To contain multidrug-resistant Plasmodium falciparum, malaria elimination in the Greater Mekong subregion needs to be accelerated while current antimalarials remain effective. We evaluated the safety, effectiveness, and potential resistance selection of dihydroartemisinin-piperaquine mass drug administration (MDA) in a region with artemisinin resistance in Myanmar.NCT01872702.We did a cluster-randomised controlled trial in rural community clusters in Kayin (Karen) state in southeast Myanmar. Malaria prevalence was assessed using ultrasensitive quantitative PCR (uPCR) in villages that were operationally suitable for MDA . Villages were eligible to participate if the prevalence of malaria  in adults was greater than 30% or P falciparum prevalence was greater than 10% (or both). Contiguous villages were combined into clusters. Eligible clusters were paired based on P falciparum prevalence (estimates within 10%) and proximity. Community health workers provided routine malaria case management and distributed long-lasting insecticidal bed-nets (LLINs) in all clusters. Randomisation of clusters (1:1) to the MDA intervention group or control group was by public coin-flip. Group allocations were not concealed. Three MDA rounds  were delivered to intervention clusters. Parasitaemia prevalence was assessed at 3, 5, 10, 15, 21, 27, and 33 months. The primary outcomes were P falciparum prevalence at months 3 and 10. All clusters were included in the primary analysis. Adverse events were monitored from the first MDA dose until 1 month after the final dose, or until resolution of any adverse event occurring during follow-up. This trial is registered with ClinicalTrials.gov, Baseline uPCR malaria surveys were done in January, 2015, in 43 villages that were operationally suitable for MDA . 18 villages met the eligibility criteria. Three villages in close proximity were combined into one cluster because a border between them could not be defined. This gave a total of 16 clusters in eight pairs. In the intervention clusters, MDA was delivered from March 4 to March 17, from March 30 to April 10, and from April 27 to May 10, 2015. The weighted mean absolute difference in P falciparum prevalence in the MDA group relative to the control group was -10·6%  at month 3 and -4·5%  at month 10. At month 3, the weighted P falciparum prevalence was 1·4%  in the MDA group and 10·6%  in the control group. Corresponding prevalences at month 10 were 3·2%  and 5·8% . Adverse events were reported for 151 (3·6%) of 4173 treated individuals. The most common adverse events were dizziness (n=109) and rash or itching (n=20). No treatment-related deaths occurred.In this low-transmission setting, the substantial reduction in P falciparum prevalence resulting from support of community case management was accelerated by MDA. In addition to supporting community health worker case management and LLIN distribution, malaria elimination programmes should consider using MDA to reduce P falciparum prevalence rapidly in foci of higher transmission.The Global Fund to Fight AIDS, Tuberculosis and Malaria. However, the emergence and spread of artemisinin resistant P. falciparum malaria in the region 4 and the subsequent loss of partner drugs 10 pose a major threat to the control and elimination of P. falciparum malaria. In Cambodia, Thailand, Laos and Vietnam artemisinin resistance was followed by partner drug resistance. Fitter multidrug-resistant parasites have spread across the Eastern GMS causing high ACT treatment failure rates. In Myanmar, which bears the majority of the malaria burden in the GMS, molecular markers associated with artemisinin resistance have also been found in many parts of the country 11 but artemether-lumefantrine, the first line ACT, has remained highly effective so far 12. Although artemisinin resistance in Myanmar has a different genetic origin to that in the Eastern GMS, widespread artemisinin resistance places selective pressure on the partner antimalarial (lumefantrine) threatening malaria control. In the past Myanmar was the likely gateway for the spread of first chloroquine and then sulfadoxine-pyrimethamine resistance in P. falciparum parasites from the eastern GMS to India, and then on to Africa. This cost millions of lives. Preventing the further spread of ACT resistance is a high priority and, as elimination of artemisinin resistance requires elimination of all falciparum malaria, all countries in the GMS have committed to malaria elimination by 2030 13.Substantial reductions in malaria morbidity and mortality have been observed over the past 15 years where highly effective artemisinin-based combination therapies (ACTs) and vector control measures have been deployed. In the Greater Mekong sub-region (GMS), where forest and forest fringe malaria transmission predominates, and conventional vector control interventions  are less effective, the increased deployment of ACTs is likely to have contributed decisively to the 50% reduction in malaria cases between the years 2000-2014 14 and it reduces malaria deaths but it does not necessarily eliminate the disease. Using ultra-sensitive quantitative PCR methods (uPCR) for the detection of low parasite densities has shown that even in low transmission settings, the majority of people infected with malaria parasites are asymptomatic and therefore do not seek treatment 15. Most of these infected individuals have low parasitaemias, below the detection threshold of routine testing methods 17. These parasitaemias can persist for many months 17 and, left untreated, fluctuate intermittently producing transmissible densities of gametocytes 20. This explains how malaria is sustained during the dry season each year when vector densities are very low.Early diagnosis and treatment with effective antimalarial medicines reduces symptomatic malaria 24, whereas in others the impact has been transient. The popularity of MDA decreased with concerns over operational feasibility, long term effectiveness and the potential, under some circumstances, to increase drug resistance. Despite this, calls for urgent malaria elimination before artemisinin resistance spreads outside the GMS and a better understanding of the risks and benefits have renewed interest in MDA as an elimination accelerator. As people with asymptomatic parasitaemia have already controlled their infections, therapeutic responses are better than in symptomatic malaria. As with intermittent preventive treatments in higher transmission settings efficacy is greater than in the treatment of symptomatic malaria. Provided that MDA reduces the number of symptomatic malaria cases, then effective MDA with high coverage can reduce the emergence and spread of antimalarial resistance rather than increase it 25.Mass drug administration (MDA) targets this reservoir of malaria parasites. MDA aims to treat all people able to receive antimalarial drugs in a community regardless of their symptoms. The treatment clears the asymptomatic infections and prevents reinfection for the period that the slowly eliminated partner drug provides post treatment prophylaxis. The objective is to reduce malaria prevalence and transmission rapidly. Over the past century MDA has been used in many situations; sometimes resulting in sustained malaria elimination 26. Although efficacy has declined markedly in the Eastern GMS it remains a very effective treatment for uncomplicated malaria in Myanmar and elsewhere 27. DP provides a long period of post-treatment prophylaxis and can be used safely over multiple rounds 30. Primaquine (PQ) is a potent gametocytocide that has very low risk of toxicity when used at the recommended single low dose of 0.25mg/kg 32. We performed a cluster-randomised controlled trial to assess the operational effectiveness and impact of MDA with DP and PQ in rural communities with a high prevalence of asymptomatic malaria carriage (“hotspots”) in a region with artemisinin resistance in Southeast Myanmar. After providing routine CHW community case management and distributing long-lasting insecticidal nets (LLIN) we assessed MDA safety and its impact on P. falciparum malaria incidence, prevalence, and on artemisinin and piperaquine resistance markers in a cluster randomised comparison.Dihydroartemisinin-piperaquine (DP) is a safe and well tolerated antimalarial P. falciparum is prevalent in this area; a recent survey found kelch13 mutations in 46% of P. falciparum infections 12. Most of the population are Kayin (Karen) farmers. Villages are generally remote and small, with an average of approximately 600 people. The infrastructure is poor and access to formal health care is limited. In 2012, Medical Action Myanmar (MAM) instituted a network of community health workers (CHWs) in remote communities in this region to provide community-based malaria case management, in cooperation with the Myanmar National Malaria Control Program, as described previously 14.The study was conducted in Southern Kayin (Karen) state, in East Myanmar near the Myanmar-Thai border . The ter15. Three to four field teams each started at a house in a different corner of the village, and visited every fourth household. If that household was empty the team moved to the nearest house backward or forwards. From each selected household, a minimum of two persons over 18 years of age were invited to participate. After written informed consent was obtained, participants were asked questions about potential malaria symptoms, allergies and recent travel history, and a venous blood sample was taken and tested for malaria  using a Rapid Diagnostic Test (RDT) (SD Bioline). This RDT detects P. falciparum HRP2 and plasmodium lactate dehydrogenase. In total 2,647 participants were sampled. Participants with a positive RDT were treated immediately according to national guidelines. Blood samples were transported to a field laboratory, where they were processed and stored at -20°C for a maximum of two weeks before transfer on dry ice to Bangkok, Thailand, for uPCR analysis (described previously 15). If there was sufficient DNA the propeller domain of kelch13 (k13), a marker of artemisinin resistance, was sequenced and the Pfplasmepsin2 copy number (a marker of piperaquine resistance) was quantified using RT-PCR on all P. falciparum positive samples.We obtained a list of 58 villages from the Kayin state health authorities in a 1600 sq km area . In eachP. falciparum malaria prevalence exceeded 10% in the adult population tested by uPCR. 2.The survey baseline malaria prevalence exceeded 30% and/or the They were discrete geographically.P. falciparum prevalence.If the cluster could be paired to another cluster nearby with similar Villages were eligible to participate in the MDA study if they fulfilled the following criteria: falciparum prevalence (estimates within 10%); b) proximity to each other; and c) distance to the main road. Matching was done in order to minimise confounding by environmental differences, likely to influence vector populations, malaria transmission intensities and population mobility. For each pair, one cluster was allocated randomly to the intervention arm by coin toss.Eighteen villages were deemed eligible to participate in the study . In one 33. Community engagement was undertaken before each MDA round and uPCR survey. If the community agreed to participate, house visits were conducted to allow community members to discuss issues directly with the study team, to collect census data, to invite eligible participants, and to take participants’ informed consent. At each interaction it was reiterated that individuals could choose to participate or opt out at any time, regardless of the community decision.Community leaders were first informed about the study and if they agreed study staff subsequently interacted with the communities in a series of community engagement activities described elsewhere P. falciparum) or chloroquine plus the radical curative regimen of primaquine nationally recommended at the time; 0.75mg/kg once weekly for 8 weeks (P. vivax).Patients presenting to the community health worker with signs or symptoms of malaria were tested using a rapid diagnostic test (SD Bioline). If the test was positive the patients was treated