Background: Artemisinin and partner drug-resistant falciparum malaria is expanding over the Greater Mekong Sub-region (GMS). Eliminating falciparum malaria in the GMS while drugs still retain enough efficacy could prevent global spread of antimalarial resistance. Eliminating malaria rapidly requires targeting the reservoir of asymptomatic parasite carriers. This pilot trial aimed to evaluate the acceptability, safety, feasibility and effectiveness of mass-drug administration (MDA) in reducing malaria in four villages in Eastern Myanmar.Methods: Villages with ≥30% malaria prevalence were selected. Long-lasting insecticidal bednets (LLINs) and access to malaria early diagnosis and treatment (EDT) were provided. Two villages received MDA immediately and two were followed for nine months pre-MDA. MDA consisted of a 3-day supervised course of  dihydroartemisinin-piperaquine and single low-dose primaquine administered monthly for three months. Adverse events (AE) were monitored by interviews and consultations. Malaria prevalence was assessed by ultrasensitive PCR quarterly for 24 months. Symptomatic malaria incidence,entomological indices, and antimalarial resistance markers were monitored.Results: MDA was well tolerated. There were no serious AE and mild to moderate AE were reported in 5.6%(212/3931) interviews. In the smaller villages, participation to three MDA courses was 61% and 57%, compared to 28% and 29% in the larger villages. Baseline prevalence was higher in intervention than in control villages (18.7% (95%CI=16.1-21.6) versus 6.8%(5.2-8.7), p<0.0001) whereas three months after starting MDA, prevalence was lower in intervention villages (0.4%(0.04-1.3) versus 2.7%(1.7-4.1), p=0.0014). After nine months the difference was no longer significant (2.0%(1.0-3.5) versus 0.9%(0.04-1.8), p=0.10). M0-M9 symptomatic falciparum incidence was similar between intervention and control. Before/after MDA comparisons showed that asymptomaticP. falciparum carriage and anopheline vector positivity decreased significantly whereas prevalence of the artemisinin-resistance molecular marker remained stable.Conclusions: This MDA was safe and feasible, and, could accelerate elimination ofP. falciparum in addition to EDT and LLINswhen community participation was sufficient. Historically, the Greater Mekong sub–region (GMS) has been the source of antimalarial drug resistance, and artemisinin resistantPlasmodium falciparum has also emerged and recently spread in this region2. Artemisinin resistance has led to the failure of artemisinin based combination treatments (ACT)4, the cornerstone of uncomplicatedP. falciparum malaria therapy worldwide. Without alternative medicines in the near future, the best option to prevent the spread of resistant parasites to Africa is the rapid elimination ofP. falciparum parasites from the GMS. Early detection and treatment of clinical cases at the community level is effective in preventing mortality and reducesP. falciparum transmission and the associated morbidity7. However this approach alone is unlikely to halt the rapid spread of artemisinin resistance because it fails to directly address foci of asymptomatic carriage ofP. falciparum. This persistent reservoir represents the main obstacle to the rapid elimination of falciparum malaria in the GMS, where transmission is low, seasonal and unstable9.Recent progress in the control of malaria is now under threat because the two pillars of malaria control – antimalarial drugs and insecticides – are falling to resistance12. Mass drug administration (MDA) in malaria remains controversial because of its history of success and failure13. MDA in the ACT era often relies on piperaquine, which provides approximately a one-month protection from reinfection. Adding a single low-dose of primaquine rapidly sterilisesP. falciparum infections14. Promising short term results following MDA were recently obtained in Zambia, albeit in a context of generally decreasing malaria prevalence15. We present a 24-month pilot study of the safety and effectiveness of MDA in reducingP. falciparum incidence and prevalence in four villages with high prevalence of sub-microscopic infections located on the Thailand–Myanmar border, an area where artemisinin resistance is firmly established17.Systematic antimalarial drug treatment of the entire population of these foci may be the only method to achieve a rapid elimination of the parasite reservoir in the absence of a point-of-care test sensitive enough to allow mass screening and treatment of submicroscopic carriersNCT01872702.This study was the pilot phase of a multicentre cluster-randomized control trial conducted in several sites in the GMS and therefore limited to a sample size of four villages. It is registered at ClinicalTrials.gov:P. falciparum andP. vivax malaria17. In early 2013, 12 villages were screened using a high volume ultra-sensitive qPCR assay method (uPCR) with the original intention of identifying villages with >30% malaria prevalence, of which >30% wasP. falciparum  was set up at month 0 (M0) in all four villages and long lasting insecticidal nets (LLIN) were provided. Two villages (referred to as A1-KNH and A2-TOT) were randomized using a flipped coin to receive anti-malarial MDA for three consecutive months (M0 to M2) immediately. The other two villages (referred to as B1-TPN and B2-HKT) were monitored for a 9-month control period with MP and LLIN only. The control period was planned originally for 12 months, but MDA had to be expedited because of accessibility concerns during the rainy season, and was given from M9 to M11. All four villages were followed for 24 months in total.The study was conducted in rural Eastern Kayin (Karen) state of Myanmar, a region endemic forciparum . However18, and by village committees. Participation in surveys and MDA activities was voluntary. In addition to group information during community engagement activities, participants received individual information in their language (Karen or Myanmar) and provided written informed consent before inclusion in a survey or MDA. Participants under 18 years provided an assent in addition to the consent of their parent or guardian.This study protocol was reviewed and approved by OxTREC (reference no. 1017–13 and 1015–13), by the Tak Community Advisory BoardParticipants were defined as individuals living in the village who provided information during home visits at any time point. Each participant was followed using a unique identification code to collect information on participation, adverse events, malaria carriage during surveys, clinical episodes of malaria, and mobility.20. The different activities conducted were: workshops with authorities and local gatekeepers, meetings and activities with different population groups, health-related community incentives 19. Through this mutual understanding, the study activities could be conducted while respecting the life of the community and ensuring maximal benefit for the participants.Before and during the study, community engagement (CE) activities were conducted , and geographic references (latitude and longitude) were recorded for each house. Population movement in and out of villages was monitored by census during the quarterly surveys and by home visits. Newcomers intending to stay in the village for more than two weeks were enrolled after consenting to participate in the follow-up surveys. All newcomers arriving after MDA were offered a single curative treatment course of DP-primaquine, irrespective of disease or infection status.7. Fever cases were tested with SD Bioline Pf/Pv rapid diagnostic test (RDT). UncomplicatedP. falciparum cases were given weight-adjusted doses of dihydroartemisinin-piperaquine  for three days and one 0.25mg base/kg dose of primaquine . UncomplicatedP. vivax cases were given weight-adjusted doses of chloroquine  for three days. Severe illnesses were referred to the nearest clinic. LLINs were distributed to all households at M0.A MP was set up at M0 in all four villages to provide early detection and treatment of clinical cases21.Blood sampling surveys of the entire village population were conducted at months 0, 3, 6, 9, 12, 15, 18, and 24. Samples of 3 and 0.5 mL of blood were taken by venous puncture from survey participants over and under 5 years respectively. Malaria infection was detected in the laboratory by RDT, microscopy and by a uPCR method with an approximate limit of detection of 20 parasites/mLEach MDA comprised a standard 3–day treatment course of DP (dihydroartemisinin (7mg/kg) and piperaquine (55 mg/kg) and one 0.25mg base/kg dose of primaquine given orally under supervision. Treatments were given three times at one-month intervals (M0 to M2 or M9 to M11).Infants <6 months, individuals with a known allergy to any of the antimalarial drugs, and pregnant women in their first trimester were excluded from MDA participation. Pregnant women in their second or third trimester and breastfeeding mothers were eligible for DP, but excluded from primaquine treatment. Women of childbearing age were asked if they were or could be pregnant and a pregnancy test was offered if unsure. Infants <6 months were excluded from prevalence surveys.Adverse events (AE) were monitored using a structured questionnaire on the second, third, and seventh day after the start of the MDA treatment course. During the three months of MDA, a mobile clinic staffed with a medical assistant was available in the village to provide free consultations to all villagers and to assess MDA participants presenting with AE. Serious adverse events were reported centrally.Participant follow-up. The individual follow-up time was defined as the number of days spent within the catchment area of the MP, i.e. the village and the surrounding farms.Participation in MDA. Participation in MDA was measured as the total number of individuals completing one, two or three 3-day DP treatment courses divided by the total number of inhabitants present in the village at least once during the 3-month period, after excluding individuals who visited the village for two weeks or less. Individual participation in MDA was categorized in three groups: no MDA, one or two rounds , and three rounds MDA (i.e. infection cured and 3-month protection).Malaria infection prevalence. Prevalence ofP. falciparum orP. vivax infection was defined as the proportion of individuals with a positive uPCR for each parasite divided by total number of individuals sampled. When the parasite species could not be determined (because of low DNA content) samples were attributed to species based on the falciparum/vivax ratio measured in clinical cases in the village during the previous period8.Incidence of symptomatic malaria. Symptomatic malaria cases were defined as individuals with fever (temperature ≥37.5°C) or history of fever in the past two days and confirmed falciparum or vivax infection by RDT or microscopy. During the period between surveys, cases were detected passively at the MP and confirmed by RDT. During survey periods, individuals with fever or history of fever and a positive RDT or microscopy slide (with uPCR confirmation when available) were considered clinical cases. Incidence of clinical malaria episodes in the village was expressed in cases per 1000 persons per month . Patients residing in nearby villages and coming to the MP for diagnosis were excluded from the analysis. Mixed infections contributed to bothP. falciparum andP. vivax incidence.Assessment of mutations in PfKelch13. Polymorphisms in thePfKelch gene were assessed by nested PCR amplification covering the full length of the gene 2, and followed by sequencing of the gene by ABI Sequencer . Cross contamination was monitored by adding negative control samples in every run. Sequencing results were aligned againstPfKelch13 of reference strain 3D7 (putative 9PF13_0238 NCBI Reference Sequence (3D7): XM_001350122.1), using Bioedit software . Polymorphic patterns were assessed by two individuals blinded to the origin of the sample.PfPlasmepsin2 gene amplification.Pfplasmepsin2 copy number was quantified using Relative-quantitative Real-time PCR based on Taqman probe on a Corbett Rotor-GeneTM Q . Primers and probes have been described previously22. Amplification was performed in triplicate on a total volume of 25 μL as multiplex PCR using Quantitec Multiplex PCR no ROX . Every amplification run contained 9 replicates of calibrators and triplicates without template as negative controls.