the GMS. Despite this, the greater sensitivity of the secondary outcome measure of Plasmodium spp. infection detected by PCR, which yielded 8 times the number of events, combined with our study design and analytical approach, permitted effect estimation for repellent distribution with markedly greater accuracy in terms of inference.Interpretation of the effectiveness of repellent from trials in low transmission areas is challenging because insufficient power may result from low event rates, compounded by the low sensitivity of conventional malaria parasite detection methods. Previous trials in low transmission areas , which did not integrate repellent into established health systems, reported that repellents were not associated with reductions in RDT-detectable infections  , but couificance –25. Only44–1.56) . Our triPlasmodium spp. infections are observed repeatedly and frequently in both the control and intervention states . This repeat sampling frequency allowed us to explore delayed effects of repellent distribution—no delayed effect was found for PCR-detectable infections, but a 2-month delayed effect for RDT-detectable infections was present—and permitted both within- and between-village differences to be incorporated into statistical modelling. The estimation of crossed random effects  permitted more effective partitioning of any variance in risk of infection from unmeasured factors impacting at both temporal and village-specific levels, resulting in increased precision of the average independent effect of repellent distribution. In addition, it enabled the quantification of heterogeneity in both village-specific baseline and time-specific risk of infection, as well as village-specific heterogeneity in the treatment effect. Interestingly, the effect of repellent on reducing PCR-detectable infections was consistent across villages, suggesting that repellent distributed by VHVs may be an effective intervention across a range of transmission settings and populations. Further studies are warranted across different transmission settings and populations, from the GMS and beyond, in order to inform WHO public health policy on the deployment of topical repellents for malaria prevention [A strength of our trial, compared to previous trials, was the stepped-wedge design, whereby village evention .Plasmodium spp. infection where daily or weekly use (compared to monthly) was observed—but it should be noted the effect here was not statistically significant. Differences in compliance rates may explain the poor effectiveness of repellents when translated into field settings—possibly indicated by heterogeneity in levels of effectiveness across trials [As an effectiveness trial, this study applied a principally ‘intention to treat’ approach by design, measuring the effect of repellent distribution by VHVs on malaria at a village level. Participants were not obliged to use repellent, and, given the population size, compliance and the usage of repellent by individuals was not strictly monitored. However, analysis based on VHVs’ estimates of the average frequency of repellent use in their village indicated lower risk of s trials . Whethers trials ,25,26. NPlasmodium spp. infection detected by RDT) was limited by the lower than expected number of infections observed during the study period. Despite this, the greater sensitivity of the secondary outcome measure (Plasmodium spp. infection detected by PCR) and the subsequent higher number of events permitted modelling with increased precision for the estimate of the intervention effect. The geographical proximity of some villages may have resulted in contamination of some villages with repellent while in the control state. If this did occur, then this would bias results towards the null, meaning that the true protective effect of repellent may be larger than we observed. In the village, repellent was made available to high-risk groups (migrants and forest dwellers) as well as village residents, and we found that these groups were balanced in control and intervention periods and that the protective effect of repellent was similar across these groups. More targeted distribution campaigns for high-risk groups in areas where high-risk groups harbour the vast majority of infections may be beneficial because individuals in these high-risk groups are less likely to use ITNs or LLINs than the general population [There were limitations to the findings of this study. As mentioned previously, inference for the primary outcome of our study Click here for additional data file.S1 FigRed circles indicate adjusted odds ratios; blue diamonds indicate adjusted relative risk ratios.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S5 Text(DOCX)Click here for additional data file.S6 Text(DOCX)Click here for additional data file.


With a decline in malaria burden, innovative interventions and tools are required to reduce malaria transmission further. Mass drug administration (MDA) of artemisinin-based combination therapy (ACT) has been identified as a potential tool to further reduce malaria transmission, where coverage of vector control interventions is already high. However, the impact is limited in time. Combining an ACT with an endectocide treatment that is able to reduce vector survival, such as ivermectin (IVM), could increase the impact of MDA and offer a new tool to reduce malaria transmission.The study objective is to evaluate the impact of MDA with IVM plus dihydroartemisinin-piperaquine (DP) on malaria transmission in an area with high coverage of malaria control interventions.The study is a cluster randomized trial in the Upper River Region of The Gambia and included 32 villages (16 control and 16 intervention). A buffer zone of ~2 km was created around all intervention clusters. MDA with IVM plus DP was implemented in all intervention villages and the buffer zones; control villages received standard malaria interventions according to the Gambian National Malaria Control Program plans.The MDA campaigns were carried out from August to October 2018 for the first year and from July to September 2019 for the second year. Statistical analysis will commence once the database is completed, cleaned, and locked.This is the first cluster randomized clinical trial of MDA with IVM plus DP. The results will provide evidence on the impact of MDA with IVM plus DP on malaria transmission.ClinicalTrials.gov NCT03576313; https://clinicaltrials.gov/ct2/show/NCT03576313DERR1-10.2196/20904 Between 2000 and 2015, the burden of malaria decreased substantially in sub-Saharan Africa following the scale-up of insecticide-treated bed nets (ITNs), indoor residual spraying (IRS), and artemisinin-based combination therapy ,4. NeverMass drug administration (MDA), a full antimalarial treatment to all inhabitants of target communities regardless of their infection status, has been identified as a potential tool to further reduce transmission where coverage of vector control interventions is already high ,9. MDA wAnopheles gambiae s.s. lasting approximately 28 days post treatment [Combining an antimalarial treatment (DP) with an endectocide treatment able to reduce vector survival, ivermectin (IVM), could increase the impact of MDA and offer a new tool to reduce malaria transmission ,16. IVM The community administration of IVM could reduce malaria transmission ,21, provThe mass drug administration of ivermectin and dihydroartemisinin-piperaquine as an additional intervention for malaria elimination (MASSIV) study is a cluster randomized trial that aims to evaluate the impact of MDA with IVM plus DP on malaria transmission in an area with high coverage of malaria control interventions, eg, ITNs, IRS, and seasonal chemoprevention (SMC). The trial was implemented in Basse, Upper River Region (URR) in The Gambia.The clinical objective is to determine whether three monthly rounds of MDA with IVM plus DP implemented over two malaria transmission seasons will reduce malaria transmission in communities with high coverage of malaria control interventions.The entomological aim is to determine whether three monthly rounds of MDA with IVM plus DP implemented over two malaria transmission seasons will reduce vector parity, a proxy for vector survival, in communities with high coverage of malaria control interventions.The social science and health economics aims are to (1) identify the most socially acceptable and sustainable way of achieving and maintaining high coverage of MDA with IVM plus DP, and (2) determine the costs and cost-effectiveness of this intervention compared to standard malaria control measures.The study is a cluster randomized trial  that included a total of 32 villages (16 control and 16 intervention), located at least 3 km from each other. A buffer zone of ~2 km was created around all intervention clusters in a modified fried-egg design . No buff2 [The study was conducted in the eastern part of The Gambia in the Upper River Region (URR). The region is open Sudanese savanna and covers an area of 1995 km2 . Most of2 ,24. MalaA cross-sectional survey was carried out in November 2017, at the peak of malaria transmission, in 47 medium-sized (200-600 inhabitants) villages to select study villages with a baseline malaria prevalence of at least 10%. Following the survey, 32 villages with malaria prevalence determined by qPCR of at least 10% were included in the trial and randomly assigned to either the intervention or control arm using STATA, version 15; randomization was constrained such that the mean baseline prevalence in the intervention