according to National guidelines with a standard regimen of artemether-lumefantrine  was from the day of administration of the first dose of study drugs until one month after final dose or until resolution of any AE that had occurred during the follow-up period. Participants were screened for AEs on each day of drug administration using open and closed questions designed to detect relevant AEs . Some serious adverse events were reported on other days or identified during home visits to participants. All SAEs were to be assessed independently by the study medical safety monitor and reported to the ERB of the Department of Medical Research in Myanmar.kelch13 sequencing and Pfplasmepsin2 copy number quantification.Cross-sectional prevalence surveys were conducted in adults , in intervention and control villages 3, 5, 10, 15, 21, 27 and 33 months after the beginning of MDA. Field teams attempted to sample the same individuals where possible to allow for longitudinal analysis. If they were unavailable , another adult participant from the same household  was selected based on similar characteristics . As in the baseline prevalence survey, consent was obtained, RDTs were performed and venous blood was collected for uPCR analysis, PfKelch gene was examined by nested PCR amplification covering the propeller region of the gene as described previously 3 followed by sequencing of the gene by ABI Sequencer . The sequencing results were then aligned against the PfKelch gene of reference strain 3D7 (putative PF13_0238 NCBI Reference Sequence (3D7): XM_001350122.1) and analysed with Bioedit software . Pfplasmepsin2/3 copy number was quantified using Relative quantitative Real-time PCR based on Taqman real time PCR on a Corbett Rotor-Gene™ Q . Primers and probes have been described previously 34. Amplification was performed in triplicate on a total volume of 10 μL as multiplex PCR using Quantitec Multiplex PCR no ROX . Copy numbers were calculated using the formula: copy number =2-ΔΔct; with ΔΔ Ct denoting the difference between Δ Ct of the unknown sample and Δ Ct of the reference sample. A cut-off copy number of 1.5 was used to define Pfplasmepsin2/3 and pfmdr1 amplification. Reactions were repeated whenever the profile did not conform to exponential kinetics, or ΔΔCt spread was >1.5, or the Ct value was >35. To confirm amplification and resolve indeterminate results, samples passing these criteria but with an estimated copy number >1.3 were also re-tested once, the second result counting as final.Polymorphism in the P. falciparum infections in the intervention and control arms one month after completion of MDA (Month 3) and 12 months after the baseline survey. MDA coverage was defined as the number of people who received MDA as a proportion of the total population physically present in the cluster on any day of MDA distribution. Incident malaria cases were defined as persons who attended a CHW and tested as malaria positive by RDT. Field data were collected on paper reporting forms and entered afterwards into an OpenClinica  electronic database. For baseline malaria surveys, the sample size was calculated to estimate a 10% prevalence of P. falciparum with 6% precision, alpha 0.05 and 90% confidence after applying a finite population correction. Later surveys had increased sample sizes to increase the precision of the prevalence estimates.The primary outcome of the study was the prevalence of uPCR detected t test 35. Incidence density of P. falciparum and P. vivax infections was defined as number of incident malaria cases per 1,000 person years at risk, using total cluster population at census to estimate total person years. Incidence was compared between arms for each year after MDA was administered. For seven months during which one of the MDA clusters had no active CHW, incidence from the paired control village was not included in the comparison. For calculation of the weighted prevalence of P. falciparum and P. vivax in the intervention and control arms at each time point, clusters were weighted by the reciprocal of the sampling fraction with application of a finite population correction using the total adult population. Finite population corrections were also applied to the calculations of 95% confidence intervals for the cluster-specific prevalences. Where zero infections were observed in a cluster survey, exact/Clopper-Pearson binomial 95% confidence intervals were calculated.For the randomised comparison the cluster was the unit of randomisation and the unit of analysis. Cross-sectional prevalence survey data and incidence data were aggregated at cluster level for analyses to account for the cluster randomised trial study design. We compared the prevalence and incidence of malaria in the MDA clusters to control clusters using a weighted paired P. falciparum infection, multilevel mixed-effects logistic regression models were used with study cluster specified as a random effect. Exposures were sex, age , reported recent forest stay, reported recent overnight stay outside of the village, received MDA drugs, and presence in village during MDA administration. An estimate and 95% confidence interval of the unadjusted odds ratio was presented for each exposure variable.For the exploratory analysis of individual-level factors associated with P. falciparum and P. vivax infections occurring after the intervention we assumed that positive uPCR infections detected within the same individual without an interim uPCR negative result were a single continuous infection 36. We assumed that all instances of a P. falciparum positive uPCR test that were preceded either by a negative uPCR test, or were the first uPCR test conducted in that individual were new infections. Individual exposure time for this analysis was defined as the number of days in the catchment area following the beginning of the MDA. We assumed an individual who was present at two consecutive qPCR surveys was present for the entirety of the time between the surveys. If a resident was missing during a qPCR survey we assumed they remained under exposure time for 45 days after their last survey and 45 days before the next survey they were present for 37. We assumed a newly tested individual began their exposure period 45 days before their first test. Data were analysed using Stata .For the post-hoc exploratory analysis of the incidence of new uPCR detected This study was approved by the Department of Health and ethical approval was given by the Ethics Review Committees of the Department of Medical Research in Myanmar and the Oxford Tropical Research Ethics Committee. The trial was registered with clinicaltrials.gov (id: NCT01872702). The funding source had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.P. falciparum, 501 (19%) for P. vivax, 73 (2.7%) for P. malariae, 3 (0.1%) for P. ovale; and 75 (2.8%) for unspeciated malaria (insufficient DNA for speciation) (P. knowlesi and P. cynomolgi were not detected. 80 persons had mixed infections of 2 (78) or 3 species (2).The baseline uPCR malaria surveys were performed in January 2015 on 2,674 individuals in 43 villages  . Overallciation) . P. knowP. falciparum prevalences were similar in the intervention (15.9%) and control (16.9%) arms and the intra-cluster correlation coefficient was 0.056.During the census 8,721 people were present in the selected study clusters, 5,481 in the MDA clusters and 3,240 in the control clusters. The age and sex distributions were similar for the MDA and the control villages . Baselinth percentile, 75th percentile, maximum) village positivity rates of 4%  for P. falciparum and 16%  for P. vivax respectively. RDTs were positive in 30 (13%) of the 234 people who tested positive for P. falciparum by uPCR and 3 (0.6%) of the 501 people who tested positive for P. vivax by uPCR (p<0.0001). Of the individuals who tested positive for P. falciparum and P. vivax by RDT and uPCR 87% (26/30) and 67% (2/3) respectively did not have fever or a complaint of fever. RDTs were positive in 7 (0.3%) of the 2437 people negative for P. falciparum by uPCR and 3 (0.1%) of the 2170 people negative for P. vivax by uPCR. None of these RDT positive uPCR negative patients had fever or a history of fever.Malaria prevalence varied widely with median . P. vivax infections had lower densities; geometric mean 5,935 parasites/mL .The geometric mean estimated parasite density, based on the uPCR, of st dose, 8 refused further medication because of side effects after the 1st dose, and one became ineligible for medication during their course).Of the 5,481 people present in the MDA intervention villages at any time during the census or during the MDA delivery, 863 (15.7%) were present only at the census. This reflected substantial population mobility. The MDA cluster population varied markedly during the 3 MDA rounds with 4,000, 4,104, and 4,176 people present at months 0, 1 and 2 respectively. In total, 4618 individuals were seen across the three MDA rounds and 3,544 (76.7%) were present for all three rounds. MDA uptake was high; of 4,618 people present during the intervention period, 4,156 (90.0%) received at least one full course of treatment, 292 (6.3%) refused MDA while 153 (3.3%) were ineligible for medication because of age <1 year , pregnancy  concomitant illness  or previous suspected adverse reaction to the drugs  . Across Acceptance of MDA among people present was 3,432/4,000 (85.8%), 3,542/4,104 (86.3%) and 3,647/4,176 (87.3%) for each MDA round respectively . One clua)P. falciparum prevalence estimated by uPCR declined rapidly after MDA from 14.5%  at baseline to 1.4%  at month 3, a 90% reduction  or P. vivax (93) and who took MDA drugs in the 3rd round (month 2) and were tested subsequently at month 3, were negative. Twelve people had P. falciparum infections detected by uPCR at month 3, of whom eight had not received MDA. The other four individuals had received three full courses of MDA. Three had infections with K13 mutations indicating artemisinin resistance (two C580Y and one F446I). Thus among 626 individuals who were tested at month 3 after receiving a full course of DHA-piperaquine in month 2, 4 (0.6%) had a P. falciparum infection detected by uPCR.eduction . All indP. falciparum prevalence in the MDA arm remained low in successive surveys, with estimated prevalences ranging from 1.5% to 3.2% . Prevalence trends in most intervention clusters were consistent with the notable exception of MDA cluster 1, the only cluster without an active community health worker by the time of MDA administration. In this village there was an immediate reduction in prevalence after the MDA but a substantial rebound then followed  at baseline to 10.6%  at month 3 and 2.7%  at month 33  . By month 21 the OR was 1.8  (2.1% versus 1.1%). By contrast there was no consistent association between reporting of a recent  (10% versus 0.6%) at month 3 than at later periods ) at month 3 than at later periods  per 1000 person years in the control arm and 53.4  in the intervention arm, a weighted mean difference of -35.8  new cases.After MDA 87 new b)P. vivax prevalence declined immediately following MDA in the intervention arm from 23.3%  at baseline to 4.5%  at month 3  at baseline to 5.5%  at month 33  had a P. vivax infections detected by uPCR in the control arm and 422 new P. vivax infections detected in the intervention arm after the MDA. The incidence of newly detected P. vivax infections was 257.7  per 1000 person years in the control arm and 202.9  in the intervention arm, a weighted mean difference of -79.8  new cases.There were 271 new P. malariae prevalence in the intervention arm was 4.2  pre-MDA and ≤1% at all timepoints post-MDA  was 90  in the intervention arm and 94  in the control arm. P. falciparum incidence was lower in the subsequent years in both MDA and control arms. P. falciparum incidence (95% CI) in the MDA arm was 25 , 33  and 32  cases per 1000 person years for the first, second and third year post-MDA respectively. Corresponding incidences in the control arm were 38  in year 1, 29  in year 2 and 29  cases per 1000 person years in year 3. In the first year after MDA was concluded, most new symptomatic infections detected by the CHWs in the MDA clusters were among people who did not receive MDA (57%: 31/54).Fifteen clusters had active trained CHWs in place before the first baseline survey (Month -2). In MDA cluster 1, there were problems recruiting and the CHW started malaria activities only in October 2015 (Month 7). In the year preceding MDA, the incidence of symptomatic rol arms . Among tP. falciparum incidence was lower in the MDA intervention arm than in the control arm in the first year after the MDA , though the difference was not statistically significant (p=0.26). In the second and third years post-MDA the difference in P. falciparum incidence between study arms had narrowed  and -2.3  cases per 1000 person years respectively). There was considerable heterogeneity in cluster-level falciparum malaria incidence post-MDA; MDA cluster 1 had the highest post-MDA incidence  (n years) P. vivax incidence was lower in the intervention arm than in the control arm in the first year post-MDA , though the difference was not statistically significant (p=0.38)  cases and -0.1  respectively). Overall malaria incidence was lower in the intervention arm than in the control arm in the first year post-MDA , though this difference was not statistically significant (p=0.17). In the second and third years post-MDA malaria incidence was similar in both study arms  cases and -2.2  respectively).