β-tubulin served as an internal standard for the amount of sample DNA added to the reactions. Copy numbers were calculated using the formula: copy number=-ΔΔCt2 withtΔΔ C denoting the difference between Δ Ct of the unknown sample and Δ Ct of the reference sample.24. Briefly, mosquitos were collected using indoor and outdoor human landing catch (HLC) in five sites per village, yielding a total of 50 human.nights per survey. Infection of primary malaria vectors  by plasmodium parasites was determined using a quantitative real-time PCR (qPCR) assay, confirmed with a second assay amplifying a different DNA target25. The limit of detection of the whole procedure was 6P. falciparum sporozoites per mosquito25. Main entomological indices - human biting rate (HBR), sporozoite index (SI), and entomological inoculation rate (EIR) - were calculated.Entomological surveys to identify malaria vectors species and abundance were conducted monthly in each village for five nights per month, as described elsewhereStatistical analyses were performed using Stata 14.1 . Binomial 95% confidence intervals (CIs) were calculated for prevalence and SI, and Poisson 95% CIs for incidence of clinical episodes, HBR, and EIR. Intervention and control group prevalence were compared using chi-square or Fisher’s exact test, as appropriate.P. falciparum infection 3 and 9 months after MDA. The secondary endpoints were defined as 1) safety and 2) acceptability of MDA, as evaluated by questionnaires. Additional outcome measures reported here are the incidence of clinical malaria over the first 9 months, number of people participating in MDA, and proportion of artemisinin-resistantP. falciparum infections.Because the MDA in control villages was conducted earlier than originally planned (M9 instead of M12), the revised primary endpoints were defined as the prevalence of asymptomaticP. falciparum carriage  was estimated using Generalised Estimating Equations (GEE) with a logit link to estimate Odds Ratios (ORs), an exchangeable correlation structure and robust standard errors to account for repeated measurements at individual level. The change in odds of P. falciparum carriage compared to baseline survey was estimated in the control period and in the after MDA period. To assess the duration of MDA effect, time-dependent changes in the odds ofP. falciparum carriage were estimated separately for the control and after MDA periods. Demographic characteristics, village, age group (≤ & >10 years), sex and season, were included as covariates. The analysis was repeated to assess the effect of individual MDA participation. A direct comparison of control and after MDA periods was also conducted in which baselineP. falciparum infection status was added as a covariate in addition to the demographic characteristics.A post-hoc before/after analysis of the entire duration of follow-up was conducted to understand the factors influencing the impact of antimalarial MDA and the overall intervention. The effect of the intervention onThe study was conducted between May 2013 and June 2015 . Of 3238MDA coverage. The target population was 2392 residents of the four villages at the time of MDA. Overall 78% (1866/2392) received at least one 3–day course of DP and 38% (911/2392) received the full course of three treatments. Coverage was higher in the two small villages compared to the two larger villages, for uptake of at least one treatment  and uptake of three treatments  (80/977) .Safety and tolerability of MDA. MDA was well accepted and well tolerated. In 3719 of 3931 (94.6%) complete structured interviews, no AE were reported. Reported AE were in majority dizziness (N=192) and pruritus (N=17). There were three reports of black urine. One G6PD-deficient male reported black urine before primaquine and one G6PD-normal male reported black urine 48h after primaquine. Neither were anaemic and both remained well. The third case, a G6PD heterozygote woman with normal G6PD activity, became anaemic but concomitant HIV and TB were considered contributory, as reported previously26. The percentage of participants reporting AE declined after each MDA round from 8.2% (114/1394) to 3.7% (49/1336). Among the 200 patients who presented at the mobile clinic over the 3-month MDA period, all AE were mild or moderate and most  were rated as unrelated or unlikely to be related to treatment  versus 6.8% (58/848); p<0.0001).There was a significant difference in baselineP. falciparum, 17 (8%) were still positive in the following survey 3 months later, three were positive after 6 months, and one after 9 months. DP was highly efficacious; no participant receiving at least one complete 3-day DP course was positive forP. falciparum in the survey following MDA (M3 or M12), irrespective of previous infection status  and 70% were not detected by microscopy or RDT (1432/2059) . Overall status . During Pilot trial endpoints: MDA+MP versus MP only comparisonPrevalence. At M3 the prevalence ofP. falciparum infection had fallen to 0.4% , in intervention villages A1-KNH and A2-TOT, and 2.7%  in non-intervention control villages, B1-TPN and B2-HKT (p=0.001). Comparing M0 to M3, prevalence decreased by 98% in MDA villages compared to a 57% decrease in control villages. At M9, the difference in prevalence was no longer significant: 2.0%  in MDA villages, and 0.9%  in control villages (p=0.10). Comparing M0 to M9, MDA villages exhibited an 88% decrease in prevalence, compared to an 85% decrease in control villages.Incidence. Between M0 and M9, falciparum malaria incidence was 1.0 case/1000 person-months (95%CI=0.4-1.9) in MDA villages (8/8352) compared with 1.3 case/1000 person-months (0.6-2.5) in control villages .P. vivax malaria incidence was 8.6 cases/1000 person-months (6.8-10.9) in MDA villages (72/8352), compared with 5.6 cases/1000 person-months (4.1-7.6) in control villages .Before versus after MDA comparisonP. falciparum infection. The impact of MDA onP. falciparum carriage was different in the two intervention villages. In A1–KNH, there was a rapid >95% reduction inP. falciparum prevalence, which was sustained from M3 to M24  compared to baseline (OR [95%CI]=0.08 [0.05–0.11])   . The con3–0.7]) . This trP. falciparum carriage was 30-fold immediately after MDA (0.03 [0.02–0.06]). Each additional month after MDA was associated with a minimal increase in odds ofP. falciparum carriage . For example, 12 months after MDA, a 15-fold reduction persisted (0.06 [0.04–0.09]). Similar results were obtained when estimating odds for each quarter instead of assuming a monthly linear trend  . In contrast, individuals who did not receive MDA in villages allocated to it had no decrease in the odds ofP. falciparum carriage (0.4 [0.1–1.9]) compared to the control period  . Irrespe period .P. falciparum clinical malaria. A total of 113P. falciparum symptomatic cases were detected among study participants, of which 91 were detected passively by the MP and 22 were detected during surveys. An additional 19 cases were detected among non-participants .P. falciparum clinical cases, which was close to zero after 12 months  was available for 107 of 247 detectedP. falciparum infections. The prevalence of PfK13 mutations was 77.6% (83/107) (Data on the artemisinin resistance marker83/107) . The mosNo plasmepsin 2 amplification (the molecular marker of piperaquine resistance) was found in the 69 samples that could be analysed (53 before MDA and 16 after) .P. vivax and 0.4/1000 forP. falciparum  compared to 0.2 (95%CI=0.05-0.4) after (p=0.0001). Likewise, before MDA,P. falciparum EIR was 0.25 infective bites/human/month (95%CI=0.13-0.43) compared to 0.06 (95%CI=0.02-0.15) after (p=0.005).The main anopheles vectors in the villages belonged to the Minimus Complex , Maculatus Group (11%) and Dirus Complex (1%). Overall, the SI of primary malaria vectors was 2.2/1000 forciparum . During P. falciparum parasites in the GMS leaves few options to prevent worsening malaria and spread of resistance westwards, as has occurred before. This preliminary evaluation of the feasibility, safety and effectiveness of MDA in accelerating the elimination of artemisinin-resistantP. falciparum in communities with a high prevalence of asymptomatic malaria shows that MDA with DP and a single low dose of primaquine was safe and well accepted26. Most participants did not report any AE, and those who did reported only mild and transient AE which declined in frequency after each successive round. Access to diagnosis and treatment, an essential component of malaria control, was deployed at baseline and had a substantial impact on malaria, which was augmented by MDA. When coverage was high, MDA provided a marked and long-lasting impact onP. falciparum malaria prevalence and incidence, but these benefits were transient in one village (A2-TOT). Overall, the odds ofP. falciparum carriage decreased by >95% immediately after MDA in comparison with baseline and an 80% decrease was maintained for 24 months.The rapid emergence of artemisinin resistance followed by ACT partner drug resistance inP. vivax malaria was substantially less important, as expected. The MDA did not target hypnozoites, so relapses of vivax were not prevented andP. vivax incidence and prevalence rose rapidly after the period of post treatment prophylaxis. Because of the relatively high prevalence of G6PD deficiency in this population and the unavailability of rapid testing, radical cure with primaquine was not used inP. vivax malaria treatment. Interestingly, in village B1-TPN only, the prevalence ofP. vivax infection fell and did not rebound.The effect of these interventions onThe main limitation of this study is that, being a pilot investigation, it is underpowered to draw definitive conclusions on the overall benefit of this approach. The comparison between intervention and control is indeed strongly influenced by individual village characteristics: although the four villages had similar malaria prevalence in the pre-survey , they weP. falciparum prevalence over a nine month observation period until MDA was conducted. As the two control villages had a relatively low baselineP. falciparum prevalence, the impact in a higher prevalence setting of instituting a malaria post and distributing LLIN without providing MDA cannot be predicted from these observations. Nevertheless providing MDA in these control villages after nine months reduced prevalence by 100% immediately after MDA, showing an additional effect of MDA to enhanced control measures, as shown recently in Zambia15.A second limitation was that the beneficial effects of MDA could not be evaluated independently from the continuing effects of the malaria post and the LLINs. Their benefits are illustrated in the control villages where there was three-fold reduction inThird, those who refused MDA were less likely to participate in surveys (as in village A2-TOT). This led to underestimation of the true prevalence of malaria, which was close to zero in the general survey population but 5% in the group of survey participants who did not take MDA. Individual participation in MDA was therefore included in the statistical models of impact to take this into account.P. falciparum prevalence in village A1-KNH, where MDA coverage was high, suggests a reduction of malaria transmission. This is supported by finding reductions of similar magnitude inP. falciparum SI and EIR, while HBR remained stable. By contrast, in A2–TOT,P. falciparum prevalence and incidence increased as vector abundance rose during the rainy and cold seasons of 2014, 14 to 20 months after MDA  in this group. In addition, the MP was not functioning properly during the first year, with frequent interruptions of activity and the lowest rate of consultations among the four villages .Importantly this intervention does not appear to have modified the resistance profile ofP. vivax returns rapidly, presumably because of relapse, which occurs frequently in the GMS. These preliminary findings need confirmation and exploration in further studies. Scaling up to provide MDA to larger populations