clusters was within ±10% of the prevalence in the control clusters.After explaining the study objective and methods of the trial to the local authorities and the populations of the study villages through sensitization meetings in the local language, a census of the study population was carried out in November 2017. This was followed by individual consent procedures to obtain written informed consent for all willing residents in the study villages. Consent for children was provided by their parents/guardians; assent was sought for adolescents 12-17 years old.Consent and enrolment procedures were carried out throughout the trial to obtain written informed consent from new residents and individuals missed previously by the research team due to absence at the time of the initial consenting and enrolment procedures.A list of all residents in the study villages, by compound, including consent status, were generated and made available to the study team.Loa loa endemic countries ; for DP, these were: (1) first-trimester pregnancy, (2) hypersensitivity to DP, and (3) taking drugs that influence cardiac function or prolong QTc interval.The target population for MDA was all eligible individual residents in the intervention villages. During each MDA round, residents were (re)assessed for eligibility to receive IVM and DP. Participants had to meet all inclusion criteria and none of the exclusion criteria. Inclusion criteria were: (1) age/anthropometry, for IVM: weight ≥15 kg; for DP: age >6 months, (2) willingness to comply with trial procedures, and (3) individual written informed consent. The exclusion criteria for both IVM and DP were known chronic illnesses such as HIV, tuberculosis, hepatitis, and severe malnutrition. Additionally, for IVM only, exclusion criteria were (1) pregnancy (any trimester) or breastfeeding, (2) hypersensitivity to IVM, and (3) travel to DP  was available as tablets of 320/40 mg and 160/20 mg piperaquine/dihydroartemisinin per tablet. DP was administered orally, once daily for three days, according to body weight per manufacturer’s guidelines. For participants unable to swallow the tablets, such as infants and young children, DP was crushed and mixed with water. The mixture was used immediately after preparation. If a participant vomited within 30 minutes of taking DP, the full dose was readministered; if a patient vomited within 30-60 minutes, half the dose was readministered. IVM  was available as 6 mg tablets. It was given at 300-400 μg/kg/day once daily for three days . Therefore, the total dose of IVM for each round was 900-1200 μg/kg. Before treatment, women of reproductive age (15-49 years) were asked to provide a urine sample to test for pregnancy. All treatments were supervised.MDA was implemented each year and for two consecutive transmission seasons as three-monthly rounds starting from July (end of the dry season) to August and September (rainy season). The choice of implementing the intervention for two consecutive years was taken to estimate its cumulative effect and to monitor community participation over repeated intervention rounds.Standard malaria control interventions were implemented in both intervention and control villages. These consisted of ITNs, IRS with pirimiphos-methyl (Actellic 300CS) done in mid-July 2019, just before the first MDA round of IVM and DP, prompt diagnosis and treatment with artemether-lumefantrine, SMC, and intermittent preventive treatment during pregnancy (IPTp). During the three rounds of MDA, SMC was not administered to children in the intervention villages eligible for MDA with IVM plus DP to avoid double antimalarial treatment. In these villages and during the MDA period, only children aged 3-6 months received SMC as they were not eligible for DP treatment. Nevertheless, all children aged 3-59 months received SMC one month after completing the last MDA round in intervention villages. The implementation of these malaria control interventions was done by the Regional Health Team and documented at the level of individual participants. Eligible children in control villages received SMC as planned by the NMCP.The primary clinical endpoint was the prevalence of malaria infection measured by qPCR in all age groups  via a yeSecondary endpoints included (1) incidence of clinical malaria estimated by passive case detection by recording all clinical cases from study villages (both intervention and control) attending the local health facilities or the community health worker when present; (2) prevalence of drug-resistant markers such as Pfcrt and Pfmdr-1 mutations , in malaTertiary endpoints were the prevalence of bedbugs, headlice, scabies, and soil-transmitted helminths. Surveys were done yearly, before and after the MDA campaign.An. gambiaes.l. females in each study group, determined on extracted ovaries [An. coluzzii after feeding on IVM-treated individuals on days 7, 14, and 21 post treatment. An. coluzzii mortality was recorded over 14 days after feeding.The primary entomological endpoint was the parous rate of  ovaries . The secThe economics and social sciences endpoints included: (1) MDA participation and acceptability, and (2) MDA costs and cost-effectiveness. For participation and acceptability, quantitative and qualitative research methods were used. For the former, in each intervention village, a cross-sectional survey was conducted between MDA years 1 and 2. For qualitative methods, in-depth interviews and focus group discussions with MDA participants, MDA decliners, village leaders, MDA field staff, and others were carried out throughout all trial activities. Information on the costs of malaria episodes to households and their management and the intervention costs was collected to estimate costs and cost-effectiveness. From these, the incremental cost-effectiveness ratio of the intervention versus control was calculated as costs per malaria-related DALY averted. Moreover, policy considerations around translating and scaling up the intervention were explored.The sample size was based on two primary endpoints: malaria prevalence determined by qPCR at the peak of the malaria transmission season and the vector population’s parous rate. For malaria prevalence, we assumed the mean malaria prevalence in the control arm at peak transmission would be 15%; this is a conservative assumption, as, in November 2013, the prevalence in villages in eastern Gambia varied between 21.3% and 44.3% . Based oAn. gambiae s.l. would be 85% because, in 2013, it was 90.7% and 81.1% in the North Bank and South Bank of the URR, respectively [An. gambiae s.l per village would have 90% power to find a significant difference between arms.For the vector parous rate, the assumption was that the parous rate of ectively . TherefoThe unit of randomization was the village. Randomization to the intervention or the control arm was done by computer-based randomization. To prevent imbalance for potential confounding factors, restricted randomization to balance the arms on factors such as baseline prevalence, ITN coverage, and distance from health facilities was used .Considering the nature of the trial (cluster randomized), the primary endpoint , and the logistical and ethical  challenges, we chose not to blind participating individuals. All laboratory staff involved in processing samples and evaluations contributing to any clinical or entomological endpoints were blinded to the allocation arm.Passive case detection was established at local health facilities and the village level if community health workers were present. All suspected malaria cases, such as patients with fever or history of fever in the last 24 h without any other apparent illness, were investigated with an RDT ; if positive, the patient was treated with artemether-lumefantrine, the first-line treatment in The Gambia. Axillary body temperature was measured using an electronic thermometer; a blood slide and blood spots were collected for later molecular analysis. Randomly selected individuals, regardless of temperature or history of symptoms, were included in the cross-sectional surveys to collect blood samples by finger prick and administer a short questionnaire on demographic characteristics, area of residence, recent travel history, and use of malaria control interventions and recent history of clinical malaria.Strongyloides stercoralis, Trichuris trichiura, Necator americanus, Ancylostoma duodenale, and Ascaris lumbricoides using PCR.A cross-sectional survey to estimate the prevalence of ectoparasites, scabies, and helminths was carried out. In each cluster, 30 children aged 4-13 years, the age group at highest risk of these infections, were selected. Bedbugs were detected by visual inspection of the child’s bed using torchlight, and headlice were detected by visual search of the scalp. Scabies was identified by a complete physical examination of the body. A stool sample was taken to identify the presence of helminths, including An. gambiae s.l. (10 per trap per village) in droplets of phosphate-buffered saline (PBS) and read with a microscope at 10× field magnification. Sporozoite rate was determined by ELISA on the head and thorax of An. gambiae s.l.Both intensive and routine entomological surveys were performed. Intensive surveys were carried out 7-14 days after each MDA in all the intervention villages and for a similar period in 8 control villages randomly selected at the beginning of the study. The same control villages were sampled throughout the transmission season. Collections were carried out over three consecutive nights in