(p=0.38) . In the Pf kelch 13 genotyping was successful in 385 of 529 (73%) P. falciparum positive blood samples from all survey rounds  and at the conclusion of the study . F446I was the most prevalent Pf-kelch mutant identified comprising 149 of 247 (60%) of K13 mutant infections. Other frequently identified K13 mutations included C580Y (13%), G449A (7%) and N537I (4%). Plasmepsin 2 copy number genotyping was successful in 299 of 529 (56%) P. falciparum positive samples from all survey rounds (plasmepsin 2 parasites were observed at any time in the intervention (0/157) or control arms (0/142). Thus there was no evidence that MDA had selected either artemisinin or piperaquine resistant parasites.y rounds . K13 proy rounds . No multThere were 151 adverse events (AEs) reported  during t40. There is therefore a race to eliminate malaria in the GMS before resistance prevents it. Fortunately in most of Myanmar, which bears the main burden of malaria in the region, ACT partner drug (lumefantrine) resistance has not yet emerged 11. Malaria in forest and forest fringe communities is unevenly distributed with some villages having a high prevalence of asymptomatic infections. This study shows that implementation and support of community health workers (CHW) to diagnose and treat malaria in these remote malaria affected villages followed by antimalarial mass drug administration (MDA) with dihydroartemisinin-piperaquine can safely and rapidly reduce the burden of P. falciparum despite a high prevalence of artemisinin resistance. MDA reduced asymptomatic P. falciparum prevalence by 90% in the first year, then early diagnosis and treatment of symptomatic malaria cases by the CHWs plus vector control maintained low levels of P. falciparum infection for nearly three years.In the Greater Mekong sub-region (GMS), as in much of the malaria endemic world, malaria transmission is low and heterogeneous. In the GMS the main anopheline vectors often bite outdoors at dusk or dawn and conventional vector control measures  are less effective than elsewhere. This places a greater reliance on antimalarial drugs for malaria control. In recent years in the eastern GMS, and along the Thailand-Myanmar border, the efficacy of the first line ACTs has declined alarmingly as first artemisinin resistance, and then partner drug resistance, has increased and spread 33 in order to achieve a high population coverage and concomitant vector control. The properly supported CHW is the key to malaria control in this region. In this long prospective study conducted in remote areas of South-Eastern Myanmar well-supported and well-functioning CHWs reduced malaria by a factor of four over a three year period. These reductions in malaria prevalence are faster than reported previously with implementation of community based EDT and vector control programmes 43.The rapidity of the malaria decline in our study may have been increased by the intermittent RDT screening and treatment provided (i.e. active case detection of people with higher parasitaemias) at each survey time point, although this only accounted for a total of 37 treatments given across eight surveys. Spillover benefits from ACTs delivered to the neighbouring MDA villages could also be a contributory factor. The paired study design meant that the intervention and control clusters were in close proximity and so control clusters would have experienced less importation of P. falciparum parasites from neighbouring MDA clusters than before. However the most isolated control village, far from any MDA village also experienced a protracted reduction in malaria prevalence indicating that community based EDT and LLIN interventions reduce the asymptomatic reservoir as well. Notably the cluster with the poorest results  was without an active CHW until 7 months after MDA had begun.This combination of malaria control activities is necessary for sustained success. It requires uninterrupted support for the CHWs to provide early diagnosis and appropriate antimalarial treatment (EDT) and a strong community engagement programme P. falciparum prevalence after MDA were the more isolated clusters. Isolation presumably guarded them from reintroduction of P. falciparum from neighbouring untreated communities. Reintroduction confounds small studies but in a programmatic implementation of MDA, ideally all malaria hotspots in a target area would be treated. This would reduce reintroduction form adjacent areas and so the benefit conferred by MDA should be greater than shown here. Systematic large scale MDA distribution is feasible. In part of Eastern Myanmar where a similar approach has been prosecuted there has been no significant rebound of P. falciparum prevalence for 12 months after implementing MDA 45.MDA is a malaria elimination accelerator. Two factors determine MDA efficacy – coverage and efficacy. In this study 10% of the population did not receive MDA  providing an important residual reservoir of infection. Driving low levels of falciparum malaria to elimination requires prevention of reintroduction. Even in these remote forested areas people movement was extensive. MDA clusters with the lowest 46.We delivered >10,000 courses of DP to healthy subjects using DOT, of which >99% were completed. MDA with this ACT proved well tolerated safe and was widely accepted in this evaluation. This confirms previous reports that large scale administration of multiple courses of DP is acceptable and safe 34. MDA usually encounters relatively low parasite densities which have already been controlled by immunity, which together improve the chance of successful parasite clearance regardless of resistance. Piperaquine has a long terminal elimination half-life and monthly repeated courses ensure that MDA participants have only a single time period, after the final treatment round, where newly introduced malaria parasites may be exposed to sub-therapeutic drug concentrations. By that time most P.falciparum parasites have disappeared from the community because of MDA. Unsurprisingly there was no evidence for the selection of drug resistant parasites in this study.The high efficacy of dihydroartemisinin-piperaquine in artemisinin resistant infections is unsurprising as piperaquine remains highly effective in Myanmar. There was no evidence for the plasmepsin amplification associated with piperaquine resistance in the Eastern GMS P. falciparum and ten (1.6%) had P. vivax detected in blood samples one month after receiving a full treatment course of dihydroartemisinin-piperaquine. Without drug level measurement it is not possible to determine whether or not these were treatment failures. uPCR detects reliably parasite densities as low as 22/mL, which means that the parasitaemia in the first asexual cycle following liver schizont rupture could be detected. As the MDA drugs used do not affect pre-erythrocytic development, and are not hypnozoitocidal, it is possible that some of the parasitaemias detected one month after MDA resulted from recent pre-erythrocytic schizogony.Four people (0.6%) had P. falciparum, and as observed elsewhere, the initial substantial reduction of P. vivax prevalence at month 3 was not maintained. This was anticipated as P. vivax hypnozoites were not treated by the MDA regimen of DP and a single dose of PQ and relapses of vivax malaria were expected. On the other hand, the P. malariae prevalence reduction after MDA was sustained reflecting the absence of hypnozoites in this species. The gradual decline in the prevalence of uPCR detectable P. vivax in the control arm mirrors results reported recently from an observational study in Myanmar that described substantial declines in clinical P. vivax incidence and positivity rates in communities receiving community based case management by CHWs 14.In contrast to A limitation of this study is the small number of randomised clusters which meant that an assessment of the impact of cluster-level covariates was not feasible. By its nature, mass drug administration cannot be studied in an individual randomised control trial. The paired design in this trial, where control hotspots were left untreated, limits the generalizability of this study as programmatic implementation of MDA would likely take place across a contiguous and large region. Prevalence surveys were conducted in adults only as we were not permitted to take blood repeatedly from children.22. MDA regimens have been successful in eliminating malaria in island settings in Vanuatu and the Comoros 47 and recent studies from elsewhere in the GMS have provided promising results 48. Effective MDA requires a significant political, logistical and financial commitment. It should be considered an accelerator of elimination, and not a substitute for community based EDT. The poor long-term outcome in the MDA cluster with no active CHW in the seven months post-MDA highlights the limits of conducting MDA without the inclusion of routine control measures. While the drugs remain effective MDA is a safe and effective tool to accelerate P. falciparum elimination, alongside routine malaria control measures.Historically, MDA has often led to an initial reduction in malaria outcomes, but reductions are often not sustained, or there has been no long-term follow up Supplementary Materials