will introduce new challenges of feasibility, but may improve overall effects as human migration from untreated areas becomes less likely. From a community perspective, elimination of malaria means elimination of all malaria, and to achieve that rapidly will require targeting the vivax hypnozoite reservoir.This pilot study shows that MDA with three rounds of a treatment course of DP and a single low dose of primaquine at one month interval is well tolerated, safe and feasible once the community has been successfully engaged in participation to activities and ownership of the program. It provides evidence that in a remote malaria endemic area, following implementation and support of a functioning MP, and distribution of LLIN, MDA eliminates the sub-microscopic parasites in those who take it. If a sufficiently high proportion of the community participates and if early diagnosis and treatment of clinical episodes remains available, this has a long lasting effect on falciparum malaria reservoir. By contrast,The data referenced by this article are under copyright with the following copyright statement: Copyright: © 2017 Landier J et al.http://www.tropmedres.ac/data-sharing) and following the Mahidol Oxford Tropical Medicine Research Unit data access policy (http://www.tropmedres.ac/_asset/file/data-sharing-policy-v1-0.pdf).The data is available upon request to the Mahidol Oxford Tropical Medicine Research Unit Data Access Committee . MDA appeared not to affect the incidence of clinical malaria episodes when compared to the control (‘pre-MDA’) villages. However, in the control villages, early diagnosis and treatment provided by MP was associated with reductions in malaria transmission, comparable to that seen in villages with MP and MDA. The relative contribution of MDA over MP is a key question for malaria control programmes as they seek to decide whether ensuring basic diagnostics and treatment is sufficient to eliminate malaria. The data are interesting but fall short of answering this comprehensively given the small sample size - presumably this will be a focus of the main study.Abstract:vivax infections. It seems from the methods section that the criterion was >10% falciparum prevalence. Also, it is not clear by what method this prevalence is assessed. It is not clear what refers to falciparum and what refers to What population size is village size small or large? Vector positivity presumably means sporozoite.Introduction: The evidence for the effect of treatment of the reduction of transmission is quite sparse and typically from areas with good health systems and low transmission. Context could be improved here. It might help to cite studies that show mosquito feeding experiments in SE Asia have shown that most infections come from symptomatic infections. Primaquine does not strictly sterilize infections – it has little demonstrable effect on asexual parasites.Methods: Were the numbers malaria posts proportional to population?P.vivax? What was the rationale for no radical cure for Why was parasite prevalence was not assessed in infants? One might expect that the overall transmission effect of MDA (and MP) on parasite prevalence would also be seen in this group. Does malaria infection prevalence represent for each or for either ? Why the change in time for MDA from M12 to M9? this seems irrational and inconsistent with the rigour of the general context of the study. It does not allow similar follow up periods and confounds any potential seasonal effects.Results: How did population movement relate to coverage? Were those individuals who spent less time in villages less likely to receive MDA? What was the prevalence of infection in this group? There is a lack of spatial analysis. The authors could use the georeferenced data to estimate whether MDA coverage predicted residual parasite prevalence spatially. Similarly, it would be interesting to understand if compliance was spatially aggregated (presumably it was). Also, were AEs related to coverage? The prevalence of AEs declined after each round, but that could be related to individuals most likely to develop AEs refusing to receive additional treatment. What was the prevalence of AEs in the different rounds in those individuals who received all 3 treatments? The most informative analysis is the comparison in the control villages before MDA  versus after MDA (combined MDA and MP effects). The GEE model estimated that the post-MDA period was associated with 5 fold reduction in falciparum carriage. However there was a clear trend of declining falciparum prevalence during the pre-MDA period. How much of this 5-fold reduction is related to the continued reduction in transmission due to MP.P. falciparum infections had a significantly increased odds of subsequentP. falciparum carriage…” – is this related to age? To occupation? Residence? “During the pre-MDA control period, individual with baseline  It would be interesting to see a graph that shows temporal variation in the entomological parameters together with human prevalence and comments on the effect of seasonality.Discussion The authors argue that access to diagnosis and treatment had an important effect on transmission that was “augmented by MDA”. The design of the study and the limited number of villages does not really justify this conclusion. In the control villages, where MDA was implemented 9 months after improved access to diagnosis and treatment, there was a clear trend of decreasing transmission before MDA. It is not clear how the authors expect to quantify effectiveness of MDA, that is independent of MP, using this design. This needs to be mentioned early in the discussion as this comparison is the main question the study wants to address: the added value of MDA in area with improved access to diagnosis and treatment. Authors could discuss what additional interventions should be considered to sustain vivax transmission reduction. It might be worth speculating how malaria elimination can be delayed by not treating first trimester pregnant women and infants.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Suggest line numbers be added to future manuscript versions to facilitate peer review. Page 3, right column, para 1, line 2: Suggest mentioning the net-to-person ratio for bed net distribution. Were all individuals provided a net, was each household provided a single net to share, etc.? Page 3, right column, para 2, line 8: Suggest mentioning the lower age at which assent was obtained. Page 5, right column, para 2: Can the authors explain why uptake was higher in the smaller versus the larger villages? Did the larger villages experience more out-migration during the study period, were there logistical problems in tracking down all previous participants in the larger villages, etc.? Page 5, right column, para 3, line 1: Is the acceptability of MDA simply inferred because AEs were mild or moderate? Or was a specific assessment of acceptability made? Page 5, right column, para 3: Suggest mentioning whether any of the AEs were treatment-limiting. Page 8, left column, para 1, line 1: Suggest mentioning why the other 31% of newcomers could not be included in the first survey. Page 8, Figure 2, panels A, B and D: The reappearance of Pv parasites within 3-6 months are obviously due to relapsing Pv from the liver, but do the authors know whether this rate of reappearance is typical following DP/MDA in this area? Also, can they speculate why there was very little Pv relapse in B1-TPN? This finding is mentioned twice in the manuscript text, but no speculation is offered. Page 10, right column, para 2, line 6: Is the difference in K13 mutation prevalence before and after MDA significant? Had the authors hypothesized a priori that MDA would have a selective effect on reducing the prevalence of K13-mutant infections? Page 10, right column, para 4, line 3: Suggest adding a citation to support the notion that these vectors are the contemporaneous primary malaria vectors in this area.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Anopheles culicifacies on malaria transmission among a cohort of children in villages of the Keshkal sub-district in Chhattisgarh state.Subclinical (asymptomatic) cases of malaria could be a major barrier to the success of malaria elimination programs. This study has evaluated the impact of long-lasting insecticidal nets (LLINs) on the prevalence of subclinical malaria in the presence of pyrethroid resistance in the main malaria vector A cohort of 6582 children ages less than 14 years was enrolled from 80 study clusters. Post monsoon survey was carried out at baseline before LLIN distribution, and 5862 children were followed up in the subsequent year. Study outcomes included assessment of subclinical malarial infections and use of LLINs among the study cohort in the presence of varied levels of pyrethroid resistance.In the baseline survey, the proportion of subclinical malaria was 6·1%. LLIN use during the previous night was 94·8%. Overall, prevalence of subclinical malaria was significantly reduced to 1% (p < 0·001) in the second survey. LLIN users were protected from malaria  and subclinical malaria  despite the presence of pyrethroid resistance in the study area.In this low transmission area, sleeping under LLINs significantly reduced the burden of malaria among children. In the presence of pyrethroid resistant malaria vector, a high LLIN use of 94·5% was observed to have significantly brought down the proportion of subclinical malaria among the cohort children. Vector control strategies including universal coverage of long-lasting insecticidal nets (LLINs) during the last decade and a half have significantly contributed to overall reduction in global malaria morbidity by 30%.Most malaria endemic countries, including India, have expanded their focus from malaria control to elimination, and India has recently launched a framework for a national level malaria elimination programme 2016–2030. This plan envisages scaling up of existing interventions, appropriate vector control measures, capacity building and strengthening drug compliance among malaria positive cases.7Under the existing health care system, malaria positive cases are captured through active and passive fever surveillance mechanisms. Persons with confirmed blood parasitemia through either microscopy or rapid diagnostic test (RDT) receive anti-malarial treatment. However, a sizeable proportion of the population has been observed to harbour malaria parasites without presentation of any symptom. Such cases are not captured through the routine surveillance system. As carriers, they pose a serious challenge to efforts towards malaria elimination.9Several studies from malaria endemic countries have reported that irrespective of the intensity of transmission, a large proportion of malarial infections remain asymptomatic and contribute about 20–50% of all malaria transmission.In endemic areas asymptomatic parasitemia may confer partial immunity on repeated exposure and may provide protection against clinical manifestations.There is a dearth of Indian studies evaluating impact of LLIN use on subclinical malarial infection and malaria transmission in pyrethroid resistant areas. Here, we present the results of a study on the impact of community-wide use of LLIN on the prevalence of subclinical malaria in a cohort of children under age 14 years in the presence of pyrethroid resistance in malaria vectors on the protective effectiveness of LLINs.The study was carried out during the post-monsoon seasons (August − December) of 2014 and 2015 among a cohort of children living in tribal forested villages in southern Chhattisgarh state, India.Anopheles culicifacies is the principal vector in the region.2 and twice a year) has been used in IRS as a major vector control measure by the state health department in the study area. With a consideration on year-round accessibility, 80 clusters were selected for the study. In 2014, 31,000 LLINs were distributed in collaboration with the state health department to cover a population of nearly 74,000 people living in these 80 clusters. In the remaining non-study clusters, the state health department has distributed LLINs covering nearly 16,000 population.Keshkal (20° 5' 1 N and 81° 35' 12 E), a