six randomly selected houses. Monthly human landing catches (indoor and outdoor) were carried out in three houses for two nights (6 nights/month) to determine the landing rate, parity rate, sporozoite rate, and the entomological inoculation rate, and their potential reduction by IVM. Four collectors rotated between indoor and outdoor sites every 2 hours, from 7:00 pm to 7:00 am, in four selected villages per study arm. Routine surveys were carried out monthly, after the intensive surveys, between October and December, in all intervention and control villages using CDC light traps. . In eachAn. gambiae s.s. mosquitoes, were presented to the membrane feeder for 20 minutes [An. coluzzii at different age ranges.To monitor the duration of the IVM mosquitocidal effect and potential host characteristics associated with lower efficacy, 40 randomly selected adults and 40 randomly selected children (2-10 years old) were recruited in one intervention and one control village near Basse field station, which has an insectary. Children were included in this component of the study because linear dosing based on total body weight may not achieve similar drug exposure in children as in adults, possibly a consequence of age-related changes in gastrointestinal motility, pH, or prehepatic expression of metabolic enzymes or transporters, resulting in increased absorptive capacity with age ,30. Only minutes . The numA mixed-methods social science study was carried out prior to and throughout the trial. This study used ethnographic, qualitative, and quantitative methods to assess MDA coverage, potential bottlenecks to implementation, adherence, and acceptability among potential MDA participants. Before the initial MDA campaign, potential bottlenecks for the intervention were assessed, and recommendations were made to improve implementation in the first phase. These data helped fine-tune the intervention to local realities and engaged stakeholders to assure participation and long-term sustainability. In the second phase, exploratory research was conducted in all intervention and control villages to assess the MDA’s socio-cultural effects. Methods included observations of all trial components, in-depth and key-informant interviews, and focus group discussions. These methods were carried out in both the first and second years of the MDA. A quantitative household survey was conducted between years to assess findings from the first intervention year in more detail; these findings were then explored by additional qualitative and ethnographic work, including surveys, interviews, and focus group discussions in the second year.P. falciparum parasitemia, three 6-mm punches of dried blood spot, each representing approximately 8 μL blood, were added to a 96-deep well plate and used for DNA extraction. Extracted DNA was analyzed with varATS qPCR using 5 μL of extracted DNA per assay [P. falciparum standard dilution, used to make the quantification curve and allow quality control. For the serological analysis, serum was eluted from one 6-mm punch (~4 µL serum) from the filter paper. This punch was eluted in 200 µL PBS/azide to have one 1:50 predilution from which 10 µL were used on the Luminex plate. As controls for the immunoassay, BSA-coupled beads, a pool of negative sera , and positive sera (highly exposed Gambian sera from previous surveys) were added to each plate. Plates were analyzed and read using the MAGPIX [A finger prick blood sample was collected from all selected individuals; 40-50 µL (3 droplets) were blotted onto Whatman 3 MM filter paper and stored with a desiccant at 4°C. For the molecular detection of er assay . All qPCe MAGPIX . ResultsData for each participant were captured on electronic case report forms. Informed consent, demography, medical history, physical examination, vital signs, drug administration, adherence to study drugs, adverse events including serious adverse events, and laboratory evaluations were recorded.All trial data were stored and managed within the REDCap trial database system, an application specifically designed to collect and store clinical trial data and customized for electronic data capture at the trial site. Analytical results for samples from the prevalence surveys were linked to the main trial database. When inconsistencies were found, these were checked against the original forms and subsequently amended in the dataset. All staff involved in the trial was trained on their specific tasks.-effects logistic regression, and a random effect for study village will be included to account for clustering. An odds ratio, 95% CI, and P value will be presented. Malaria prevalence at the end of the first transmission season will be analyzed separately and in the same way.The primary clinical endpoint, malaria prevalence at the end of the second transmission season, will be compared between arms using randomP value will be presented. Incidence will be compared between arms using random-effects Poisson regression, and a random effect for study village will be included to account for clustering. An offset for village population and a fixed effect for the study year will be included. A rate ratio, 95% CI, and P value will be presented. Mosquito mortality, a secondary entomological endpoint, will be determined by Kaplan-Meier estimates. Kaplan-Meier plots of mosquito survival will be presented for each age group and timepoint. A comparison between the arms will be made using survival analysis with an exponential survival model. A random effect (shared frailty) will be included for each human participant to account for correlated outcomes on mosquitoes fed on the same participant.The primary entomological endpoint, vector parity, will be compared between arms using random-effects logistic regression, and a random effect for study village will be included to account for clustering. A fixed effect for study month and year will be included. An odds ratio, 95% CI, and -effects negative binomial regression, and a random effect for study village will be included to account for clustering. A fixed effect for study month and year will be included. An odds ratio, 95% CI, and P value will be presented; (2) Sporozoite rates in field-caught mosquitoes will be compared between arms using random-effects logistic regression, and a random effect for study village will be included to account for clustering. A fixed effect for study month and year will be included. An odds ratio, 95% CI, and P value will be presented.Other entomological endpoints: (1) Mosquito density will be compared between arms using randomAn independent data and safety monitoring board (DSMB) was appointed to advise the sponsor and investigators on the trial’s safety issues. Study physicians and nurses were trained to monitor and report all adverse events, including serious adverse events associated with treatment. Before each dose, adverse events that occurred were systematically documented by medical terms, start and end date, severity and seriousness, relation to the study drug, and outcome. The relationship to the study drugs was determined based on temporal association and clinical judgment. The study nurses visited each household in intervention villages 7 days after starting treatment to monitor and document any adverse events in the study population. Serious adverse events were reported to the DSMB, the local ethics committee, and the London School of Hygiene and Tropical Medicine (LSHTM) Ethics Committee. Analysis of adverse events was restricted to the intervention arm because the study population in the control arm was not treated with the investigational products.The protocol, informed consent documents, and patient information sheets have been reviewed and approved by The Gambia Government/MRC Joint Ethics Committee (ref number: 1593) and the London School of Hygiene and Tropical Medicine Ethics Committee (ref number: 15823).The MDA campaigns were carried out from August to October 2018 for the first year and from July to September 2019 for the second year. Statistical analysis will commence once the database is completed, cleaned, and locked.IVM is a promising tool to complement the current effort to interrupt malaria transmission. It targets malaria vectors regardless of biting behavior  and reduces vectorial capacity ,33. ReseCluster randomized trials are the gold standard design to assess the community-level effect of an intervention . Key conMeasuring and detecting changes in malaria transmission requires appropriate metrics to assess the community-wide impact of an intervention . SeveralParasite prevalence, the metric designed to measure the proportion of individuals found with parasites in their blood, varies by the methods used , includiSuccessful MDA campaigns require high coverage and good compliance . UnderstTo our knowledge, this is the first cluster randomized trial with a 3-day regimen of 300 µg/kg IVM (high-dose regimen). Strengths of the study include its design as an adequately powered cluster randomized trial, the setting in an area with high coverage of ITNs, IRS, and SMC, and where there is prompt and effective treatment with ACTs. Another major strength is the provision of safety data for the high dose IVM administered with DP. The trial is limited by its inability to determine the impact of IVM alone as this treatment is administered with DP, and there is no DP-only arm.This study will be the first cluster randomized clinical trial of MDA with IVM plus DP. The results will provide evidence on the effect of MDA with IVM plus DP on malaria transmission.