In Uganda, childhood anemia remains a health challenge and is associated with malaria infection as well as iron deficiency. Iron deficiency is intertwined with nutritional status, age and other comorbidities including helminths and Lead toxicity. Environmental Lead levels accounts for one’s blood Lead (BL) levels. Blood Lead competitively blocks iron absorption, inhibits hemoglobin (Hb) biosynthesis and elevates free erythrocyte protoporphyrin (FEP) levels. Lead toxicity’s contribution towards anemia pathogenesis, especially during malaria infection has not been studied. Concomitant exposure to both malaria infection and Lead pollution, exacerbates the anemia status. This study therefore aimed at expounding the anemia status of these Ugandan children aged under 5years who are exposed to both malaria infection and environmental Lead pollution.Briefly, venous blood samples from 198 children were microscopically assayed for malaria parasite density (PD), and hemoglobin (Hb) concentrations using the cyanmethemoglobin method, while BL and FEP levels were determined by the standard atomic absorption spectrophotometric and fluorometric methods respectively.3 parasites / µL, while eight (4%) were severely anemic (<5 g/dL). Regression analysis and statistical correlation between PD and Hb  was negative and weak as compared to that between FEP/Hb and Hb .One hundred and fifty-one (76.3%) of the children analyzed had moderate anemia (Hb <10>5 g/dL) with Means of BLL=8.6 µg/dL, Hb =7.5 g/dL, FEP/Hb =8.3 µg/g and PD =3.21×10Based on the study’s findings, we conclude that BL significantly contributes to the pathogenesis of anemia and therefore its co-existence with malaria infection in the host exacerbates the anemia status. Like many malaria holoendemic developing countries, Uganda is faced with health-threatening diseases including anemia. Majority (60%) of the Uganda’s urban poor live in social disadvantaged slums like Katanga, Kampala , 2. In sMalaria and Lead pollution geographically overlap, with water acting as sinks to Lead contaminated runoffs as well as bleeding ground for mosquitos. According to a study by , malariaAlthough, malaria infections remain the key cause of anemia in Uganda, other neglected yet significant confounders contributing to its pathogenesis include Lead poisoning, nutritional status, and helminths among others –9. Lead Blood Lead further triggers a reduction in red blood cells (RBC) production by specifically inhibiting Ferrochelatase a mitochondrial enzyme that catalyzes the insertion of iron into protoporphyrin during heme formation. Aminolevulinic acid synthetase (ALAS) a mitochondrial enzyme that catalyzes the formation of aminolevulinic acid (ALA) from succinyl CoA and glycine is also affected by BL –16. In aHigh malaria burden, overproduction of protoporphyrin, iron deficiency, inhibited ALAD and impaired ferro chelatase activity accounts for anemia pathogenesis. During malaria infection, intrinsic and extrinsic challenges are elevated and they induce iron deficiency anemia whose persistence elevates the rate at which Lead is absorbed –19. In aReduced cellular iron concentrations on the other hand enhances Lead’s effects on major synthesis reactions. For example, ferrochelatase enzyme which is specifically sensitive to low iron levels is affected by lead toxicity . Also ziSince red cells are at the centre of anemia pathogenesis, as well as home to both the malaria parasites and BL, their co-existence propagates the host cell’s survival challenges. The severity of anemia is dependent on the host’s age and nutrition status , 26. In 0C awaiting various analyses and determinations.This was a cross-sectional study on children aged 6 -60months living in Katanga area- Kampala city. The children were first screened for Plasmodium parasite infection before recruitment using rapid diagnostic kits. Children with malaria negative test, HIV positive test, blood transfusion history and signs of malnutrition were excluded from the study. Five (5) mls of venous blood from 198 malaria positive children were collected into EDTA by Qualified Nurses and Technicians. The samples were then transported on ice to Makerere University, Biochemistry Department Laboratory, stratified according to malaria parasite density and kept at 4Thick smears were prepared as described by , air dri4)2HPO4 solution were added to 1.8 ml of deionized water and 1.5 ml of 20% Trichloroacetic acid (TCA) and vortex mixed. The samples were centrifuged for 20 min at 5000 rpm and 10 µl of the supernatant injected onto the graphite tube. The calibration curve was drawn using a standard addition method as described in M-572 of INSPQ’s Toxicology Laboratory method.Blood Lead levels were determined following a method described by  using anThe FEP was measured following a method described by  using a Where Fz is the sample fluorescence, Cs concentration of the standard, Fs is the fluorescence of the standard, 2.7 is the final volume of HCl phase, 100 is the conversion factor to 100 ml of extract, 1.1 conversion factor for protoporphyrin measured against a coproporphyrin standard and 0.2 (20 µl) is the original blood volume measured. The FEP/Hb ratio expressed as microgram Hb per gram Hb was calculated by dividing the FEP/dL RBC by gram Hb/dL.Hemoglobin levels were determined by cyanmethemoglobin reaction method . AliquotWhere OD = optical density or absorbance at 540 nm.3 parasites/ µL, mean BLL = 9.2 ± 4.3 µg/dL, an FEP/Hb = 7.4 ± 2.9 µg/g. Thirty nine (19.7%) of the study population were not anemic with a mean value of Hb > 10 g Hb/dL (WHO cut off reference), mean parasite density = 1.7 × 103 parasites/ µL, mean BLL < 2 µg/dL, Mean FEP/Hb = 7.4 ± 2.9 µg /g. The details of the distribution are shown in Table Of the 198 children enrolled in the study, 8/198 (4%) were severely anemic with < 5 g/dL, mean parasite density = 7.4 × 10Several cellular biomarkers are used to measure the extent and effects of blood Lead (BL) on various biochemical systems. This study explored the levels of Free erythrocyte porphyrins (FEP) as a biomarker of heme biosynthesis disorder among the study participants. The heme biosynthesis process is inactivated by either iron deficiency, failed iron regulatory system or inhibited δ-aminolevulinic acid dehydratase (δ-ALAD) enzyme .Free erythrocyte porphyrin levels of ˃50 µg/ dL are important indicator of heme biosynthesis disorder. Common causes of this disorder include iron deficiency due to malnutrition and poor iron absorption, elevated hepcidin levels, and ALAD inhibition by blood Lead , 22, 29.3 parasites/µL of blood), and no detectable BLL, there was a perfectly functioning heme biosynthesis system (FEP <10µg/ dL), with no anemia (mean Hb=10.4 g/ dL). It is likely that low PD by its own may not induce anemia especially to people living in malaria endemic areas. However, coexistence with another confounding factor like BL heightens the anemia pathogenesis. We can therefore hypothesize that progression to a severe anemia status is multifactorial and is exacerbated by having both malaria infection and blood Lead simultaneously.Considering the group of 39 participants Table  with parTable This study reports a lower mean BLL (= 8.6 µg/dL) compared to literature –37. FurtA moderate interaction between the three variables  after a multivariate analysis Table  is reporIt can therefore be speculated that progression to severe anemia during malaria infection and Lead toxicity involves destruction of erythrocytes, inhibition of heme synthesis inhibition (ineffective erythropoiesis) and interference of hepcidin iron regulatory system. The increased erythrocytic clearance due to extrinsic and intrinsic challenges increase the susceptibility to phagocytosis and hence anemia. Reduced serum iron (substrate) availability further complicates the heme synthesis mechanism by disturbing the enzyme /substrate enzymatic reaction equation.However, this study did not find a direct relationship between high blood Lead concentrations with parasite density as previously reported. It is likely that as BLL increases, hepcidin expression is upregulated decreasing the available serum iron important for parasite survival. Blood Lead levels and malaria parasites Table ; Fig. 3 Children who participated in the study had no co-morbid diseases like HIV, sickle cell traits which are known confounding factors of anemia pathogenesis. This study therefore represents the first reported significant association between malaria and Lead poisoning during the anemia pathogenesis among the pediatric population in Uganda.This study did not assess the nutritional status of the participants which is an important confounding factor. We therefore recommend that future studies incorporate nutritional status of participants for a better understanding of the relationship between blood Lead levels and anemia during malaria infection.Based on this study findings, low blood Lead is a key confounding factor of anemia pathogenesis especially in children infected with malaria. Since both blood Lead and plasmodium infection geographically overlap with similar hematological consequences, their co–existence heightens the anemia status of the host.We recommend a detailed study involving a bigger sample size to properly understand the effects and anemia pathogenesis during Lead exposure and Plasmodium malaria infection over a long period of time.


Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as ‘test-and-treat’ policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM.Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case–control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle–Ottawa scale, and all studies were ranked as ‘Good’, scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility . Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79  for a delay of 2–3 days and 5.46  for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes . In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify.A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment. In a systematic review and meta-analysis, Andria Mousa and colleagues investigate associations between treatment delays and outcomes in severe malaria. In 2018, severe malaria was responsible for an estimated 405,000 deaths worldwide. Patient access to first-line antimalarials in health facilities remains suboptimal in many endemic areas.The contribution of delay to treatment of uncomplicated malaria is often believed to be a risk factor for developing severe malaria, but this relationship has not been systematically quantified, and findings are not consistent across all studies. Understanding this relationship is critical to determine how quickly patients need to receive treatment and to quantify the impact of treatment interventions.We conducted a pooled individual-participant meta-analysis to estimate the association between delay from onset of symptoms to seeking treatment and risk of presenting with different types of severe malaria rather than uncomplicated malaria.The risk of severe disease was significantly higher in children and adults who had longer delays from symptom onset to treatment-seeking, and this relationship was the strongest for progression to severe malarial anaemia.We estimate that almost half of the severe anaemia cases in both children and adults could be prevented if they presented within the first 24 hours of symptom onset.The findings of this individual-participant data (IPD) meta-analysis highlight the importance of improving access to prompt first-line treatment in preventing severe malarial anaemia cases and reducing the need for potentially harmful blood transfusions.Our findings highlight that provision of timely treatment is essential in preventing severe disease and death.Providing quantitative setting-specific estimates of the benefits of timely treatment through programs such as community health workers or strengthened health systems is essential in guiding their implementation and in determining the best allocation of resources amongst all malaria interventions. Plasmodium falciparum malaria is key in preventing progression to severe complications and death. In 2018, an estimated 405,000 deaths were attributed to severe malaria (SM) worldwide, with 67% of them occurring in children aged under 5 years . Ri. Ri37]. All studies included were approved by an institutional ethics review committee  and associated 95% CIs are also reported for ease of subsequent generalisation by readers. These were obtained using a generalised estimating equations (GEE) model, allowing for correlation of observations within studies. All analyses were done separately for children aged <15 years and for ages 15 and over. A separate subset analysis included young children aged between 6 months to 5 years (excluding <6-month–olds to minimise any biases that may arise from differences in case definitions between infants and children). In all analyses, a likelihood ratio test was performed to compare the difference between a model with and without the term for illness duration.P. falciparum in 2- to 10-year–olds (PR2–10) obtained from the Malaria Atlas Project database at the upper administrative area level for the year of admission for each individual  were omitted). SM, severe malaria; UM, uncomplicated malaria.Box-and-whisker plots showing median and IQR of duration of illness/fever in children <15 stratified by severity group (UM and SM) and data set are shown below (N(TIF)Click here for additional data file.S5 FigUM = 300, SM: NSM = 226). Median is shown as a thick dark line, and outliers (observations that are 1.5 × IQR from the lower or upper quartiles) are denoted. Extreme outliers, defined as duration of illness of over 3 weeks, are omitted from these plots (1.9% of cases aged 15 or over [4 UM and 6 SM] were omitted). SM, severe malaria; UM, uncomplicated malaria.Box-and-whisker plots showing median and IQR of duration of illness/fever in those aged 15 and over, stratified by severity group (UM: N(TIF)Click here for additional data file.S6 FigPrevalence of phenotypes amongst severe cases in children aged between 6 months and 5 years (A), treatment delay and odds of presenting with any SM (B), SMA (C), and receiving blood transfusion (D) in children aged between 6 months and 5 years. Proportions were calculated for severe cases with no missing values of a given measure. Each case can present with more than one phenotype. ORs (and 95% CIs) for presentation with severe disease rather than UM (A & B) with each additional reported day of delay after initial symptoms, compared with patients receiving treatment within 1 day of symptom onset . Amongst 5 studies with information on blood transfusions during hospital admission, 29.8%  of children aged between 6 months to 5 years with available data had received a blood transfusion. ORs (and 95% CIs) for receiving blood transfusion was estimated for each additional day of illness duration amongst all uncomplicated and severe cases. All ORs shown were obtained from a mixed-effects logistic regression adjusted for age as a linear predictor and allowed for random study effects. Statistically significant ORs are denoted in red (dashed purple line: OR = 1). OR, odds ratio; SM, severe malaria; SMA, severe malarial anaemia; UM, uncomplicated malaria.