sub-district of Kondagaon district, has a population of about 90,000 living in 124 villages. A basic health care facility is available through one Community Health Centre (CHC) situated at block headquarter Keshkal, as well as four Primary Health Centres (PHCs), each catering to a population of about 20000–25000. Malaria transmission takes place mainly during the rainy season (June − October). Plasmodium falciparum (Pf): artesunate + sulfadoxine − pyrimethmine + primaquine; P. vivax(Pv):chloroquine + primaquine]. Follow up slides were prepared seven days after medication to ensure clearance of asexual parasites from the peripheral blood.A cohort of 6582 children aged under 14 years was enrolled (60–80 children/cluster) after reading out the purpose of the study and obtaining written informed consent from the parent/guardian. Peripheral blood smear of all cohort children was prepared for microscopic confirmation of parasitemia to assess parasite prevalence before inclusion. Axial temperature of all study children at the time of survey was recorded with a digital thermometer . Blood smears were stained with Geimsa stain and 100 microscopic fields of thick smear were examined at 1000 x magnification to detect malaria parasites. Parasite density (parasites/μl blood) was counted against 200 White Blood cells (WBCs) considering the average of 8000 WBC/μl. All slide positive malaria cases were treated with anti-malarial drugs according to national drug policy [A slide positive case  with no symptoms of fever  was considered as a case of subclinical malaria.For routine fever surveillance, 30 malaria surveillance workers were recruited from different clusters (villages) and trained in malaria surveillance and treatment activities. To assist them in malaria surveillance in houses of cohort children, 124 female Community Health Volunteers i.e. mitanins, were deployed. All cohort children were visited once in a fortnight. A follow up survey of all cohort children was carried out after six months of enrolment. Axial temperature was recorded using a digital thermometer. Self-reported history of fever during the preceding week, previous night and at the time of blood smear collection was recorded. Information on reported use of LLINs during the previous night was also recorded. However, in the follow up survey, ∼10% of children were lost to follow up due mainly to their exiting the study area to pursue higher studies elsewhere.An. Culicifacies adult females caught from each cluster  were exposed to deltamethrin 0·05% treated papers following WHO test procedures to determine susceptibility status . Based on median mortality the clusters were stratified into low resistance  and high resistance  clusters. Thus, 3,249 and 3,333 cohort children were allocated to low resistance and high resistance clusters respectively.In the year 2015, wild caught full-fed Data were entered in EpiData version 3·1 software. All entries were double checked and further cleaned for error and analysed using IBM SPSS version 20 . Continuous variables were described as mean and standard deviation (M ± SD), and categorical variables were described in percentages.To assess the reduction in prevalence of malaria and subclinical malaria cases in consecutive years, the data for both surveys were combined into one data set and included ‘survey year’ as a fixed effect in the generalised estimated equation (GEE) model together with all the other variables. Reduction in malaria from one survey to the next, adjusted for all the other variables in the model were analyzed.Effect of LLIN use between cluster resistance status was determined in a separate stratified analysis, and then overall cross level interaction between the two was analyzed by adding interaction term in the GEE model to assess impact of insecticide resistance on LLIN effectiveness.P-value.Univariate and multivariate analyses were carried out using a generalised estimated equation (GEE) regression model to explore the relation between subclinical malaria and exposure variables. GEE analysis was chosen so that within-cluster correlation can be adjusted and possible interaction of village resistance status can be taken into account during modelling. Response variable presence of subclinical malaria has binary events (Yes/No). Binomial distribution with logit link function was selected. Exchangeable correlation structure was chosen for the model. In the model, explanatory variables that included age group, gender, and last night LLIN use were added and village insecticide status was taken as main effect. Study cluster variable was taken at the subject level. The results of GEE regression analyses were stated as unadjusted and adjusted odds ratio (OR) with 95% confidence interval (CI) and associated The purpose of the study was described in the local language to all study participants, and study related queries such as routine surveillance and blood slide collection procedures etc. were addressed. A written informed consent was collected from the parents or guardians of the enrolled children. This study was undertaken as a part of a WHO-coordinated multi-country project and ethical clearance was obtained from the Institutional Ethics Committee (ECR/NIMR/EC/2010/75).Between September and November 2014, 6582 children  were enrolled from 80 study clusters. The mean age of the study cohort was 6·3 years (SD- 3·4); children of age group 5–9 years constituted 40% of the study population followed by 2–4 years (28·9%). Of all children, 114(1·7%) and 196 (3·3%) reported with a history of fever  in the baseline and follow up surveys respectively. Detailed demographic and clinical description of the study population is given in Pf (331) or Pv (67) parasitemia. One child had a subclinical infection with P. malariae. In all, 91 microscopy positive children (1·4%) had malarial infection  associated with clinical symptoms. Among Pf infections, asexual parasitemia was higher  in symptomatic cases as compared to asymptomatic cases . Similarly, sexual (gametocyte) parasitemia in symptomatic Pf infections was nearly two times higher than in asymptomatic children (GMD 613 vs 303 parasite/μl).In the baseline survey, peripheral blood samples from a total of 490/6582 (7·4%) children had malarial parasites . A totalPf and Pv infections, respectively.In the follow up survey carried out during 2015, blood samples of 5862 children were collected, of which 82 (1·4%) were positive for malaria parasites. Among the positives, 46 (56·1%) and 19 (23·2%) children had subclinical The prevalence of malaria was significantly reduced during the follow-up survey in August 2015 as compared to the baseline survey in November 2014  after adjusting other factors such as gender and age groups. Similarly, significant reduction was observed in the prevalence of subclinical malaria in children in baseline and follow up surveys  keeping other variables (gender and age group) constant. .An. culicifacies to pyrethroid in high and low resistance clusters was 71·8% (53·0–83·6) and 93·8% (84–100) respectively. Malaria positive cases both clinical and subclinical were stratified based on susceptibility test data of An. culicifacies to deltamethrin of year 2015. Clusters with high deltamethrin resistance An. culicifacies showed almost equal numbers of subclinical malaria cases compared to low resistance clusters (1% vs. 1·4%). Overall, no interaction between the LLIN uses and insecticide resistance of study clusters was observed . (Mean (range) mortality rate of = 0.21). .Identification and targeting of subclinical malaria cases is one of the key challenges for countries aiming for malaria elimination.P. falciparum infections (67·6%). Studies from different parts of the world have reported that subclinical malaria burden could be 4–5 times of total reported malaria cases,The findings of the baseline survey (2014) showed that overall prevalence of subclinical malaria infection was 6·1%, constituting more than three fourths of total slide positive cases 81·3%) among the enrolled children with predominance of 1·3% amonAn active case surveillance (ACS) system to monitor the fever cases and to ensure regular LLIN use in the selected houses showed that LLIN use among cohort children (under 14 years old) in the study area was 94·8%, which was comparatively higher than reported in other studies.From this study, it can be stated that use of LLINs was equally effective in protecting the cohort from clinical malaria and subclinical malarial infections. A possible hypothesis for the protective effect of LLIN against subclinical infection is that there was interruption of transmission at the household level. It has been observed that if malaria infection were diagnosed from a member of one household there would be increased chance of presence of subclinical infection in other members of the family.Malaria incidence and subclinical malaria prevalence were significantly reduced by 84% in the follow up survey relative to the baseline, which was perhaps attributable to adequate LLIN coverage and high usage rates by the cohort children. Nonetheless, ACS also substantially contributed to the control of malarial infection in the study population. A study from Kenya showed a similar decrease in subclinical malaria due to regular ACS.Parasite density, age and season are essential co-variates in risk of developing fever.The worldwide spread of insecticide resistance among malaria vectors has been well-established.40An. Culicifacies in most of the study clusters (69) based on WHO cut off ≥98% of mortality rate. Stratified data analysis showed that the occurrence of malaria infection among LLIN users and non-users was similar and resistance status was not behaving as an effect modifier for LLIN usage and malaria cases. We speculate that this could be due to higher LLIN usage among the children and no loss of effectiveness in LLIN efficacy in protecting from malaria and subclinical malaria was observed.Susceptibility tests carried out in 2015 have confirmed pyrethroid resistance in Apart from seasonal dynamics of malaria transmission, residing in malaria hotspots at micro-geographical scale in low transmission areas is an independent predictive factor for malaria infections.While this study has focused on the post-monsoon season, further assessment is required during the dry season, when LLIN utilization might reduce due to reduction in vector densities or hot climates that deters usage. Further follow up could delineate the effect in prevalence of subclinical malaria in the low transmission season as well.This study presents an ideal scenario with high LLIN use, and well-informed household and study findings could only be generalized to other tribal populated area within low transmission settings, if high LLIN coverage is maintained with a robust active surveillance activity in place.There was a high proportion of subclinical malaria infections among children in the tribal inhabited malaria endemic area of the study, which may also be the case in areas with similar populations and malaria transmission intensities, and therefore may inform appropriate malaria elimination strategies. These observations indicated that high use of LLINs can significantly reduce the number of subclinical malaria cases, despite vectors having reduced susceptibility to the pyrethroid used in the LLINs. Therefore, high LLIN coverage and usage supported by a strong active case surveillance system would be a useful strategy in low transmission settings where parasite reservoirs exist as subclinical carriers.No competing interest declaredBill and Melinda Gates Foundation (Grant number OPP 1062754) which has no role in the planning, study design, data collection or write up. This research forms part of a multi-country study coordinated by the Global Malaria Programme of the World Health Organization.Funding was provided by the The purpose of the study was described in the local language to all study participants, study related queries such as routine surveillance, and blood slide collection procedures etc were addressed. A written informed consent was collected from the parents or guardians of the enrolled children. Ethical clearance was obtained from the Institutional Ethics Committee (ECR/NIMR/EC/2010/75).