E vaccine has shown consistent but partial vaccine efficacy in a pediatric phase 3 clinical trial using a 3-dose immunization schedule. A fourth-dose 18 months after the primary vaccination was shown to restore the waning efficacy. However, only total IgG against the immunodominant malaria vaccine epitope has been analyzed following the booster. To better characterize the magnitude, nature, and longevity of the immune response to the booster, we measured levels of total IgM, IgG, and IgG1-4 subclasses against three constructs of the circumsporozoite protein (CSP) and the hepatitis B surface antigen  by quantitative suspension array technology in 50 subjects in the phase 3 trial in Manhiça, Mozambique. To explore the impact of vaccination on naturally acquired immune responses, we measured antibodies to P. falciparum antigens not included in RTS,S. We found increased IgG, IgG1, IgG3 and IgG4, but not IgG2 nor IgM, levels against vaccine antigens 1 month after the fourth dose. Overall, antibody responses to the booster dose were lower than the initial peak response to primary immunization and children had higher IgG and IgG1 levels than infants. Higher anti-Rh5 IgG and IgG1-4 levels were detected after the booster dose, suggesting that RTS,S partial protection could increase some blood stage antibody responses. Our work shows that the response to the RTS,S/AS01E booster dose is different from the primary vaccine immune response and highlights the dynamic changes in subclass antibody patterns upon the vaccine booster and with acquisition of adaptive immunity to malaria.The RTS,S/AS01 Plasmodium falciparum1. Importantly, 90% of these deaths concentrated in sub-Saharan Africa and a large proportion occurred in children under 5 years. Owing to the concerning rise of parasite resistance to antimalarial drugs and vector resistance to insecticides2 and stalling progress in reducing malaria since 20162, integration of a malaria vaccine with other preventive measures will be a useful addition to control disease burden in the future.Despite the great reduction in malaria cases in the last 15 years, thanks to the combination of multiple control measures, it is estimated that 219 million malaria cases and 435,000 deaths occurred in 2017, mostly associated with E vaccine is the most advanced, having shown consistent but partial vaccine efficacy (VE) that wanes over time and is less effective in infants compared to children3. RTS,S/AS01E contains a fusion protein including the central tandem repeat (NANP) and the C-terminal (C-term) regions of the P. falciparum circumsporozoite protein (CSP), and the hepatitis B virus surface antigen (HBsAg). It is expressed together with HBsAg, and injected in combination with the AS01 adjuvant system4. The vaccine was tested in a phase 3 clinical trial of a 3-dose immunization schedule  with a fourth dose 18 months after primary vaccination (M20)3, with the booster dose partly restoring the waning VE. Specifically, VE for the 3-dose immunization schedule was 35.2% in children and 20.3% in infants up to M32 of the study, but VE waned over time with a VE of 16.1 and 7.6%, respectively, when considering only the period from M20 to M32. In children and infants who received the booster dose, waning VE was restored to overall levels of 43.9 and 27.8%, respectively3. In order to understand why protection offered by RTS,S is suboptimal and continue efforts to improve it, there is a need to decipher the mechanisms of protection elicited by the vaccine. It has been shown that antibody levels are involved in the vaccine-induced immunity, but they do not fully explain the protective effect of the vaccine6. Thus far, the study of antibody response in trials performed in endemic areas has been largely focused on IgG levels against the NANP repeat region of CSP, with the exception of our previous work assessing more generally subclass responses to NANP and to other antigens after primary vaccination in the phase 3 trial9.Currently, the pre-erythrocytic RTS,S/AS0110. For instance, some IgG subclasses act as cytophilic while others have non-cytophilic functions10, influencing the roles of Fc-mediated functions such as complement fixation and phagocytosis11. Determining which type of response is detrimental or beneficial could further inform which responses could be modified to enhance the efficacy of the vaccine.Characterizing responses by other antibody isotypes, subclasses, and responses to different epitopes may provide in depth understanding of the immune response to the vaccine and the mode of action. Antibody levels are not the sole means to determine vaccine mechanisms of action. Characteristics like the balance between isotypes or subclasses of the antibodies are important because of their varying effector functions6 but other regions could also mediate protection. Avidity of IgG to the CSP C-term has been associated with protection in African children12, and C-term and not the NANP-repeat-specific antibodies have been reported to be the main mediators of phagocytic activity in naive adults13. Furthermore, antibodies to both C-term and NANP-repeat can mediate complement fixation in children, suggesting both regions are important for functional activity15.The epitope specificity of the antibody response is also relevant. There is clear evidence that NANP is related to VEP. falciparum blood stage antigens not present in the vaccine is relevant to determine the effect of the vaccine on naturally acquired immunity (NAI), developed from continuous parasite exposure. It has been hypothesized that vaccination could (1) decrease NAI by reducing the exposure to the parasite, which could mean individuals are left vulnerable in the long term due to the waning efficacy of the vaccine3, as predicted for other malaria prevention tools16, or (2) increase NAI by allowing subclinical exposure to the parasite due to the partial efficacy of the vaccine17.Additionally, studying the response to E booster dose on different antibody responses. We evaluated total IgM, IgG and IgG1-4 subclasses to vaccine and vaccine-unrelated P. falciparum blood stage antigens. Data were combined with those from the primary vaccine response previously assessed9 to display the kinetics from baseline (M0) until M32.Here, we used samples from the phase 3 trial at the time of the booster dose (M20) and onwards from a subgroup of subjects in Manhiça, Mozambique, to characterize the effect of the RTS,S/AS01E booster dose increased IgG, IgG1, IgG3, and IgG4 levels against all vaccine antigens 1 month (M21) after its administration (M20), but it did not increase IgG2 nor IgM levels . IgG and IgG1 (but not IgG3) levels against vaccine antigens in the R3R group remained above the R3C group, except for IgG1 NANP  to the primary vaccination response M3), the group that received the booster had a lower peak in IgG, IgG1 and IgG3 levels after the booster than after primary vaccination  while for IgG3 it was around 2.5 log10MFI. IgG2 levels remained more stable after the primary vaccination and did not increase or decrease vastly after M3. Remarkably, IgG4 levels against CSP FL and C-term at M20 were slightly higher than levels at M3 although this effect was not observed in the C3C group.The decrease in anti-CSP levels from primary vaccination to M20 was larger for IgG3 than for IgG and IgG1; the mean decreases for anti-NANP IgG and IgG1 was around 1.5 log1-4 to CSP in RTS,S/AS01E vaccination groups (R3), with or without booster, were higher for most post-vaccination time points than the levels in the comparator group. IgM levels were higher only at M3 in the R3 groups compared to the C3C group.The levels of IgG and IgGE either with or without a booster had higher IgG and IgG1 levels against CSP antigens than infants throughout the study period  in individuals who had clinical malaria. For the R3C group at M21, individuals who presented with clinical malaria before M20 had lower anti-CSP IgG and IgG1 mean levels against CSP antigens, and lower IgG3 levels against CSP FL (p < 0.05 before adjustment). In contrast, IgM levels were higher in plasma from previous malaria cases but this was not statistically significant. In the C3C group, IgG and IgG1-3 to FL CSP and NANP were higher in the subjects with previous malaria cases but this difference was not statistically significant after adjusting for multiple testing. Lower levels of IgG, IgG1, and IgG3 to HBsAg were also observed in R3C at M20, M21, and M32 in the previous malaria cases  . In most cases, there was no statistically significant difference between subjects presenting with clinical malaria after M21 and those who did not.The study was not designed to assess associations with future malaria risk but we had some provisional findings. IgG2 and IgG3 to vaccine antigens in the R3R group at M21 were higher in those subjects with subsequent clinical malaria, but this difference was not statistically significant. Only for anti-NANP IgG2 and anti-HBsAg IgG2 and IgG3 levels, differences