(TIF)Click here for additional data file.S7 FigAge-adjusted ORs (and 95% CIs) for the association between delay to treatment and presenting with (A) any severe disease, (B) prostration, (C) SMA, (D) hyperlactataemia or acidosis, (E) RDS, and (F) CM for adults aged 15 or over. Age-adjusted ORs were obtained from a mixed-effects logistic regression, with receiving treatment within 1 day of symptom onset being the reference category (dashed purple line: OR = 1). UM: N = 300; SM: N = 226; prostration: N = 103; SMA: N = 101; hyperlactataemia/acidosis: N = 40; RDS: N = 30; CM: N = 37. CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SM, severe malaria; SMA, severe malarial anaemia; UM, uncomplicated malaria.(TIF)Click here for additional data file.S8 FigAssociation between treatment delay and hyperlactataemia/acidosis (A), jaundice (B), prostration (C), and hyperparasitaemia (D) in children under 15 years. Age-adjusted ORs (and 95% CIs) for the association between delay to treatment and severe disease phenotypes for children under 15. Age-adjusted ORs were obtained from a mixed-effects logistic regression, with receiving treatment within 1 day of symptom onset being the reference category (dashed purple line: OR = 1). The equivalent plots for any SM and SMA are shown in (TIF)Click here for additional data file.S9 FigDuration of fever and severe malarial disease (A), SMA (B), hyperlactataemia/acidosis (C), prostration (D), and hyperparasitaemia (E). Age-adjusted ORs (and 95% CIs) for the association between duration of fever and severe disease phenotypes for children under 15. Age-adjusted ORs were obtained from a mixed-effects logistic regression, with receiving treatment within 1 day of fever onset being the reference category (dashed purple line: OR = 1). Six categories were used instead of 8 for duration of fever because sample size was smaller than the analysis including duration of either illness or fever. The equivalent plots for CM and RDS are shown in (TIF)Click here for additional data file.S10 FigMortality adjusted by phenotype in severe cases (A) and the association between delay to treatment and mortality in severe cases (B), in SMA cases (C), and in CM cases (D) in children. Adjusted ORs along with 95% CIs for mortality are shown obtained from mixed-effects logistic regression in severe cases. Panel A shows results of a model adjusting for age and presence of CM, severe anaemia, RDS, hyperlactataemia/acidosis, hypoglycaemia, and prostration in children aged under 15 years . ORs for Panel A show the adjusted odds of mortality associated with each phenotype relative to other phenotypes . Panels B–D show the association between duration of illness and odds of death amongst severe cases , SMA cases  and CM cases . CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SMA, severe malarial anaemia.(TIF)Click here for additional data file.S11 FigORs (and 95% CIs) for the associations between travelling time to the health facility and presentation with any severe disease and specifically for SMA, hyperlactataemia/acidosis, RDS, CM, jaundice and hypoglycaemia for children aged <15. ORs were adjusted for age and duration of illness and were obtained from a mixed-effects logistic regression, with travelling time of under 1 hour being the reference category (dashed purple line: OR = 1). Duration of illness was fitted as a categorical variable with the following 8 categories: ≤1 day, >1 to ≤2 days, >2 to ≤3 days, >3 to ≤4 days, >4 to ≤5 days, >5 to ≤6 days, >6 to ≤7 days, >7 days. Uncomplicated: N = 2,468; any severe: N = 1,274; SMA: N = 1,274; prostration: N = 899; hyperlactataemia/acidosis: N = 541; RDS: N = 590; CM: N = 261; jaundice: N = 195; hypoglycaemia: N = 206. For the studies conducted in Farafenni (The Gambia) and the earlier Tanzanian study, travelling time was reported by either the patient or caregiver, and for Yemen, this was measured by a field assistant. For the later Tanzanian study, distance between the hospital and the individual’s district/village was estimated using AccessMod, and travelling times were then computed based on reports by local residents of how they would normally undertake the journey to the hospital (detailed criteria in Manongi and colleagues ). CM, ce(TIF)Click here for additional data file.S12 FigORs (and 95% CIs) for the association between distance from the patient’s residence to the health facility and presenting with (A) any severe disease and specifically for (B) SMA, (C) hyperlactataemia/acidosis, (D) RDS, (E) CM, and (F) hypoglycaemia for children aged <15. ORs were adjusted for age and duration of illness and were obtained from a mixed-effects logistic regression, with distance <10 km being the reference category (dashed purple line: OR = 1). Duration of illness was fitted as a categorical variable with the following 8 categories: ≤1 day, >1 to ≤2 days, >2 to ≤3 days, >3 to ≤4 days, >4 to ≤5 days, >5 to ≤6 days, >6 to ≤7 days, >7 days. Uncomplicated: N = 479; any severe: N = 285; SMA: N = 102; hyperlactataemia/acidosis: N = 48; RDS: N = 145; CM: N = 24; hypoglycaemia: N = 27. In the study set in Benin, distance was measured between district of residence and the hospital using Google Maps. For Yemen, the distance between the patient’s residence and the hospital was measured by the visiting field assistant using car mileage. CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SMA, severe malarial anaemia.(TIF)Click here for additional data file.S13 FigORs (and 95% CIs) for presentation with SMA with each additional reported day of delay after initial symptoms compared with patients receiving treatment within 1 day of symptom onset. ORs were obtained from a mixed-effects logistic regression adjusted for age as a linear predictor and allowed for random study effects. Statistically significant ORs are denoted in red (dashed purple line: OR = 1). (A) Adjusting for travelling time  and (B) limiting analysis to those who live near a health facility . This includes only those who live within median distance (<17.2 km) or travelling time (<35 minutes) from the hospital. OR, odds ratio; SMA, severe malarial anaemia; UM, uncomplicated malaria.(TIF)Click here for additional data file.S14 FigORs (and 95% CIs) for presentation with (A) any SM, (B) SMA, (E) CM, and (F) RDS and requiring a blood transfusion (C) with each additional reported day of delay after initial symptoms compared with patients receiving treatment within 1 day of symptom onset. The equivalent plots for delay from fever onset are also shown for CM (G) and RDS (H). Age-adjusted change in haemoglobin (g/dl) from a mixed-effects general linear model is also shown in (D). All ORs were obtained from an age-adjusted mixed-effects logistic regression. CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SM, severe malaria; SMA, severe malarial anaemia.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableTable includes study site, study period, age ranges included, and frequencies of SM phenotype groups. Percentages with a given phenotype amongst severe cases are shown in brackets and omit missing values for that phenotype. The denominator only includes those who were assessed for that phenotype. For instance, renal impairment was not systematically assessed in many of the studies. ‘NA’ entries indicate that no information was collected for that phenotype in that study. See (DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableTable includes data from 415 individuals aged 16 or over  recruited in Northwick Park Hospital  between April 1991 and May 2006 . Table d(DOCX)Click here for additional data file.S5 TableTable includes study site, study period, age ranges included, and frequencies of UM and SM for 3 age groups . Age was missing for 1 SM and 5 UM cases. SM, severe malaria; UM, uncomplicated malaria.(DOCX)Click here for additional data file.S6 TableCM, cerebral malaria; HG, Hypoglycaemia; HL, Hyperlactataemia or Acidosis; HP, Hyperparasitaemia; JN, Jaundice; PRO, Prostration; RDS, respiratory distress syndrome; RI, Renal Impairment SM, severe malaria; SMA, severe malarial anaemia.(DOCX)Click here for additional data file.S7 TableDuration of severe symptoms obtained from 5 studies for 1,323 individuals. The median delay to admission in days after onset of severe symptoms and the difference between reported onset of severe symptoms and onset of uncomplicated disease are presented.(DOCX)Click here for additional data file.S8 TableRRs and associated 95% CI shown were obtained using a GEE model, allowing for correlation of observations within studies. Models are shown for children (<15 years). GEE, generalised estimating equations; RR, risk ratio(DOCX)Click here for additional data file.S9 TableSensitivity analysis with (A) exclusion of multiple phenotypes and (B) mother’s education as a covariate. Age-adjusted ORs (and 95% CIs) for the association between delay to treatment and SMA, RDS, and CM in children aged under 15. Age-adjusted ORs were obtained from a mixed-effects logistic regression, with receiving treatment within 1 day of symptom onset being the reference category. For sensitivity analysis A, cases with the phenotype of interest and prostration were not excluded because of prostration being a consequence of other symptoms . For sensitivity analysis B, low mother’s education was defined as not having completed at least primary education. The mothers of 1,782 (38.0%) children did not complete primary education. Mother’s education was quantified in 4 of the studies from Yemen (n = 781), The Gambia , Tanzania 2002–2003 , and Tanzania 2006–2007 . CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SMA, severe malarial anaemia.(DOCX)Click here for additional data file.S10 TablePfPR2–10 < 10%), moderate (PfPR2–10 of 10% to <35%), and high (PfPR2–10 ≥ 35%). CM, cerebral malaria; OR, odds ratio; RDS, respiratory distress syndrome; SMA, severe malarial anaemia.Age-adjusted ORs (and 95% CIs) for the association between duration of illness and SMA, RDS, and CM in children under 15. Age-adjusted ORs were obtained from a mixed-effects logistic regression, with receiving treatment within 1 day of symptom onset being the reference category. Models include an interaction between transmission intensity and the effect of duration of illness on a phenotype. Transmission intensity was categorised into low ((DOCX)Click here for additional data file.S11 TableCase fatality in UM and SM groups. Table denotes the number of individuals with available mortality status during admission and the number (and %) of deaths amongst those. SM, severe malaria; UM, uncomplicated malaria.(DOCX)Click here for additional data file.S1 Data(CSV)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.