Randomized controlled trials have established that seasonal malaria chemoprevention (SMC) in children is a promising strategy to reduce malaria transmission in Sahelian West Africa. This strategy was recently introduced in a dozen countries, and about 12 million children received SMC in 2016. However, evidence on SMC effectiveness under routine programme conditions is sparse. We aim to measure the effects of the nationwide SMC programme in Mali on the prevalence of malaria and anemia in children 6–59 months. We used data from the 2015 nationally representative malaria indicator survey. A post-test only with non-randomized control group study was designed. We fitted a generalized structural equation model that controlled for potential bias on observed and non-observed variables (endogenous treatment effect model). Having received SMC reduced by 44% (95% CI [0.39–0.49]) the risk of having a positive rapid diagnostic test for malaria. In addition, the programme indirectly reduced by 18% the risk of moderate-to-severe anemia (95% CI [0.15–0.21]). SMC in Mali has substantial protective effects under routine nationwide programme conditions. Endogenous treatment effects analyses can contribute to rigorously measuring the effectiveness of health programmes and to bridging a widening gap in evaluation methods to measure progress towards achieving malaria elimination. Drug administration usually takes place once per month during the transmission season, for a total of three or four cycles per year depending on the length of the rainy season. This strategy typically targets all children 3–59 months, and combines therapeutic and prophylactic effects. The drug regimen recommended in West African countries is one single-dose of sulfadoxine-pyrimethamine (SP) and three daily doses of amodiaquine (3AQ)3.Seasonal malaria chemoprevention (SMC) is a promising strategy to reduce the burden attributable to malaria in children under five. It consists of the repeated administration of a long-acting antimalarial drug regimen in areas where malaria transmission is highly seasonal10. A meta-analysis, pooling data from seven trials, showed a large and significant reduction in the incidence of clinical malaria  over the intervention period11. One particular trial showed that SMC reduces the prevalence of malaria infection by 85% (95% CI 73–92) during the transmission season7. Trials also suggested a beneficial effect on anemia8. Based on this evidence, the WHO has been recommending SMC since 2012, and it has been introduced in a dozen Sahelian or sub-Sahelian countries13. Thanks to this rapid scale-up, about 12 million children received SMC in 201614.SMC efficacy has been demonstrated in several randomized controlled trials15. An ecological study conducted in Mali with a similar quasi-experimental design found that SMC reduced the odds of malaria infection at the district level by two thirds  at the end of the transmission season16. Unfortunately, these studies were limited to small study areas and have limited external validity. To date, there have been no evaluations of SMC impacts at the regional, national or sub-national levels using routine data or nationally representative surveys.Outside of experimental contexts, little is known about the effectiveness of routinely implemented SMC programmes. A pre-post study with a non-randomized control group conducted in Burkina Faso showed that after the first cycle of SMC, the prevalence of malaria parasitaemia was reduced by 62% (95% CI 48–71)We aim to assess the effectiveness of SMC at reducing the prevalence of malaria and anemia in children 6–59 months, using data from the 2015 malaria indicator surveys (MIS) in Mali. Our intention is also to illustrate that nationally representative surveys, such as the MIS or the demographic and health surveys, can provide a rich resource for natural experiments and rigorous evaluation designs.A total of 5,960 children aged 6–59 months were included in the MIS survey. Their characteristics are summarized in Table 2 = 22.52 and Chi2 = 16.57, respectively, and both have P-values < 0.001). Therefore, endogenous Models II and III were preferred over Model I since they control for potential selection on unobservables (“treatment selection bias”). As a GSEM, Model III has the additional advantage of allowing mediation relationships to be included in the analysis, and therefore to measure SMC direct and indirect effects on anemia. For this reason, Model III was retained as the final model for the analysis.Three different types of models were used to measure the effects of SMC on malaria: multiple regression (Model I), multiple regression with endogenous treatment (Model II), and generalized structural equation models with endogenous treatment (Modell III). These models were fitted for the two primary study outcomes, i.e., RDT- and microscopy-confirmed malaria. Results are shown in Tables Children who received SMC had a lower risk ratio  for RDT-confirmed malaria compared to those who did not, after adjustment for covariates and for endogenous treatment effects. The treatment effect was slightly smaller for microscopy-confirmed malaria, a risk reduction of 35% . Children were at significantly higher risk of malaria if they were older and if they lived in regions other than Kayes . They were at significantly lower risk of malaria if they lived in the capital area, if their household had a higher SES, were Peulh or Toucouleur, or if their mother received secondary education (or higher). Covariates’ influence on the outcomes were very similar regardless of which test was used to detect parasitaemia, and was always in the anticipated direction.17. Children who received SMC had an 18% reduction in risk of anemia compared to those who did not .The path diagram of the mediated effects of SMC is shown in Fig. Post-test only with control group designs are subject to selection bias if exposed individuals differ from unexposed individuals in characteristics correlated with exposure and outcomes variables. This study is based on a natural experiment – we had no control over the process through which survey participants were exposed or not exposed to SMC. As such, it is plausible that participants differ according to observed or unobserved characteristics. Several measures were taken to reduce the risk of a selection bias and increase the internal validity of the impact evaluation. First, data was collected using a two-stage cluster sampling and is representative of the national population, except for the regions of Gao, Tombouctou and Kidal. Second, we used multiple regression to control for a set of observed and potentially confounding factors. Third, we fitted endogenous treatment effects models to control for unobserved residual heterogeneity between the treatment and control groups.18. Indeed, the same hypothesis was tested on three different outcome indicators, representing different components of the chain of effects19. Results were always in agreement with those expected. Furthermore, a placebo test with a fake outcome was realized by differentiating SMC’s direct and indirect effects on anaemia. These tests increase the robustness of the results and our capacity to attribute the effects to the intervention21.Another strength of this study is the use of a theoretical replication strategyAnalyses were intentionally conservative; bilateral statistical tests were used, and we evaluated the effects of being exposed to at least one SMC cycle, while the theory of intervention entails 4 cycles. Furthermore, in Mali, SMC is coupled with a test and treat strategy – if the RDT is positive, children receive Artemisinin-based combination therapy (ACTs), and if it is negative, children receive SP-AQ. Children were included in the analysis even if they had received ACTs rather than SMC. This could have diluted the estimate of SMC treatment effect.The risk of recall bias cannot be excluded, since MIS implies a retrospective recording of practices and events. When possible, self-reported information was cross-validated by direct observation. Another limit is that it was not possible to assess SMC impact depending on the adherence level, because this information was not collected during the MIS. Also, although coverage was already high in 2015, SMC was only introduced nation-wide in 2016. So survey participants could have been unexposed to SMC in 2015 because they lived in an area that was not targeted. Finally, the fitted GSEM has several assumptions, including that causal effects are unidirectional (model is recursive) and that it is fully parametric. Because traditional tests are unavailable for GSEM, goodness-of-fit was indirectly checked by comparing the GSEM to a regression with endogenous treatment effects model.22, SMC has the potential for major public health impact in terms of morbidity, mortality and use of health services attributable to malaria16. However, the effect measured in the present study was lower than the 62% reduction in period prevalence measured in Burkina Faso during a routinely implemented SMC campaign15. Several factors may have contributed to a lower treatment effect in Mali compared to the one measured in Burkina Faso, including resistance to SP and/or AQ, lower adherence levels among children, a lower validity of the instruments, and the coupling of a test and treat strategy with SMC. In addition, contrary to the MIS in Mali, the study in Burkina Faso was not nationally representative of the population, but was conducted in a small area of a single district. The SMC treatment effect for microscopy-confirmed malaria, i.e. a reduction of 35%, was more modest than for RDT-confirmed malaria. This is potentially due to the lower sensitivity of microscopy vs. RDTs24. The lower capacity of microscopy to detect positives in both groups likely diluted the treatment effect. Results for both outcomes are presented here so readers can appreciate and compare them. A previous study conducted in two Malian districts suggested a larger effect; SMC was associated with a 65% reduction in the odds ratio of being infected by Plasmodium (measured by microscopy). However, this larger effect can partly be attributable to the fact that odds ratios overestimate risk ratios25. Also, the previous study was conducted after the full SMC campaign, while prevalence in the present study was measured during a SMC campaign.This study shows that exposure to at least one cycle of the 2015 SMC campaign in Mali had positive and significant  effects on malaria and anemia in children aged 6–59 months. The risk of RDT-confirmed malaria was reduced by 44%. With a national prevalence over 35% in children under five during high transmission seasonHowever, even if we conservatively estimate that SMC effectiveness at reducing prevalence is 35–44%, results of such magnitude at the national level are promising for a routinely implemented programme. While approximately 45% of the children belonged to the treatment group in this nationwide natural experiment, it is important to sustain the efforts to increase SMC coverage in Mali.27. In this study, moderate to severe anaemia in children under five was defined as haemoglobin concentration <10 g/dl, as per WHO guidelines28. However, many SMC impact studies use a cut-off of 8 g/dl instead29. Had this lower cut-off been used here, SMC indirect effect on anaemia would have been larger , and comparable to the results obtained from previous studies conducted in high transmission settings16.The study also showed that SMC has no direct effect on moderate to severe anemia, but indirectly reduced its likelihood by 18%. The indirect effects on anemia were smaller than the direct effects on malaria parasitaemia. This is consistent with the fact that only a fraction of the risk of anemia is attributable to malaria, and that this fraction varies depending on the cut-off