had p < 0.05 before adjustment) in antibody levels at M3 and/or M21 for MSP5, MSP142, MSP1-BL2, Rh4.2, EBA140 and EBA175  but it increased IgG1 and IgG3 levels, which may explain how the booster led to higher efficacy overall. IgG1 and IgG3 can effectively fix complement and promote interactions with Fcγ-receptors on phagocytes10, which could be contributing to RTS,S-induced protection. IgG2 and IgG4, on the contrary, are non-cytophilic antibodies unable to fix complement and to interact with Fcγ-receptors10.This study confirms that the RTS,S/AS016, being the established immunodominant region of the vaccine antigen4. However, there is evidence that antibodies against C-term are involved in phagocytic activity in US naive adults13, and RTS,S vaccine-induced antibodies to the C-term among children can promote complement fixation14. Also, in our previous work we have found that post-primary vaccination, the avidity of the IgG response to CSP C-term was associated with protection12. Here, we show that the booster dose increases levels of antibodies against both NANP and the C-term, and that the responses against these two regions may behave differently. Antibody levels to NANP were higher compared to C-term, but the proportional increase 1 month after the booster dose was not different.The profile of antibody responses seems to be epitope-specific. Previously, the bulk of studies had only evaluated NANP antibodies and have provided clear evidence that NANP antibodies are associated with protection6. In this study, we found similar results for IgG subclasses—there was boosting but levels overall were lower than following primary vaccination7. Remarkably, IgG4 levels against CSP kept increasing with time. The RTS,S pattern differs to other vaccines in which the peak for the booster response is higher than the peak for the primary vaccination18. The unusual response to the booster dose could be caused by different factors. These factors include the response to primary vaccination19, but also the booster dose or the primary vaccination schedule21. For instance, it has been reported that for some vaccines high residual levels of vaccine antibodies have a negative effect on the post-booster response19; however, our study did not find evidence for a negative correlation between M20 and M21 antibody levels  to non-cytophilic (IgG2 and IgG4) CSP antibodies was associated with protection. The association between IgG4 levels and protection is consistent with findings of Chaudhury et al.27 who reported that only IgG4 levels were positively associated with increased vaccine efficacy in malaria-naive adults under the fractional dose regime. We have also previously found an association between IgG4 responses to non-RTS,S antigens after primary vaccination and protection8. IgG4 antibodies are associated with repeated or long-term exposure to antigens and have been linked to induction of tolerance, for instance higher IgG4/IgE ratios are associated with better food tolerance, as IgG4 competes with IgE28. In the context of helminth infections, high IgG4 is associated with asymptomatic infection for some parasites10. However, there is conflicting evidence on the role of IgG2 and IgG4 on protection against malaria, whilst more information exists on the protective role of IgG1 and IgG3. In the context of naturally acquired immunity, the ratio of cytophilic (IgG1 + IgG3) to non-cytophilic subclasses (IgG2 + IgG4) is generally higher in subjects with uncomplicated malaria compared to subjects with complicated malaria, and higher in subjects protected from malaria29. Additionally, it has been reported that the IgG2/IgG4 ratio is higher in subjects with uncomplicated malaria30. However, it has also been observed that IgG2 and IgG4 with high avidity are found in subjects with uncomplicated malaria compared to complicated malaria29. In contrast, the Chaudhury et al.27 study assessing avidity and opsonization reported that RTS,S protection was mediated by IgG4 against the C-term of CSP. All of this evidence indicates that not only antibody levels are important for protection but also the balance between subclasses.The study was not powered to assess association of antibodies with malaria risk but we obtained some preliminary findings. Interestingly, in children with malaria episodes post-booster dose, the IgG2 and IgG3 levels at M21 were higher than non-malaria controls, while IgG, IgG1, and IgG4 levels were lower, although with the available sample size this was not statistically significant for most comparisons. These results are in line with the findings of our previous studyWe note that the pattern of higher IgG, IgG1, and IgG4 levels to CSP FL and C-term in non-malaria cases was not apparent for NANP.12. On the contrary, 1 month after receiving the RTS,S booster dose, anti-HBsAg IgG2 and IgG3 levels appeared as risk factors for future malaria episodes, further indicating that the nature and role of responses may differ following a primary and a booster immunization.We previously observed that after primary vaccination, HBsAg antibody responses were associated with malaria protection3. It was hypothesized that the primary vaccination had prevented vaccinees from acquiring natural immunity, as has been predicted for other malaria prevention tools16, increasing the risk of severe malaria in those individuals in whom infection reached the erythrocytic stage. However, on a longer follow-up study of up to 7 years on 3 of 11 sites, no increased risk was found for severe malaria between those groups that received the RTS,S/AS01 vaccine and the control group31. Antibody responses to asexual blood stage antigens have been studied previously with samples from phase 2 clinical trials and showed a reduced antibody response in RTS,S vaccinees, but these trials did not include a booster dose33. We detected a decreased antibody response to certain vaccine-unrelated P. falciparum antigens after primary vaccination in the phase 3 trial as well9. However, we also observed an induction of antibody responses to other P. falciparum antigens following RTS,S vaccination , which could contribute to malaria protection9. Here, we did not find consistent differences in the NAI response between booster groups except for Rh5. Interestingly and in line with our previous results, Rh5 IgG and IgG1-2 levels were higher in the RTS,S booster group than in the comparator vaccine groups at M21 and M32, and R3C levels were either higher or did not differ from C3C. This finding requires future investigation to understand the basis and clinical relevance of this effect, especially since Rh5 is a leading vaccine candidate34, and because Rh5 antibody concentrations need to be very high to actually confer protection35. However, these observations are important as they may explain why an anti-sporozoite infection vaccine also protects against clinical disease in the parasite blood stage, considering that Rh5 plays an essential role during erythrocyte invasion by P. falciparum merozoites38. Additionally, IgG to MSP5 showed higher levels after RTS,S booster dose compared to the comparator booster group, but this was not statistically significant.The analysis of the phase 3 clinical trial found that children who did not receive the booster dose were at higher risk of severe malaria than the comparator  group39, (2) there were unexpected results of VE in the phase 3 clinical trial, i.e., VE was lower in the R3R than in the R3C group, contrary to most sites, and (3) Manhiça has a high HIV prevalence40. HIV infection was associated with a reduced immunogenicity to the vaccine in a phase 3 trial exploratory analysis but it was concluded that HIV-infected children should not be excluded from RTS,S vaccination41.Our findings are limited because of a small sample size and because data were obtained only for Manhiça. Therefore, a larger longitudinal study with samples from different sites is necessary to corroborate these data. This is particularly important in our case because there are some special considerations about Manhiça that limits the generalization of these findings: (1) at the time of the study malaria transmission was lowDespite the constraints, our study provides new and interesting clues to the immune response elicited by the RTS,S booster dose. Additionally, avidity and functional antibody responses should be assessed, and these results integrated with cellular data to address memory responses induced by the booster. This information is necessary for a deeper understanding of the mechanisms of action of the vaccine, as well as the determination of the factors causing partial and short VE. Results of these studies are required for the rational design and deployment of improved CSP-based vaccines and other malaria vaccines with an increased and long-term efficacy.39, as part of the MAL067 study ancillary to the phase 3 randomized clinical trial MAL055 (NCT00866619)3. A subset of 50 individuals (24 children 5–17 months and 26 infants 6–12 weeks) was