Surveillance of low-density infections and of exposure to vectors is crucial to understand where malaria elimination might be feasible, and where the risk of outbreaks is high. Archived rapid diagnostic tests (RDTs), used by national malaria control and elimination programs for clinical diagnosis, present a valuable, yet rarely used resource for in-depth studies on malaria epidemiology.Plasmodium falciparum and Plasmodium vivax infections, and by ELISA for Anopheles salivary gland antibodies as a marker for exposure to vectors.1022 RDTs from two sub-Districts in Bangladesh  were screened by qPCR for low-density Anopheles was significantly higher in Kamalganj despite low transmission, which could be explained by low bed net use.Concordance between RDT and qPCR was moderate. qPCR detected 31/1022 infections compared to 36/1022 diagnosed by RDT. Exposure to Archived RDTs present a valuable source of antibodies for serological studies on exposure to vectors. In contrast, the benefit of screening archived RDTs to obtain a better estimate of clinical case numbers is moderate. Kamalganj could be prone to outbreaks. In many countries, scaled up malaria control has resulted in reduced morbidity and mortality. Between 2000 and 2015, malaria incidence worldwide fell by 37%, malaria mortality by 60% . This liIn pre-elimination settings, surveillance is crucial to monitor the impact of control interventions and adapt strategies as needed. As alternative to case number routinely reported by health centres, molecular screening can be applied to screen for low-density infections, either among febrile patients, or among the general population to identify asymptomatic infections. Ideally, such screening is accompanied by entomological surveys to measure vector density and composition.Molecular surveillance in malaria elimination settings across large geographical scales is challenging. Intensive sampling activities are required to obtain sufficient specimens to identify risk factors and determine spatial heterogeneity . As an aAnopheles vector bites can be assessed by Anopheles biting biomarkers [Anopheles mosquito injects saliva containing immunogens into the host. Proteomic studies lead to the identification of small salivary proteins specific to Anopheles species. The human IgG responses to the gSG6 protein and the gSG6-P1 peptide were validated as serological indicators of exposure to Anopheles gambiae, Anopheles arabiensis and Anopheles funestus [Anopheles saliva has been showed to gradually increase with increasing intensity of exposure to An. gambiae sensu lato (s.l.) bites, as assessed by conventional entomological methods [As an alternative to entomological surveys, exposure to omarkers . During funestus –15. The  methods . A drop  methods . The bio methods –22, and  methods .Plasmodium falciparum and Plasmodium vivax are endemic along the north-eastern and south-eastern borders. In recent years, a significant decrease of malaria cases and deaths was achieved, with only 10,523 confirmed cases in 2018 compared to 39,719 cases in 2015 (NMEP MIS report). Transmission within Bangladesh is heterogenous. Most cases are reported from the three districts collectively known as Chittagong Hill Tract (CHT) in the south-east of the country [In Bangladesh Anopheles mosquitoes using an antibody-based salivary biomarker.Each year, over 300,000 RDTs are used by the Bangladesh Malaria Elimination Program (NMEP) to screen febrile patients. qPCR screening was applied to 1022 archived RDTs from two Upazilas (sub-districts) to estimate the proportion of infections missed by routine diagnosis. For the first time, RDTs were used to study heterogeneity in exposure to Archived RDTs and routine demographic data collected during diagnosis were obtained from the NMEP from 2 Upazilas in Bangladesh: Alikadam , and Kamalganj  , 24. In P. falciparum at densities of 20,000 and 10,000 parasites/µL. After 10 days of storage at 4 °C, the RDT cassettes were opened, the entire RDT strip was removed, and fragments of the following components were sampled: sample pad and conjugate pad, nitrocellulose strip 1 (between the conjugate pad and the 1st test band), nitrocellulose strip 2 (between the two test bands), nitrocellulose strip 3 (between the 2nd test band and the filter paper), and filter paper. DNA was extracted with the NucleoMag whole blood kit (Macherey–Nagel) and screened by qPCR [Plasmodium falciparum DNA was detected in almost all regions of the RDT, with the highest concentration DNA found in the first half of the RDT strip between the sample pad and the first test band. Thus, this part was used for screening of archived RDTs.For initial testing, RDTs were prepared with whole blood spiked with cultured  by qPCR . PlasmodP. falciparum using the varATS assay [P. vivax using the cox1 assay [DNA was eluted in 25 µL volume, and 4 µL was used for screening for TS assay  and for x1 assay . Both asFor antibody elution, the second half of the RDT strip was cut into small pieces, placed in 1.5 mL microcentrifuge tubes, and immersed in 250 µl PBS supplemented with 0.05% (v/v) Tween-20  (PBS/T). RDT eluates were equivalent to a 1/100 dilution of whole blood or 1/200 of serum (assuming a haematocrit of approximately 50%). Tubes were incubated overnight at 4 °C, after which RDT eluates were separated from solid RDT components and stored at − 20 °C until assayed.ELISA conditions were standardized as described elsewhere . Testing2O2), and absorbance (OD) was measured at 405 nm. All samples were tested in duplicate. Each microplate contained in duplicate: positive control, negative control, and blank wells. The positive control was a pool of dried blood spots of people with recent travel to a malaria endemic country, and their result was always above the cut-off for positivity. The negative control was a sample of people from US (n = 2) with no exposure to malaria vectors.Nunc-Maxisorp 96-well plates were coated with 100 µL/well of gSG6-P1 peptide (2 μg/mL) diluted in 1 × PBS. Plates were incubated at 37 °C for 2:30 h and blocked with 3 × 300 µL of SuperBlock (TBS) Blocking Buffer. 100 µL of 1:10 diluted sample was added to each well. Plates were incubated overnight at 4 °C, then washed three times and incubated 1:30 h at 37 °C with 100 µL/well of a 1/500 dilution of mouse monoclonal anti-human IgG conjugated with biotin (BD Pharmingen). Peroxidase conjugated extravidine  was added at a 1/1000 concentration and incubated 1 h at 37 °C. After four final washes, colorimetric development was carried out using ABTS (2.2-azino-bis (3 ethylbenzthiazoline 6-sulfonic acid) diammonium (Sigma) in 50 mM citrate buffer . Age, gender, bed net use, and upazila were analysed as risk factors for infection and exposure. The Chi square test was used for the comparison of number of positive samples between sites and age groups. After checking the normality distribution using Kolmogorov–Smirnov and Shapiro–Wilk tests, the non-parametric Mann–Whitney test was used to compare medians of specific IgG Ab levels between two independent groups. The non-parametric Kruskal–Wallis test was used for the comparison of more than two groups. Significance was assigned at p < 0.05.Analysis by age-groups were considered using two groups: under 18 years-old and 18 years and older owing to the low number of infections recorded in smaller age-groups.P. falciparum and P. vivax combined was 3.52% (36/1022) by RDT and 3.3% (31/1022) by qPCR screening of archived RDTs. The majority of infections by RDT  and qPCR  were P. falciparum; the remaining three were P. vivax. All positive RDTs were from Alikadam. RDT positivity was comparable between individuals aged 18 years and older  and those younger .Table P. falciparum. Among the 33 positive RDTs, only 20 were positive by qPCR (≥ 1 gene copy/µL eluted DNA). On the other hand, qPCR detected P. falciparum DNA from 11 negative archived RDTs. While all RDTs from Kamalganj were negative, qPCR diagnosed 1 low-density P. falciparum infection (3.9 copies/µL eluted DNA).Concordance between RDT and qPCR was low for P. falciparum density of RDT-negative/qPCR-positive tests  9.4–35.3) was significantly lower than of RDT-positive tests .Eight out of eleven RDT-negative/qPCR-positive tests were from adults aged 18 years and older. As the majority of RDTs were from adults, the prevalence of sub-patent infection did not differ between age groups . Likewise, the proportion of sub-patent infections among all infections did not differ . The mean P. vivax. All 3 positive RDTs were confirmed by qPCR and no additional infections were detected.Results were in full concordance for Anopheles gSG6-P1 salivary peptide did not vary significantly between Alikadam  and Kamalganj , the median antibody titre was higher in Alikadam .