used to define anemia30, they can also lead to natural experiments designed to evaluate the impacts of new health interventions or policies33. This study illustrates how endogenous treatment effects models can be fitted with sectional survey data to evaluate the effectiveness of interventions using a post-test only (with control group) design. Despite its pitfalls (see limits above), this type of design is useful when there is no pre-test observation, or when pre-test data was collected too long before the intervention to constitute an adequate counterfactual21. This is often the case with MIS or nationally representative surveys, usually repeated every three years or so only.Finally, this study also contributes to the development and promotion of rigorous evaluation designs using nationally representative surveys, such as the MIS or the demographic health surveys. While these surveys are commonly used for descriptive purposes and comparisons across countries or over time34. Indeed, there has been an exceptional increase recently in funding to decrease malaria transmission worldwide35. About 35 countries are now on the path to eliminate malaria, and reinvigorated control interventions are taking place in the remaining endemic countries to envision elimination in the near future37. With this renewed attention and effort, it is essential to evaluate the health impacts attributable to routinely implemented national programmes40. We argue that, even if they were mainly developed and used in econometrics44, endogenous treatment effects analyses can contribute to bridging a widening gap in evaluation methods needed to measure progress towards achieving malaria elimination.The need to measure the impacts using rigorous quasi-experimental studies is particularly salient regarding malaria programmes introduced in resource-constrained countries3. The latest Malaria Indicator Surveys (MIS) revealed a parasitaemia prevalence detected by RDT of 32% in children 6–59 months during the high transmission season, and a prevalence of moderate to severe anemia of 63%22. In 2013, the WHO estimated that Mali was the country with the highest malaria incidence in the world, with 460.9 cases per 1000 persons at risk45.Malaria is one of the single most important public health issues in Mali. In 2016, about 2,311,000 malaria cases were confirmed out of a total population of 18 million, with an estimated number of deaths attributable to malaria close to 21,00046, which is the threshold used by WHO to recommend SMC in an area47. Because of this epidemiological profile, and based on WHO recommendations, Mali introduced SMC as a pilot project in 2012 and progressively expanded it to 48 health districts in 201522. Unfortunately, data on the precise population coverage within these districts is not available.Malaria transmission is highly seasonal in Mali. In 52 of the 60 health districts, >60% of the annual rainfall occurs within three consecutive months 48. All children 3–59 months are eligible to receive SMC unless they received ACTs, are febrile, allergic to SP or AQ, or under HIV treatment. The first dose of treatment is administered by health workers at fixed locations in villages. The two remaining doses of AQ are given to the caregiver to be administered at home. Treatments administered are recorded on an SMC card that is given to all children at the first cycle of distribution. Four cycles are organized each year, one per month between August and November.In Mali, SMC is coupled with a test and treat strategy. In the presence of fever or history of recent sickness, or upon request by the parents, children are tested for malaria using RDTs. If the RDT is positive, children receive malaria treatment (ACTs) and are excluded from SMC. If the RDT is negative, they do not receive ACTs and can be included in the SMC campaign. The regimen used for SMC combines one dose of SP and three daily doses of AQ. This treatment is still effective in Mali, where the prevalence of markers of resistance to SP and AQ is lowData used for this study come from the MIS conducted in Mali from September to November, 2015. The MIS is a nationally representative survey, but for security reasons, the three northern regions  were excluded from the sampling frame. Households were selected using a two-stage cluster sampling approach. First, 177 enumeration areas were selected based on probability proportional to size. Then, 24 households per enumeration area were randomly selected, for a total of 4,243 households identified and 4,240 that were actually surveyed .Fieldworkers administered a standardized questionnaire developed by the Demographic and Health Surveys Programme and approved by Roll Back Malaria that included questions at the household and individual levels. All women 15–49 years of age were eligible for the individual section of the questionnaire. At the household level, the MIS collects data on the household’s composition, socio-economic characteristics, and possession of bed nets. At the individual level, the MIS records individuals’ characteristics, malaria-related practices and knowledge, and self-reported exposure to interventions against malaria.A section specific to children 6–59 months collects data on recent episodes of fever, treatment-seeking behavior, and exposure to SMC. For these children, biological tests are performed using whole blood via finger-prick to detect anemia and malaria parasitaemia. Anaemia is detected by measuring hemoglobin using a HemoCue® portable photometer. A rapid diagnostic test (RDT) for malaria is also performed to detect the presence of pLDH and HRP-2 antigens (SD Bioline Pan/Pf ®). Finally, thick blood smears are prepared in the field, then stained and read by trained microscopists at the Laboratory of Parasitic Diseases, National Institute for Public Health Research in Bamako.We designed a post-test only with non-randomized control group study. The population study consists of all children aged 6–59 months nested in the 4,240 households surveyed. The intervention group includes children who received at least one SMC treatment during the 2015 campaign, while the control group includes children who had not received an SMC treatment at the time of the MIS.Plasmodium parasites detected by microscopy and the detection of Plasmodium antigens (either pLDH or HRP-2) by RDT. Both tests have limited sensitivity and specificity when compared to polymerase chain reaction as the gold standard49. A common issue with HRP-2 based RDTs is that the antigen can persist in the bloodstream for 4–5 weeks after parasite clearance52, and therefore the result is more indicative of a period prevalence54. On the other hand, microscopy detects only current infections, but has poor sensitivity, especially in low transmission areas24. The secondary outcome variable is the presence of a moderate to severe anemia, defined according to the WHO as a hemoglobin concentration under 10 g/dL28.The primary outcome variables are the presence of 56. We categorized mothers’ education as none, primary, or secondary and higher, and size of the household as 1 to 5, 6 to 10, or 11 and more. Quintiles of SES were constructed using principal component analysis based on a list of household assets and physical characteristics58. A malaria infection detected by RDT or microscopy was examined as a potential mediator between exposure to SMC and anemia.Self-reported exposure to SMC was assessed at the time of the survey. Because of the partly overlapping dates of the survey and the intervention, children were not exposed to the full SMC campaign (i.e. the four cycles). The exposure variable was categorized and defined as having received an SMC treatment at least during one cycle of the 2015 SMC campaign (yes/no). When available, self-reported exposure was cross-validated by reviewing the children’s SMC cards (85% of the exposed children showed a SMC card). Potential confounding factors (as captured by the MIS) were use of bed nets, age and sex of the child, mother’s education, the household’s size, region, ethnic group, religion, number of bed nets, socio-economic status (SES), and the cluster altitude21. Multiple regression modeling was used to control for observed confounding factors. However, unobserved heterogeneity may also be correlated to both the outcome and the probability of receiving SMC – a situation in which the treatment is endogenous44. To mitigate such selection bias on unobservables, we used a treatment effects approach by adding a latent variable related to the likelihood of receiving SMC and the outcome59. All analyses were performed using Stata® v14.The biggest threat to internal validity with post-test only designs comes from selection bias, i.e., the possibility that observed differences in outcomes between the exposed and control groups reflect differences in their histories or characteristics, rather than being attributable to the intervention60. This allowed us to simultaneously model the exposure and the outcome (treatment effects), and to measure direct and indirect effects of SMC61. Indeed, RDT-confirmed malaria was considered as a potential mediator between SMC treatment and anemia. The working hypotheses are that: (i) by reducing the risk of having been recently infected with malaria, SMC indirectly reduces the risk of presenting a moderate to severe anemia, and that (ii) SMC has no direct effect, positive or negative, on anemia (placebo test on fake outcome20).Based on the Rubin-Neyman potential outcome framework, we used a generalized structural equation model (GSEM) to assess the effects of SMC on malaria parasitaemia and on anemia62.Therefore, three multiple regression equations were simultaneously computed, whose outcomes are: (i) exposure to SMC, (ii) result from malaria tests, and (iii) presence of moderate to severe anemia. All observed potential confounding factors were entered in the three equations. Parsimony was achieved through the backward stepwise method; variables that did not alter the main coefficient by 5% or more were gradually removed, and excluded variables were re-tested in the final model. Akaike information criterion (AIC) and Bayesian information criterion (BIC) were used to inform model selection65. The linearity of the relationship between each outcome and continuous covariate was assessed by adding quadratic terms. Multicollinearity was ruled out by using the collin package  and verifying that variance inflation factors did not exceed 4. SMC indirect effects were calculated using the product-of-coefficients method, which is standard procedure for Log-link functions61.For each equation, the Poisson distribution with robust variance estimators was preferred over the logistic distribution to obtain model-based risk ratio estimates, as recommended in the literature on impact assessment66. Sensitivity analysis was therefore conducted by fitting a Poisson regression model that includes an endogenous binary treatment variable. Estimates obtained from the GSEM and from the endogenous treatment-effects model were compared, and goodness-of-fit assessment was performed on the latter. Children who had received an artemisinin-combination therapy in the two weeks prior to the survey were excluded from the analyses. The threshold for statistical significance was set at 0.05 .GSEM is an exceptional tool for causal mediation analysis, but with strong assumptions and few tests, if any, to assess goodness-of-fitAll survey protocols, questionnaires, and datasets are publicly available online. The protocol for the 2015 MIS received approval from the Research Ethics Committee of the National Institute for Public Health Research in Mali. All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all the participants as described in the protocol. Approval to use the MIS data was obtained from the Demographic and Health Surveys Programme.