selected from those previously analyzed9 who had available antibody data from M0 (baseline) and M3 (one month after third dose) and plasma samples for M20 (booster dose), M21, and M32  at M20, three doses of RTS,S/AS01E and a comparator booster , or three doses and a booster of a comparator vaccine   in the US, Hospital Clínic in Spain (CEIm) and the CNBS in Mozambique, and written informed consent was obtained from parents or guardians before recruitment.This study was performed using plasma samples previously collected from subjects in Manhiça, Mozambique, a site of low malaria transmission intensityP. falciparum blood stage antigens  that were shown to be affected by vaccination in our previous studies9 and/or that are leading vaccine candidates; and Glutathione S-transferase (GST) as a control for antigens co-expressed with a GST tag . MAGPlex beads were coupled separately to: three CSP constructs  and HBsAg that are antigenic components of the RTS,S vaccine; seven Antigen-coupled beads were added to a 96-well μClear® flat bottom plate (Greiner Bio-One) in multiplex  resuspended in 50 μL of PBS, 1% BSA, 0.05% Azide pH 7.4 (PBS-BN). Fifty microliters of sample, negative or positive control were added to wells and incubated overnight at 4 ºC in a shaker protected from light. Plates were washed three times with 200 μL/well of wash buffer (PBS-Tween 20: 0.05%) using a manual magnetic washer. Then, 100 μL of biotinylated secondary antibody were added diluted in PBS-BN: anti-human IgG 1/2500 (B1140 Sigma), anti-human IgG1 1/4000 (ab99775 Abcam), anti-human IgG3 1/1000 (B3523 Sigma), and anti-human IgM 1/1000 (B1265 Sigma). For IgG2 and IG4, mouse anti-human IgG2 1/500 and IgG4 1/500 (MA1-34755 and MA5-16716 Thermo Fisher), respectively, were added, followed by biotinylated goat anti-mouse IgG 1/40,000 for IgG2 and 1/10,000 for IgG4 (B7401 Sigma) in PBS-BN. All antibody incubations were performed for 45 min, at room temperature, in agitation and protected from light. Again, plates were washed as before and 100 μL/well streptavidin-R-phycoerythrin 1/1000 (42250 Sigma) in PBS-BN was added to all wells and incubated 30 min, at room temperature, in agitation and protected from light. Plates were washed as before and resuspended in 100 μL/well of PBS-BN. Plates were stored at 4 °C overnight protected from light and read the next day using the Luminex xMAP® 100/200 analyzer; at least 50 microspheres per analyte were acquired per sample and Report Gain was set as High PMT.P. falciparum human serum (NIBSC code: 10/198)44 at 1:50 mixed with a pool of plasmas from RTS,S/AS02 vaccinated children45 with high IgG levels against CSP at 1:200. Blanks were added to each plate in duplicates and two negative controls samples from malaria-naive adults were added in each plate. Test samples were assayed at three dilutions for IgG , IgG1, IgG3  and IgM  to ensure that at least one dilution lie in the linear range of the respective standard curve, i.e., close to the highest slope between two dilution points. Owing to the low levels previously observed in these samples for IgG2 and IgG4 in Ubillos et al.7 only one dilution (1/50) was used. Sample distribution across plates was designed to ensure a balanced distribution of vaccination groups, sex, age cohorts, and malaria cases. The three time points for each individual and the respective dilutions were placed on the same plate. Data were captured using xPonent software, and antibody levels were measured as MFI.For IgG, IgG1, IgG3, and IgM, 20 serial dilutions 1:2 of a positive control were used to perform antigen-subclass-specific standard curves. For IgG2, 16 serial dilutions 1:2 were used. For IgG4, no standard curve was performed and only one positive control dilution was included. The positive control consisted of a WHO Reference Reagent for anti-malaria 46, fitted in a 4- or 5-parameter logistic (4-PL or 5-PL) regression model, and data points logarithmically transformed. To select the sample dilution for IgG, IgG1, and IgG3 in the linear part of the sigmoidal curve , an algorithm that detects the two points with the highest slope between them was used. The slope was computed as: m = (MFIi – MFIi+1)/(dilution_factori – dilution_factori+1). The mean MFI value of the two points was computed and used as the reference value, but the standard curves were visually inspected and if the model did not converge, the R2 < 0.9 or the curve maximum values were < 15,000 MFI, a 15,000 MFI reference value was set instead of the highest slope criteria. The nearest MFI of the test sample to the reference value was determined and the corresponding dilution was selected. Since only one dilution was used for IgG2 and IgG4, the standard curves were not used to select a dilution. The MFI measurement of the selected dilution was corrected multiplying by its corresponding dilution factor and transformed to log10 scale to stabilize the variance. Blank and GST signals were not subtracted. Blanks were used to measure background signal, and GST to assess for unspecific binding to the GST-fused antigens . Background values were below 500 MFI, and no correlation was found between IgG to GST and IgG to GST-fused antigens  at each visit were done by boxplots with log10(geometric mean), medians, interquartile ranges (IQR), upper whisker as the smallest between maximum × value and Q3 + 1.5*IQR and lower whisker as the largest between minimum × value and Q1 – 1.5*IQR. Wilcoxon-Signed Rank Test between M20 and M21 for the R3R vaccination group, and between M21 and M32 for the R3R and R3C vaccination groups were performed to determine if the antibody levels changed significantly 1 month and 1 year after the booster. Additionally, Mann–Whitney tests were performed to compare the R3R and R3C groups at each timepoint. p-values were adjusted for multiple testing using p.adjust on R47 by the Holm approach for IgG and for IgM to control for family wise error, and by the Benjamini–Hochberg approach for IgG1-4, altogether to control for the false-discovery rate since there were more tests. The comparisons between time points were corrected separately from the comparisons between vaccination groups, likewise the comparisons for vaccine antigens were corrected separately from blood stage antigens. Data for M0 and M3 from our previous study7 was used to analyze the kinetics of the antibody response throughout the 5 time points in the clinical trial. Adjusted p-values were considered significant when <0.05. The qSAT assay of M20, M21, and M32 samples was performed in the same laboratory using the same reagents and under similar conditions as the assay of the M0 and M3 samples, but they were not executed at the same time and a smaller set of antigens was used.Descriptive comparisons of Ig isotype/subclass levels to specific antigens  – log10MFI(M20) and compared between individuals who either did or did not present with clinical malaria after M21. All data analysis and plots were performed using R packages gridExtra48, dplyr49, ggplot250, tidyr51, and psych52.Stratified analyses and Mann–Whitney tests for independent groups and Wilcoxon-Signed Rank Tests for paired samples were performed between age groups and between malaria cases and controls, for each timepoint. Further information on research design is available in the Supplementary Informationreporting summary


Plasmodium falciparum infections. Numerous genotypes of haptoglobin have been reported in malaria endemic populations. In this study, the relationship between haptoglobin genotypes and incidence of uncomplicated malaria in a cohort of children living in a malaria-endemic area of Uganda was determined.Haptoglobin (Hp) is an acute phase protein that takes part in systemic regulation of haem during This is an extension of a longitudinal study comprising of 423 children aged between six months and nine years, who were actively followed up for one year. Malaria episodes occurring in the cohort children were detected and the affected children treated with national policy drug regimen. Haptoglobin genotypes were determined by an allele-specific PCR method and their frequencies were calculated. A multivariate negative binomial regression model was used to estimate the impact of haptoglobin genotypes on incidence of uncomplicated malaria in the children’s cohort. In all statistical tests, a P–value of < 0.05 was considered as significant.The prevalence of the Hp 1–1, Hp 2–1 and Hp 2–2 genotypes in the children’s cohort was 41%, 36.2% and 22.9%, respectively. The overall frequency for the Hp 1 allele was 59%, while Hp 2 allele occurred at a frequency of 41%. After adjustment of incidence rates for age, insecticide treated bed net (ITN) use and malaria history, the incidence of uncomplicated malaria for children carrying the Hp 2–2 genotype and those with the Hp 2–1 genotype was statistically similar (P = 0.41). Also, no difference in the incidence of uncomplicated malaria was observed between children carrying the Hp 1–1 genotype and those having the Hp 2–1 