The malaria prevalence has declined in western Kenya, resulting in the risk of neurological phenotypes in older children. This study investigates the clinical profile of pediatric malaria admissions ahead of the introduction of the RTS,S/AS01 vaccine.Malaria admissions in children aged 1 month to 15 years were identified from routine, standardized, inpatient clinical surveillance data collected between 2015 and 2018 from 4 hospitals in western Kenya. Malaria phenotypes were defined based on available data.There were 5766 malaria admissions documented. The median age was 36 months : 15% were aged between 1–11 months of age, 33% were aged 1–23 months of age, and 70% were aged 1 month to 5 years. At admission, 2340 (40.6%) children had severe malaria: 421/2208 (19.1%) had impaired consciousness, 665/2240 (29.7%) had an inability to drink or breastfeed, 317/2340 (13.6%) had experienced 2 or more convulsions, 1057/2340 (45.2%) had severe anemia, and 441/2239 (19.7%) had severe respiratory distress. Overall, 211 (3.7%) children admitted with malaria died; 163/211  and 48/211  met the criteria for severe malaria and nonsevere malaria at admission, respectively. The median age for fatal cases was 33 months  and the case fatality rate was highest in those unconscious (44.4%).Severe malaria in western Kenya is still predominantly seen among the younger pediatric age group and current interventions targeted for those <5 years are appropriate. However, there are increasing numbers of children older than 5 years admitted with malaria, and ongoing hospital surveillance would identify when interventions should target older children. See the Editorial Commentary by Castelli on pages 381–2.) are recorded when requested; however, children may receive blood transfusions based on a clinical diagnosis of severe anemia or a documented severe pallor. Blood glucose, creatinine, lactate, bilirubin, and blood gas tests are rarely performed, and microbiology remains unreliable at all the hospital settings , 20.Malaria was defined based upon a reanalysis of characteristics documented in medical notes at admission, in-patient notes, and information recorded at discharge. A malaria diagnosis was classified as (1) the presence of fever (defined as a history of fever or an axillary temperature ≥ 37.5°C) plus a positive malaria slide and a primary discharge diagnosis of malaria; (2) the presence of fever plus a primary clinical discharge diagnosis of malaria where results of a malaria test were not documented; or (3) if both a history of fever and a temperature were not recorded at admission but the patient had a positive malaria slide and a primary discharge diagnosis of malaria .It was not possible to define the broad classification of severe malaria according to the WHO definition , 24, whiData were analyzed for 2 complete years on either side of a national health worker strike, from December 2015 to November 2016 and from November 2017 to October 2018 . The anaA total of 14 999 children aged <15 years were admitted to the 4 hospitals over the 24 months of surveillance between 1 December 2015 and 31 October 2018 . In brieAmong 5766 malaria admissions aged <15 years, complete records were available for 5219 (90.5%) on the AVPU score, 5147 (89.3%) on the ability to drink/breastfeed, and 5308 (92.1%) on the history of convulsions. Convulsions were reported in 1650 children and the number of convulsions were recorded in 1292 (78.3% of those with a history of convulsions). Hemoglobin results were available for 2085 (36.2%) of malaria admissions and, where this was not available, information on an assessment for severe pallor  or whether they received a blood transfusion  was available to identify those patients with severe anemia. Documented evidence of indrawing and/or deep breathing was available for 5307 (92.0%) malaria admissions. A total of 2340 (40.6%) children admitted with malaria had severe malaria, defined by the presence of any criteria in Among malaria admissions, the strict definition of cerebral malaria  was present in 81 (1.6%) of children at admission. A wider definition of cerebral involvement  was present in 299 (5.7%) children. There were 122 (2.3%) regarded as conscious but with an inappropriate response to voice (AVPU score = V). The inability to drink/breastfeed was present in 665 (12.6%) of children at presentation to the pediatric ward . Among aOverall, 211 (3.7%) children admitted with malaria died during hospitalization and 163 deaths occurred in children with severe malaria . The median age for fatal cases was 33 months (interquartile range [IQR] 12–72) and surviving children were of a similar age .Case fatality rates were highest among unconscious children ; however, fatality rates were also high for children who had an AVPU score of either P or U (20.7%) or of V 7.4%; . The casThe mortality rate in children with severe anemia plus impaired consciousness  was 15.3% (n = 35); it was 17.0% (n = 19) in those with severe anemia plus severe respiratory distress, 23.9% (n = 39) in those with impaired consciousness plus respiratory distress, and was highest, 25% (n = 14), in those children with a combination of severe anemia, severe respiratory distress, and impaired consciousness . OtherwiThere were 48 deaths in children who did not have any of the severity features shown in The median age for all malaria admissions was 36 months (IQR 18–60). Of all malaria admissions to the pediatric ward, 15% were aged between 1–11 months of age, 33% were aged 1–23 months of age, and 70% were aged 1 month to 5 years . Only 1.Pediatric malaria and severe malaria hospitalizations in western Kenya still predominantly affect younger pediatric age groups ; howeverThe case fatality rates for severe malaria remain high, despite the adoption of improved case-management guidelines in these hospitals , 18, 20 Preventing severe disease progression and improving timely access to emergency care must remain a priority across all settings in Africa. Access to hospital care in the area studied here is comparatively better than other areas of Kenya ; howeverHospital surveillance of severe malaria and its outcomes provide important insights into the age profiles of life-threatening diseases in the surrounding communities, enabling policy-makers to redesign disease prevention targets. Despite a reduction in malaria transmission in western Kenya and a coincidental increase in the mean ages of patients with severe malaria, hospitalization, and deaths in hospital, malaria continues to be concentrated in children aged less than 36 months 60%; . The useThe surveillance system described here has been developed in partnership with the national and county-level ministries of health , 20. TheIn an area of western Kenya where the RTS,S/AS01 malaria vaccination is proposed as part of routine EPI, malaria admissions, severe malaria, and hospital malaria deaths continue to predominantly affect the younger pediatric age group. Case fatality rates are high among patients with cerebral malaria, which was an infrequent presentation in this part of Kenya 25 years ago. Neurological complications may increase with declining malaria transmission and may involve increasingly older patients outside of the EPI protection range. Hospital surveillance provides a routine, sustainable means to track the severe malaria phenotype, as well as future impacts of community-based controls.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz844_suppl_Supplementary_FigureClick here for additional data file.


Malaria in pregnancy, including asymptomatic infection, has a detrimental impact on foetal development. Individual patient data (IPD) meta-analysis was conducted to compare the association between antimalarial treatments and adverse pregnancy outcomes, including placental malaria, accompanied with the gestational age at diagnosis of uncomplicated falciparum malaria infection.A systematic review and one-stage IPD meta-analysis of studies assessing the efficacy of artemisinin-based and quinine-based treatments for patent microscopic uncomplicated falciparum malaria infection  in pregnancy was conducted. The risks of stillbirth (pregnancy loss at ≥ 28.0 weeks of gestation), moderate to late preterm birth , small for gestational age , and placental malaria  after different treatments of uncomplicated falciparum malaria were assessed by mixed-effects logistic regression, using artemether-lumefantrine, the most used antimalarial, as the reference standard. Registration PROSPERO: CRD42018104013.n = 5015), IPD from16 studies were shared, representing 95.0% (n = 4765) of the women enrolled in literature. Malaria treatment in this pooled analysis mostly occurred in the second  or third trimester , with gestational age confirmed by ultrasound in 91.5% (4120/4503). Quinine (n = 184) and five commonly used artemisinin-based combination therapies (ACTs) were included: artemether-lumefantrine (n = 1087), artesunate-amodiaquine (n = 775), artesunate-mefloquine (n = 965), and dihydroartemisinin-piperaquine (n = 837). The overall pooled proportion of stillbirth was 1.1% (84/4361), PTB 10.0% (619/4131), SGA 32.3% (1007/3707), and placental malaria 80.1% (2543/3035), and there were no significant differences of considered outcomes by ACT. Higher parasitaemia before treatment was associated with a higher risk of SGA  and deposition of malaria pigment in the placenta .Of the 22 eligible studies (The risks of stillbirth, PTB, SGA, and placental malaria were not different between the commonly used ACTs. The risk of SGA was high among pregnant women infected with falciparum malaria despite treatment with highly effective drugs. Reduction of malaria-associated adverse birth outcomes requires effective prevention in pregnant women. Malaria during pregnancy has adverse impacts on the foetus including increased risks of pregnancy loss (i.e. miscarriage or stillbirth) , 2, pretThe most widely reported adverse impact of malaria in pregnancy is low birthweight (LBW), commonly defined as birthweight less than 2500 g and used as a proxy for foetal growth , 12. HowPlacental malaria  is one of the primary pathogenic mechanisms by which malaria during pregnancy can cause adverse effects to the foetus , 17. ManArtemisinin-based combination therapies (ACTs) are highly effective with expected ≥ 95% treatment success  in pregnancy , 24. HowIn this study, individual patient data (IPD) from treatment efficacy studies have been pooled to describe the risks of adverse pregnancy outcomes (i.e. indirect consequences) and placental malaria (i.e. a direct consequence) after the treatment of uncomplicated falciparum malaria during pregnancy accompanied with reliable gestational age, mostly estimated by ultrasound, at the times of diagnosis of uncomplicated falciparum malaria infection and delivery. This study aims to compare the risks of stillbirth, PTB, SGA, and placental malaria after different treatments for patent microscopic falciparum malaria (uncomplicated or asymptomatic) mainly in the second and third trimester, using artemether-lumefantrine (AL), the most used ACT, as the reference standard.ClinicalTrials.gov) on 26 April 2019 without any restrictions on language or publication year: malaria, pregnancy, treatment or names of antimalarial drugs, study design , and outcome types (efficacy). Studies were included if Plasmodium falciparum parasitaemia was confirmed by microscopy before treatment, the length of active follow-up was ≥ 28 days, polymerase chain reaction (PCR) was used to classify recurrence of falciparum malaria, gestational age at detection of parasitaemia and delivery was verified, and pregnant women were followed up until delivery for assessing pregnancy outcomes. Investigators were invited to join this project and share the IPD with the WorldWide Antimalarial Resistance Network (WWARN), which were then standardized for the IPD meta-analysis as described in the published protocol [A systematic review and IPD meta-analysis on the efficacy of artemisinin-based and quinine-based treatments on uncomplicated falciparum malaria in pregnancy was conducted with registration to PROSPERO (CRD42018104013). Briefly, a combination of five components were searched in seven databases  and two clinical trial registries  , 27. PTBPlacental histopathology was categorized according to the malaria parasites and malaria pigment as follows : no infeOnly singleton