We evaluated the incidence of all-cause and malaria-specific clinic visits during follow-up of a recent trial of iron therapy. In the main trial, Ugandan children 6–59 months with smear-confirmed malaria and iron deficiency [zinc protoporphyrin (ZPP > = 80 μmol/mol heme)] were treated for malaria and randomized to start a 27-day course of oral iron concurrently with (immediate group) or 28 days after (delayed group) antimalarial treatment. All children were followed for the same 56-day period starting at the time of antimalarial treatment (Day 0) and underwent passive and active surveillance for malaria and other morbidity for the entire follow-up period. All ill children were examined and treated by the study physician. In this secondary analysis of morbidity data from the main trial, we report that although the incidence of malaria-specific visits did not differ between the groups, children in the immediate group had a higher incidence rate ratio of all-cause sick-child visits to the clinic during the follow-up period (Incidence Rate Ratio (IRR) immediate/delayed = 1.76; 95%CI: 1.05–3.03, p = 0.033). Although these findings need to be tested in a larger trial powered for malaria-specific morbidity, these preliminary results suggest that delaying iron by 28 days in children with coexisting malaria and iron deficiency is associated with a reduced risk of subsequent all-cause illness. Malaria is endemic in many regions where iron deficiency is prevalent. The conditions frequently co-occur in the same child. The World Health Organization standard-of-care regimen for treating children with malaria and iron deficiency is to give antimalarial treatment and iron therapy concurrently ,2. HowevIncreased concentrations of the hepatic protein hepcidin that accompany a malaria episode may explain the findings of unresolved anemia. Hepcidin rises with malaria infection, impairing intestinal iron absorption and release of iron from reticuloendothelial stores . Becausein vitro to be preferred by P. falciparum [Whether a 28-day delay in the start of iron therapy might affect subsequent morbidity is unknown. Iron that is better absorbed would result in less iron trapped in the intestine and available to pathogenic bacteria. Alternatively, better iron absorption may result in more iron-rich blood cells, shown lciparum . Either To determine whether delaying iron until 28 days after antimalarial treatment in children with coexisting iron deficiency and malaria is associated with a difference in the risk of subsequent illness compared to the standard of care concurrent iron therapy, we analyzed morbidity data from our previous trial to investigate the frequency and incidence of physician-diagnosed episodes of illness over the 56-day follow-up period.As described previously , 100 chiDuring the 56-day follow-up period, all children were under active and passive surveillance for illness. In addition to scheduled clinic visit on Days 28 and 56, home visits were made to each enrolled child’s home by study staff on Days 14 and 42. Any ill child was brought to the study clinic for assessment and treatment. Additionally, all caregivers were instructed to contact the study office in the event of illness that occurred between study visits. Mobile phone airtime was reimbursed for these calls, and transportation to and from the study clinic was provided directly or reimbursed. All medical examination and treatment was provided free of charge. All ill children, whether identified during a home visit or self-referred, were given a physical examination by a study medical officer in the Paediatric Acute Care Unit at Mulago Hospital.Diagnoses for common illnesses were made according to Mulago Hospital guidelines, including: 1) Uncomplicated malaria: positive Giemsa smear or Rapid Diagnostic Test (RDT), with fever or history of fever, in absence of any of the World Health Organization’s Clinical Signs of Severe Malaria ; 2) SeveFor any visit to the study clinic, the findings from the child’s clinical exam, the primary diagnosis and any additional diagnoses were recorded on a sick child visit form and entered into the study’s database.Caregivers of all enrolled children provided written informed consent. The study was approved by the Institutional Review Board of the University of Minnesota, the School of Medicine Research Ethics Committee at Makerere University, the Uganda National Council of Science and Technology, and the National Drug Authority of Uganda.The original trial was powered to compare the primary outcome of percentage iron incorporation into red blood cells between the immediate vs. delayed groups . This seThe groups did not differ with regard to age, sex, hemoglobin concentration, anthropometry, or malaria parasite density at baseline . During The period prevalence, or number of children with at least one illness in the follow-up period divided by the number of children enrolled, did not differ significantly between the groups , malaria , and gastroenteritis n = 14, . The numWe did a secondary analysis of morbidity outcomes in a recent randomized trial of iron treatment in which children began a 27-day course of an oral iron supplement concurrently with or 28 days after antimalarial treatment. Our primary finding was that children in the delayed-iron group had lower incidence of all-cause sick-child visits to the study clinic in the 56-day follow-up period compared to children in the immediate-iron group, despite ending the 56-day period with equivalent iron status [Three recent Cochrane reviews –13 foundIt is unclear why the incidence of all-cause sick-child visits was greater in our study's immediate group. Perhaps lower absorption of iron at the time of supplementation, which we observed in the immediate iron group , translaLimitations to this secondary analysis include its small sample size and resulting insufficient power for malaria outcomes. Further, it is important to note that nine children in the immediate group as compared to three children in the delayed group had two episodes of illness. Thus, the difference in incidence rates of all-cause illness between the groups was driven largely by these six children. Although these data are preliminary, they suggest that delaying iron by 28 days after antimalarial treatment is associated with a reduction in all-cause morbidity while not harming iron status. Longer-term studies powered for iron status, morbidity, and neurocognitive developmental outcomes are needed to verify these findings and to determine the safest and most effective management options for children with malaria and iron deficiency.S1 Table1IRR for all sick-child visits adjusted for age, sex, malaria parasite density, hemoglobin, or height-for-age z-score; 2Standard deviation of the adjuster. HAZ = Height-for-age Z-score, IRR Incidence Rate Ratio.(DOCX)Click here for additional data file.S2 Table1IRR for malaria-specific visits adjusted for age, sex, malaria parasite density, hemoglobin, or height-for-age z-score; 2Standard deviation of the adjuster. HAZ = Height-for-age Z-score, IRR Incidence Rate Ratio.(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.


Many of these applications are To obtain a quantitative estimate of the serial interval for falciparum malaria, the sum of the components of the falciparum malaria transmission cycle was taken based on a combination of mathematical models and empirical data. During this process, a number of factors were identified that account for substantial variability in the serial interval across different contexts.Treatment with anti-malarial drugs roughly halves the serial interval due to an abbreviated period of human infectiousness, seasonality results in different serial intervals at different points in the transmission season, and variability in within-host dynamics results in many individuals whose serial intervals do not follow average behaviour. Furthermore, 24.5 % of secondary cases presenting clinically did so prior to the primary cases being identified through active detection of infection.These results have important implications for epidemiological applications that rely on quantitative estimates of the serial interval of falciparum malaria and other diseases characterized by prolonged infections and complex ecological drivers. R0, defined as the expected number of secondary cases arising from a single primary case in a susceptible population, is well known and of fundamental importance in infectious disease epidemiology. Despite extensive efforts to model, measure, and map R0 globally for falciparum malaria [The basic reproduction number  malaria , 2, litt malaria . Probabi malaria , for ass malaria , 6, for  malaria , 8, and  malaria .For directly transmitted diseases, the serial interval can be measured through contact tracing or with household data , 11. ForFor falciparum malaria, one analysis  has useddetection of sequential infections, it is defined as the sum of the GI, the IDP for the primary infection, and the IDP for the secondary infection. This approach was applied to quantifying GI and SI distributions under a variety of scenarios to describe variability in the GI and SI across a wide range of conditions typical of falciparum malaria transmission in different settings.To obtain random variables describing the generation interval (GI) and serial interval (SI) of falciparum malaria, random variables were first derived describing components of the GI and SI: the liver emergence period (LEP), the human-to-mosquito transmission period (HMTP), the extrinsic incubation period (EIP), the mosquito-to-human transmission period (MHTP), and the infection-to-detection period (IDP). The exact length of time required for each of these events to occur is inherently random, which is why each must be treated as a random variable rather than as a period of fixed length. Furthermore, because these events must occur sequentially for transmission to occur, the sum of these random variables must be calculated to describe the GI and SI as random variables. The GI is defined as the time between sequential infections, so it is therefore equal to the sum of the LEP, HMTP, EIP, and MHTP Fig. . BecauseThe first period comprising the generation interval for falciparum malaria was defined as the LEP. Consistent with empirical findings , this inTo simulate the trajectory of blood-stage parasites following their emergence from the liver, a simulation model developed by Johnston et al.  was usedPlasmodium falciparum infections in humans [P. falciparum in a controlled setting to induce a fever and an immune response. Consequently, the dynamics described by the model by Johnston et al. [P. falciparum infection dynamics of infections in adult males with no prior exposure. Elaborating on the model by Johnston et al. [P. falciparum.The dynamics of gametocytaemia, the immune response, and the effect of anti-malarial drugs were simulated with the model by Johnston et al. . This mon humans –22. Thesn et al. , 18 are n et al. , 18 to bm(t). The default setting for m(t) was a constant, but for some analyses a time-varying function was used,A is peak amplitude, Φ is a Normal probability density, and σ corresponds to the width of the seasonal peak. To obtain a random variable describing the timing of a mosquito being infected by an infectious human, the time-varying infection probabilities were multiplied by m(t) and the resulting curve was normalized. The sum of the LEP and HMTP for a constant m(t) is shown in Fig. Because the number of mosquitoes blood-feeding on a given day can be highly variable , the timP. falciparum gametocytes have been transmitted from an infectious human to a susceptible mosquito, a period of time known as the extrinsic incubation period (EIP) must elapse before sporozoites are produced and disseminated to the mosquito’s salivary glands, where they can then be transmitted to a human. It was assumed that the EIP can be reasonably described by a Normal random variable with mean estimated from any of four sites [Φ is a Normal cumulative density  were taken from Killeen et al. [After a mosquito has become infectious, the final step in the transmission cycle is for the mosquito to transmit parasites to a human. To make this