genotype (P = 0.84) or between Hp 2–2 Vs Hp 1–1 genotypes (P = 0.50).P. falciparum malaria severity are needed to understand the role of these genotypes in malarial protection.This study showed that the Hp 1–1 and Hp 2–1 genotypes each occur in nearly 4 in 10 children and the Hp 2–2 genotype occurs in 2 of every 10 children. No association with incidence of uncomplicated malaria was found. Additional studies of influence of haptoglobin genotypes on  Plasmodium falciparum malaria infections involves interplay of both parasite and host factors such as host genetic variability . About 88.2% of the participants’ guardians (373 / 423) reported owning and using an insecticide treated bed net (ITN) within their households. Four hundred and three participant’s guardians (95.3%) reported having ever administered an anti-malarial drug to the enrolled child. Throughout the 1-year of longitudinal follow up in this study, malaria episodes were not registered among 217 out of 423 children (51.3%). Among those who experienced malaria episodes  during the 1 year of follow up, the range of annual episodes per child was 1 to 9.Haptoglobin genotyping was performed successfully for 398 samples by determining the presence or absence of 935 bp, 1.2 kb and 1.4 kb DNA fragments corresponding to the Hp2, Hp1S and Hp1F genotypes, respectively. The Hp1-1, Hp2-1 and Hp2-2 genotypes were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency for the Hp1 allele was 59%, while Hp2 allele occurred at an allele frequency of 41%. The distribution of the Hp genotypes in the study cohort is presented in Table P. falciparum infection, were included in the final multivariate negative binomial regression model to determine the extent to which they were affected by the haptoglobin genotype after adjusting for other independent determinants of malaria incidence identified in an earlier study in the same children’s cohort [This study is an extension of a longitudinal study carried out previously on the same cohort of children . In thiss cohort ; namely There was no statistically significant difference in the distribution of the Hp genotypes among children who did and did not experience uncomplicated malaria throughout the year as shown in Table P. falciparum infection in Dogon, but not in Fulani tribe [Haptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. However, no association with incidence of uncomplicated malaria was found. The present findings differ from results of prospective cohort studies carried out among Kenyan children, in which the Hp 2–2 genotype was associated with lower incidence of clinical malaria , 14. In ni tribe . The finP. falciparum infections [P. falciparum malaria severity are needed to further understand the role of these genotypes in malarial protection.In addition, Hp genotypes have been shown to influence plasma Hp levels , 10 needfections , 25. Thifections , 17. AddThe Hp1 allele was present at an allele frequency of 59%, yet no influence on the incidence of uncomplicated malaria by the Hp1 allele was observed. This is also in line with some earlier studies that found no clear associations between the Hp 1 allele and malaria susceptibility , 16. InsHaptoglobin genotypes, Hp1-1, Hp2-1 and Hp2-2 were found in 41%, 36.2% and 22.9% of the cohort children, respectively. The overall allele frequency was 59% for the Hp1 allele and 41% for the Hp2 allele. Association of Hp genotypes with incidence of uncomplicated malaria was not observed. This lack of detectible association with uncomplicated malaria suggests that these alleles may have been maintained by protection from other infections. Similar studies in different settings are needed to confirm these findings.


The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome.Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv_18S rRNA genes or the multi-copy targets Pf_varATS and Pv_mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities.P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf_18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv_18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum, at 8.0% (66/828). Use of Pv_mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG.In the two low-transmission areas, P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.The proportional gains in the detection of  Plasmodium infections, persisting undetected in the population, present a silent reservoir for on-going transmission and pose a major challenge in malaria elimination . Pl. PlP. viPf_ and Pv_18S rRNA qPCR assays have been described previously for all three study sites [P. falciparum and P. vivax infections and density differed substantially between sites. To complement these former epidemiological analyses, age trends of P. falciparum and P. vivax infection in relation to parasite densities based on us-qPCR data were investigated. Parasite carriers with very low-density infections in PNG were primarily found in adolescents aged 10–20 years, whereas in Thailand and Brazil most additionally gained infections were found in older individuals (> 40 years of age). Therefore, the additional P. vivax infections detected by us-qPCR coincided with the age group that had presented the highest risk of infection in earlier analyses from Thailand, Brazil and PNG [Investigations of individuals at greatest risk of infection based on parasite detection by dy sites , 24 [W.  and PNG , 24  than in PNG . However, this difference was not statistically significant, as the absolute number of P. falciparum positive samples in the Brazilian community survey was as low as 1.7% (11/651) by P. falciparum us-qPCR. Further comparative studies are required to clarify a potentially higher gain in P. falciparum positivity in low transmission versus high transmission.The use of us-qPCR for parasite detection in the community provided a more accurate picture of low-density infections at different levels of transmission intensity. The benefits of using ultra-sensitive assays were clearly demonstrated for all study sites. When analysing the relationship of low versus high transmission intensity and the gain in P. falciparum and P. vivax transmission, the absolute number of infections additionally gained by ultra-sensitive methods seem few. However, for certain research questions arising in pre-elimination settings, such increased sensitivity might be beneficial for informing control measures. For instance, Pv_us-qPCR can play an important role in both epidemiological studies and surveillance, because those additionally detected Pv-positive individuals may experience relapses and in future contribute to onwards transmission.In areas of low Additional file 1. Supplementary Table.Additional file 2. Supplementary Figures.


Plasmodium knowlesi, a simian malaria parasite, has been in the limelight since a large focus of human P. knowlesi infection was reported from Sarawak  in 2004. Although this infection is transmitted across Southeast Asia, the largest number of cases has been reported from Malaysia. The increasing number of knowlesi malaria cases has been attributed to the use of molecular tools for detection, but environmental changes including deforestation likely play a major role by increasing human exposure to vector mosquitoes, which coexist with the macaque host. In addition, with the reduction in human malaria transmission in Southeast Asia, it is possible that human populations are at a greater risk of P. knowlesi infection due to diminishing cross-species immunity. Furthermore, the possibility of increasing exposure of humans to other simian Plasmodium parasites such as Plasmodium cynomolgi and Plasmodium inui should not be ignored. We here review the current status of these parasites in humans, macaques, and mosquitoes to support necessary reorientation of malaria control and elimination in the affected areas. Plasmodium namely Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale cause natural malaria infection in humans. However, this assumption was challenged when the first case of naturally acquired Plasmodium knowlesi infection was reported in an American army surveyor in the state of Pahang, Malaysia in 1965 [For many years, it has been accepted that only 4 species of Macaca fascicularis) was identified as a natural host of P. knowlesi, Plasmodium fieldi, Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium inui, among others [Plasmodium species in the area where the first case was reported. In contrast, Anopheles hackeri was incriminated as the vector of P. knowlesi in the state of Selangor and found to be attracted to nonhuman primates rather than humans, biting especially at canopy level [The long-tailed macaque , pig-tailed macaques (Macaca nemestrina), and banded leaf monkeys  [Plasmodium in wild macaques in Southeast Asia are listed in M. fascicularis has the highest prevalence of simian malaria. In Peninsular Malaysia, this species was positive for simian malaria in forested areas, but not in urban areas [P. knowlesi and P. inui infections