births without congenital abnormality were analysed, except for calculating the proportion of congenital abnormality, which included all pregnant women with delivery information. The proportion of miscarriage was calculated excluding malaria episodes at ≥ 28.0 weeks of gestation. PTB included only live singleton births and excluded pregnant women who had the first recorded malaria at ≥ 28.0 weeks of gestation (for extremely PTB), ≥ 32.0 (for very PTB), or ≥ 37.0 (for moderate to late PTB). Only live singleton births delivered < 43.0 weeks of gestation were included for assessing SGA. Newborns whose birthweight was assessed within 3 days were included in the birthweight analyses.mi impute mvn command to impute missing values of HIV status (~ 18% missing) and height (~ 14% missing). As recommended when MI is used, the Wald test was used for model building [p < 0.05 as the cut-off  or Stata MP 15.1 . One-stage IPD meta-analysis was conducted: univariable and multivariable mixed-effects logistic regression models were used to model risk of (i) stillbirth, (ii) PTB, (iii) SGA, and (iv) placental malaria. Study site was fitted as a random intercept in these models. Heterogeneity between studies was assessed by intraclass correlation. Multiple imputation (MI) was used for handling missing values of covariates to test the effect of exclusion of patients with missing data on our main conclusions. For the key analysis, both MI (presented as the main model) and complete case analyses are presented. MI was conducted in Stata MP 15.1 with 20 imputations using building , 34 by bAs the majority of the studies included second (≥ 14.0 to < 28.0 weeks) or third trimester (≥ 28.0 weeks) women, the gestational period between ≥ 14.0 and < 37.0 weeks was divided into four periods that had a similar number of women, and women included for a malaria episode before 14.0 weeks and after 37.0 weeks constituted a fifth and a sixth group, respectively. Gravidity (G) and previous history of pregnancy loss were jointly categorized  and always adjusted in the multivariable analyses as a confounder for assessing stillbirth, PTB, and SGA . PreviouOf the 28 identified studies in the literature with PCR-corrected efficacy, 22 studies assessed pregnancy outcomes, resulting in a total of 5015 women enrolled in those trials. Data of 4765 women from 16 studies, representing 95.0% of the total target, were shared to the WWARN repository and pooled for this IPD meta-analysis. Delivery information was available for 4503 women (3607 in Africa and 896 in Asia) and missing for 5.5% (262/4765) including 256 lost to follow-up and six maternal deaths. Of the 4501 women with estimated gestational age, 68.1% (3064/4501) had documented falciparum malaria in the second trimester and 31.6% (1421/4501) in the third trimester. Only 16 malaria episodes  were reported in the first trimester. After excluding twin pregnancies , 4440 women with singleton deliveries were included  in four studies were shared and analysed.I2 39%). However, congenital abnormalities were only reported in live born infants (n = 4335). As they are a known contributor to stillbirths, congenital abnormalities were likely to be underestimated. There was no difference among different treatments (p = 0.68). No further analyses were conducted because of the small number of cases. The 58 pregnancies with congenital abnormality were excluded from the analyses that follow.The overall proportion of congenital abnormality pooled by DerSimonian and Laird’s random effects was 0.4% . No further analyses were conducted because of the small number of cases.The overall pooled proportion of miscarriage was 0.0%  were excluded from the subsequent analysis on stillbirth.The overall pooled proportion of stillbirth was 1.1% , artesunate-amodiaquine , artesunate-mefloquine , dihydroartemisinin-piperaquine , artesunate-sulfadoxine-pyrimethamine , artesunate monotherapy , artesunate-atovaquone-proguanil , quinine monotherapy (n = 184), and quinine with clindamycin  , and gravidity was 2 (interquartile range 1–4). Malaria episodes in the included studies were mostly in the second  or third trimester . The majority of the episodes  were p = 0.03) and lower in multigravid women who had no previous pregnancy loss  compared with primigravidae. Higher haemoglobin on day 0 of the malaria episode was associated with a higher risk of stillbirth in the multiple imputation model, but not in the univariable and complete case multivariable model.In both the univariable and multivariable analyses, different antimalarial treatments were not associated with different risk of stillbirth using AL as the reference Table . In the I2 0%), 0.4% , and 10.0% , very PTB (≥ 28.0 to < 32.0 weeks), and moderate to late PTB (≥ 32.0 to < 37.0 weeks) were 0.0% , ASAQ (n = 747), ASMQ (n = 926), DP (n = 804), ASSP (n = 147), AS (n = 174), AAP (n = 76), quinine (n = 171), and QC (n = 51). Baseline characteristics , 14.0–19.9 weeks (n = 863), 20.0–23.9 weeks (n = 1023), 24.0–27.9 weeks (n = 917), and 28.0–36.9 weeks (n = 1290).The same nine antimalarial treatments were included in this analysis: AL . For each Isuccessive gestational period between 14.0 and < 37.0 weeks, the risk of PTB increased. Taller maternal height  and higher maternal BMI  were linearly associated with a lower risk of PTB. Teenagers and primigravidae were independently associated with a higher risk of PTB. Compared with primigravidae, the risk of PTB was lower in G2 women with no previous pregnancy loss  and G ≥ 3 women with no loss  or one loss . Compared with women < 20 years old, the risk of PTB was lower in 20–24 year olds , but not different in 25 and older. Compared with moderate malaria transmission areas, the risk of PTB was lower in low transmission areas but not different in high transmission areas. When only studies in sub-Saharan Africa were included , the risk of PTB was higher after ASSP than AL  . The overall pooled proportion of SGA was 32.3% , ASAQ (n = 700), ASMQ (n = 820), DP (n = 716), ASSP (n = 129), AS (n = 154), AAP (n = 55), quinine (n = 119), and QC (n = 41). Baseline characteristics  and higher maternal BMI  were associated with a lower risk of SGA. Higher baseline parasitaemia  and co-infection of other malaria species  were associated with a higher risk of SGA. Compared with primigravidae, the risk of SGA was lower in G2 women with no previous pregnancy loss  and G ≥ 3 women with no loss  or one loss .In both univariable and multivariable analyses, different antimalarial treatments were not statistically associated with SGA when compared with AL Table . For eacI2 94%) women were categorized as no infection, 49  acute infection, 1816  past infection, and 727  chronic infection. Overall pooled positivity of malaria pigment was 80.1% . The risk factors for deposition of malaria pigment (i.e. past and chronic infection) were investigated in 2987 women.Among 3033 women in four studies that assessed placental histopathology, 441 , ASAQ (n = 649), ASMQ (n = 668), DP (n = 658), AS (n = 85), and quinine (n = 80). Baseline characteristics , lower age , higher body temperature , lower haemoglobin level , higher baseline parasitaemia , and gametocytaemia before treatment  were associated with a higher risk of the presence of malaria pigment in the placenta. When the effect of parity was analysed by different malaria transmission intensities, the adjusted risk of placental malaria in pregnant women with ≥ 2 parities was lower than nulliparous women in moderate  and high  transmission areas, but not in low transmission areas   were not different following treatment for patent microscopic falciparum malaria (uncomplicated or asymptomatic) mainly in the second or third trimester of pregnancy. The results for the other treatments  need careful interpretation because of the small number of women included in the analyses, but generally, they were associated with similar risks to AL.The very low proportion of pregnancy loss from miscarriage observed in this pooled analysis is a consequence of the natural history of miscarriage, which decreases considerably after the end of the first trimester; as the mean gestation at enrolment was > 24.0 weeks, most participants were already beyond the high risk period for miscarriage. Therefore, the result of this analysis does not necessarily reflect the overall impact of malaria and antimalarial treatment on miscarriage, and likely underestimates it.P. falciparum malaria infection in sub-Saharan Africa [The low proportion of stillbirth 1.7%) in women treated with highly effective ACTs is notable compared to a previous systematic review and meta-analysis by Moore et al., reporting up to 1 in 5 stillbirths attributed to .7% in woIn this pooled analysis, the prevalence of SGA was higher than generalized regional estimates reported in the literature . This hiThe prevalence of PTB in this pooled analysis was similar to the general background population , suggestThe prevalence of pigmentation in the placenta in this pooled analysis was higher than the prevalence reported in the general population in malaria-endemic areas , which iThere are several strengths of this meta-analysis. Primarily, use of IPD, collected in prospective trials in which gestational age was predominantly assessed by ultrasound, allowed analysis of the largest number of pregnant women ever to be assembled to assess the impact of different treatments by the gestation age of falciparum malaria infection in pregnancy. With no registered trials currently recruiting pregnant women for assessment of efficacy of antimalarial treatment in pregnancy , it is uSome limitations of this study should be considered. Though confounders for each study were assessed  and adjusted in our IPD meta-analyses, it is possible that some potential confounders remained unadjusted. For example, the number of malaria episodes during pregnancy was reported to be cumulatively associated with an increased risk of SGA , 5 or plMultiple factors, including obstetric history, morbidity such as hypertensive disorders of pregnancy, gestational diabetes, sub-optimal gestational weight gain, and perinatal depression, and social factors, including employment, smoking, marital status, and literacy, can all be associated with pregnancy outcomes , 58–62, Although the use of artemisinin derivatives in the first trimester seems to be safe , 65, theThis IPD meta-analysis of over 4500 women infected with falciparum malaria from both Asia and sub-Saharan Africa demonstrated that the risks of late miscarriage and stillbirth were low, and late miscarriage, stillbirth, PTB, SGA, and placental malaria were not different between the most commonly used ACTs  in the second and third trimesters. Accurate assessment of EGA, malaria episodes beyond the current study framework (28–63 days), and thorough reporting of congenital abnormality, including abnormality in stillbirth, should be considered in future studies. Early diagnosis of parasitaemia, regardless of symptoms, and treatment with highly effective ACTs may result in improved pregnancy outcomes, but highly effective preventive measures are required to reduce the high rate of SGA.Additional file 1. Methods of multiple imputation.Additional file 2. Risk of bias assessment.Additional file 3. Sensitivity analysis.Additional file 4: Additional Figure 1. Forest plot of the proportion of miscarriage for each study site.Additional file 5: Additional Figure 2. Forest plot of the proportion of stillbirth for each study site.Additional file 6: Additional Figure 3. Forest plot of the proportion of moderate-to-late preterm birth for each study site.Additional file 7: Additional Figure 4. Forest plot of the proportion of small-for-gestational-age for each study site.Additional file 8: Additional Table 1. Summary of the studies included in the pooled analyses.Additional file 9: Additional Table 2. Baseline characteristics of pregnant women assessed for moderate-to-late preterm birth.Additional file 10: Additional Table 3. Baseline characteristics of pregnant women assessed for small-for-gestational-age.Additional file 11: Additional Table 4. Baseline characteristics of pregnant women assessed for deposition of malaria pigment in the placenta.Additional file 12: Additional Table 5. Multivariable logistic regression on the risk of deposition of malaria pigment in the placenta by parity in different malaria transmission area.