have similar rates in Peninsular Malaysia, followed by P. cynomolgi [P. inui infection is the most prevalent infection in long-tailed macaques, followed by P. knowlesi and P. cynomolgi with similar rate [P. knowlesi and P. inui have similarly high prevalence, followed by P. coatneyi and P. cynomolgi [Plasmodium parasites namely Plasmodium eylesi, P. fieldi, Plasmodium fragile, Plasmodium hylobati, Plasmodium jefferyi, Plasmodium pitheci, Plasmodium simiovale, Plasmodium silvaticum, and Plasmodium youngi [The main hosts of alophos) . The disan areas . Overallynomolgi . In contlar rate , while iynomolgi . Most ofynomolgi ,56. Besim youngi . AlthougP. knowlesi has not been detected in wild macaques in Cambodia and Indonesia even though indigenous cases of knowlesi malaria in humans have been reported there [P. knowlesi is not the most prevalent malaria parasite found in macaques in each region/country in Southeast Asia except for Malaysian Borneo. The most prevalent simian malaria parasites in the other countries is either P. inui or P. cynomolgi. Both can reach more than 70% prevalence in some regions. Based on studies done thus far . Besides, P. inui is the widest-ranging parasite, likely due to its high chronicity in the hosts [P. cynomolgi. It is also noteworthy that there is a generally high multispecies infection rate among the wild macaques. This leads to a bigger reservoir and higher risks of zoonotic transmission of P. inui and P. cynomolgi to humans.ed there ,58. Morethus far , P. inuihe hosts , followeAn. hackeri as the vector of P. knowlesi [P. knowlesi malaria in humans in Sarawak, vector studies were conducted in different ecological niches. In Kapit district, Anopheles latens was identified as the main natural vector of P. knowlesi for both macaques and humans [Anopheles balabacensis was incriminated [Most of the vector studies have been done in Malaysia  followedknowlesi . Howeverknowlesi . After td humans , while iiminated ,69. In biminated .Anopheles cracens, a more anthropophilic member of the Leucosphyrus group was identified as the vector of P. knowlesi [An. hackeri and Anopheles introlatus were found to be the vectors [An. latens and Anopheles watsonii were predominant in forested ecotypes, while in the farm areas, Anopheles donaldi and An. latens were the main species [An. latens to be positive with sporozoites and to be attracted to both humans and macaques. Thus, An. latens had been incriminated as the main vector for P. knowlesi in Kapit, Sarawak. The role of ecology was also observed by Wharton and Eyles, who incriminated An. hackeri as a vector for P. knowlesi at the coastal region of Selangor [In Pahang, Peninsular Malaysia, knowlesi , while i vectors ,71. It s species . HoweverSelangor .Anopheles like An. watsonii and An. donaldi found in sympatry with An. latens in Kapit were positive for oocysts with PCR–positive for the genus Plasmodium, but species identification by using primers for simian and human malaria parasites was not successful [Anopheles kochi in Kuala Lipis, Pahang was found in considerable numbers in monkey baited traps but was not positive for oocyst or sporozoites by dissection [An. donaldi, to be positive for DNA of P. knowlesi, P. cynomolgi, and an unknown Plasmodium species [Other species of ccessful . Anophelssection . A recen species . UnfortuAnopheles dirus, the main vector of human malaria in this region [P. knowlesi [Anopheles species found with sporozoites, and the highest number infected was detected in the forest compared to the forest fringes. P. knowlesi was the second most common Plasmodium species identified in the vectors using nested PCR assay followed by other simian plasmodia [P. knowlesi was detected in the salivary glands of An. dirus as was the case in the previous study by Marchand and colleagues in the same locality [In the forest and forest fringes of Khanh Phu commune, Vietnam, s region , was ideknowlesi . It was lasmodia . P. knowlocality .An. dirus is the main vector for human malaria besides Anopheles minimus [An. dirus could play a role in the transmission of knowlesi malaria in that region. In Palawan, Phillipines, Tsukamoto and colleagues demonstrated the presence of Plasmodium oocysts and sporozoites in An. balabacensis in an area where macaques were present. At that time, molecular tools were not available, so they carried out experiments to expose An. balabacensis, also a member of the Leucosphyrus group, to Plasmodium-positive macaques and showed that An. balabacensis could develop sporozoites [Anopheles are confirmed vectors for P. knowlesi in Southeast Asia.In the other countries of the Mekong region ,  minimus . Thus, brozoites . In concAnopheles vectors [With extensive deforestation and land exploration in Malaysia over the past decades, there have been changes in the bionomics of the mosquitoes. Deforestation has caused the migration of the long-tailed macaques from forested area to farms and semi-urban areas where they usually scavenge for food. This may have triggered mosquitoes to follow the host and adapt to forest fringes and farm areas . Fruit o vectors .An. latens prefers to bite macaques at 3 to 6 m above ground. It was demonstrated that the periodicity of the gametocyte stage in the macaques’ host coincides with the biting times of the vector mosquito [Most of these vectors tend to bite humans early, between 1900 and 2100 hours, but this varies with the geographical locations and the mosquitoes species ,67–71. Amosquito . This raAnopheles mosquitoes using PCR. Based on the compiled data  by including simian malaria in the annual World Malaria Reports. This could spur increased surveillance and research to the benefit of the populations at risk and the ultimate goal of malaria eradication.Certification of malaria-free status is granted by WHO if the transmission of human malaria in an entire country has been interrupted for at least 3 consecutive years . For SouP. knowlesi. In moving forward, it is also crucial to initiate the development of diagnostic kits for both P. inui and P. cynomolgi. In a recent case reported in Kelantan, Malaysia [P. cynomolgi was misdiagnosed as P. vivax through microscopic examinations. When later reexamined molecularly, it was found that the patient was infected with P. cynomolgi, not P. vivax. Furthermore, the knowledge of clinicians about simian malarias is insufficient [P. cynomolgi produce hypnozoites [A rapid diagnostic test with high specificity and sensitivity is yet to be developed for Malaysia , a patiefficient . It is nnozoites  and thusP. knowlesi cases poses a major challenge to malaria control. Besides P. knowlesi, priority should be given to surveillance and control of other simian Plasmodium species, especially P. inui and P. cynomolgi, which have the potential to infect human. Simian malaria could emerge and spread as a major public health problem, as the 4 classical human malarias are reduced and as environmental change brings human into increased contact with simian malaria. The early recognition and containment of transmission of these simian malaria among humans should be given high priority.Many countries in Southeast Asia are rapidly reducing indigenous human malaria transmission. However, the ongoing increase in Plasmodium knowlesi due to diminishing cross-species immunity.The reduction in human malaria cases may have exposed human populations in some areas of Southeast Asia to a greater risk of being infected by Increased number of imported cases of simian malaria in countries outside Southeast Asia shows that simian malaria is now an international concern.Plasmodium cynomolgi and Plasmodium inui are more prevalent in Anopheles vectors and macaques hosts in Southeast Asia than P. knowlesi, raising the possibility of increased natural transmission of these simian malaria parasites to human in future.P. knowlesi) necessitates rapid diagnostic tests, appropriate treatment regimes, and new control strategies for malaria caused by these parasites.The possible emergence of other simian parasites :1946–9.Grignard L, Shah S, Chua TH, William T, Drakeley CJ, Fornace KM. Natural human infections with Plasmodium knowlesi cases in a malaria elimination area, Sabang Municipality, Aceh, Indonesia. Malar J. 2018;17(1):186.Herdiana H, Irnawati I, Coutrier FN, Munthe A, Mardiati M, Yuniarti T, et al. Two clusters of Plasmodium knowlesi: Reservoir hosts and tracking the emergence in humans and macaques. PLoS Pathog. 2011;7(4):e1002015.Lee KS, Divis PCSS, Zakaria SK, Matusop A, Julin RA, Conway DJ, et al. Plasmodium knowlesi reservoir. PLoS Negl Trop Dis. 2014;8(3):e2780.Moyes CL, Henry AJ, Golding N, Huang Z, Singh B, Baird JK, et al. Defining the geographical range of the Plasmodium knowlesi within a major transmission focus in Sabah, Malaysia. PLOS Negl Trop Dis. 2015;9(10):e0004135.Wong ML, Chua TH, Leong CS, Khaw LT, Fornace K, Wan-Sulaiman W-Y, et al. Seasonal and spatial dynamics of the primary vector of S1 Table(DOC)Click here for additional data file.