a tractable quantity to model, three simplifying assumptions were made. First, no senescence or any other source of variability in mortality was assumed, such that mosquitoes are subject to a constant daily probability of survival n et al. . By settS and IDPA, respectively. Common to both was the interval between sporozoites entering the skin and asexual parasites emerging from the liver, which was assumed to always be 6 days [To calculate the serial interval distribution, one additional random variable was defined describing the interval between infection and either presentation at a clinic or detection by other means, such as active surveillance . This ine 6 days .S probability and summed to arrive at a combined IDPS + HMTP distribution that accounted for this correlation.For symptomatic cases, another random variable was added corresponding to the interval between emergence of parasites from the liver and onset of fever, which was obtained as part of the simulation output of the model by Johnston et al. . The thiA with this distribution assumes that active detection of infection is attempted only once during the infection and that its timing is random with respect to day since infection. Such would be the case in the context of reactive case detection or during a single cross-sectional survey, for example. In the event that active detection occurs at some regular interval less than the total period of infection, IDPA would be shorter. Although this may be important for accurately characterizing IDPA in certain surveillance contexts, the impact on IDPA of these and potentially many other variations on surveillance systems are beyond the scope of this work.For asymptomatic infections, it was assumed that they were identified through some form of active detection of infection at some point during their infection when their asexual parasitaemia levels exceeded 50 per μL of blood. A probability distribution describing the probability that such a level of asexual parasitaemia exceeded this threshold on a given day was obtained by directly calculating the empirical density of the number of days in excess of 50 per μL from 1000 realizations of the simulation model by Johnston et al. . Equatini, j, k, and l are dummy variables. In other words, Eq.  was set equal to the time-varying function in Eq.  GIs of 49.1 (35.0–68.0) and 101.6 (43.0–219.0) days for treated and untreated primary infections, respectively  of 16.6 (12.0–22.0) days  of 69.8 (11.0–163.0) days .Probabilistic descriptions of GIs and SIs also have an important role to play in population-level models of infectious disease dynamics. Together with an estimate of epidemic growth rate, the GI distribution can be used to estimate the basic reproduction number and related quantities , 30, 31.The study has highlighted a number of reasons why GI and SI distributions are variable for falciparum malaria and offered quantitative remedies to modelling many of these situations. To this end, code for reproducing the figures and for calculating GI and SI distributions over one or more generations of falciparum malaria cases is available online . Like ma


We have noted a recent upsurge in blackwater fever among children presenting to hospitals in eastern Uganda and speculate that this may relate to a change in policy toward new artemisinin-based combination therapies for malaria. In the Fluid Expansion as a Supportive Treatment (FEAST) trial, an unexpectedly high proportion of participants from eastern Uganda presented with blackwater fever (BWF).We describe the prevalence and outcome of BWF among trial participants and compare the prevalence of 3 malaria-protective red blood cell polymorphisms in BWF cases vs both trial (non-BWF) and population controls.Plasmodium falciparum parasitemia was less frequent than in non-BWF controls, but a higher proportion were positive for P. falciparum histidine rich protein 2 (192/246 [78.0%]) vs 811/1154 [70.3%]; P = .014), suggesting recent antimalarial treatment. Overall, 282 of 318 (88.7%) received transfusions, with 94 of 282 (33.3%) and 9 of 282 (3.4%) receiving 2 or 3 transfusions, respectively. By day 28, 39 of 318 (12.3%) BWF cases and 154 of 1554 (9.9%) non-BWF controls had died (P = .21), and 7 of 255 (3.0%) vs 13/1212 (1%), respectively, had severe anemia (P = .036). We found no association with G6PD deficiency. The prevalence of both the sickle cell trait (10/218 [4.6%]) and homozygous α+thalassemia (8/216 [3.7%]) were significantly lower among cases than among population controls , providing further support for the role of malaria.Of 3170 trial participants, 394 (12.4%) had BWF. The majority (318 [81.0%]) presented in eastern Uganda and were the subjects of further analysis. BWF cases typically presented with both clinical jaundice (254/318 [80%]) and severe anemia (hemoglobin level <5 g/dL) (238/310 [77%]). We report the emergence of BWF in eastern Uganda, a condition that, according to local investigators, was rare until the last 7 years. We speculate that this might relate to the introduction of artemisinin-based combination therapies. Further studies investigating this possibility are urgently required. Plasmodium falciparum malaria infection in nonimmune expatriate adults living in Africa, where it was frequently complicated by severe anemia, jaundice, and renal failure )  includedThe methods and outcome of the FEAST trial have been described in detail previously . In brieP. falciparum histidine rich protein 2 (pfHRP2), a sensitive marker of recent malaria infection, was quantified using methods described in detail previously ) presented in Uganda, with a particular focus in 2 hospitals in eastern Uganda (MRRH and SRRH), where BWF was recorded in 180 of 1240 (14.5%) and 138 of 680 (21.8%) participants, respectively while jaundice was recorded in 759 (61.2%) and 170 (27.0%) cases, respectively . Cases had lower median hemoglobin concentrations , had a higher prevalence of severe anemia , were more likely to be hypothermic or clinically jaundiced or to have features of impaired perfusion, and displayed greater evidence of metabolic acidosis and acute renal injury. Both mean blood urea nitrogen (BUN) and the prevalence of hyperkalemia were >2-fold higher among cases than controls. Plasmodium falciparum parasitemia, detected either by rapid test , which can remain positive for some weeks after a malaria event, or microscopy, was significantly less common in cases than controls; however, pfHRP2 positivity was significantly more common . Moreover, among those testing positive, geometric mean concentrations of pfHRP2 were lower in cases than in controls , suggesting a higher rate of recent antimalarial treatment in cases compared with controls. Plasmodium falciparum was the only species of malaria parasites detected by either microscopy or by Optimal.The baseline characteristics of BWF cases (n = 318) and non-BWF controls (n = 1552) at MRRH and SRRH are summarized in For logistic reasons, urine samples were collected and tested at admission only from a subset of case (n = 100) and control (n = 366) children . Moreove+thalassemia, and G6PD deficiency among cases and among FEAST and population controls are summarized in P < .001), providing a potential etiology in a small proportion (4.1%) of cases; however, no significant evidence was found to suggest that G6PD deficiency was a cause. Both HbAS and homozygous α+thalassemia were associated with protection from BWF , providing further evidence for the etiological role of malaria in the current case series.The prevalence of the red blood cell polymorphisms HbAS, HbSS, αP < .0001). Moreover, a higher proportion of cases than controls received a second and third transfusion . Despite a greater number of severity features in cases, 48-hour mortality was similar to that of controls. By day 28, both case fatality and the proportion with severe anemia were marginally higher in cases than controls, whereas malaria infection remained significantly lower among cases , where a history of dark urine was elicited in 14.5% and 21.8%, respectively. FEAST trial participants from these hospitals therefore form the main focus of the current report. In this series, 77.0% of BWF cases were severely anemic (hemoglobin level <5 g/dL) and in keeping with previous reports  many 80, Table 2P = .076), although the numbers affected were relatively few and reporting was limited to the period of hospital admission. The fact that our findings contrast with a number of previous malaria reports led us to question the cause for the high frequency of BWF seen in the current context.The high frequency of BWF seen in our case series from eastern Uganda contrasts with the other sites involved in the FEAST trial. Moreover, it is also at odds with observations from the an open label randomised comparison of injectable Artesunate and Quinine in patients with severe falciparum malaria in Africa (AQUAMAT) trial of severe pediatric malaria, conducted between 2005 and 2010 in 11 centers across 9 African countries, in which BWF was reported in only 4% of >5000 patients and in which only 2% presented with jaundice . Of part+thalassaemia, which were similar in degree to those previously described for severe malaria [In keeping with both historical and contemporary reports in children , 24, rec malaria . In cont malaria , 19, G6P malaria , this onIf, in our current case series, the occurrence of BWF is linked to malaria, it raises questions about why, in general, BWF has not been recognized as a major health problem in African children previously. Local investigators have indicated that childhood BWF was rare at their hospitals until relatively recently, only having grown in importance during the last 7 years. Moreover, they report that some children are recurrently admitted with the triad of dark urine, anemia, and clinical jaundice and require frequent blood transfusions . One possibility is a recent change in the local parasite population toward strains with a greater propensity to cause BWF, a hypothesis advanced by others previously , 25, 29.Of particular interest in this regard, a growing number of recent case reports from Europe have described hemolysis following intravenous therapy with artemisinin (summarized in ). In conOur study has a number of limitations of which perhaps the most important was the fact that case ascertainment was based on a clinical history of dark urine collected from a parent. We did not inquire about recent antimalarial treatments, and our study would have benefited from additional real-time laboratory investigations aimed at investigating the etiology more definitively. Urine testing was not possible in every child recruited and was only hemoglobin-positive in 65% of readable samples. This observation might suggest that hemoglobinuria was not present at the time of testing, but might also be attributable to the paroxysmal nature of this condition . Nevertheless, the hematological picture (median hemoglobin level 3.7 g/dL) is indicative of hemolysis, as is the high frequency of transfusion (88%) and repeat transfusion (36% of BWF cases receiving multiple transfusions). Despite this, severe anemia was significantly more common in cases than controls at 8 and 24 hours and at 28 days postadmission. This condition therefore is associated with a greater risk of severe and life-threatening morbidity and a marginal increase in the risk of longer-term mortality. Although the specific pathogenesis of PANDHA remains unclear, detailed investigation of patients in Europe did not implicate either drug-dependent or drug-independent hemolysis, although, to the best of our knowledge, a complement-mediated mechanism has yet to be investigated. A further limitation of the study is that we lack data on the prevalence of infections other than malaria. At the time the study was conducted, facilities for bacteriological culture were limited at the Mbale and Soroti sites, and too few samples were cultured to yield meaningful results.Historically, BWF has not been considered a common complication of malaria among children living in areas of high malaria transmission. Nevertheless, in the current report we have described a high frequency of BWF among participants of the FEAST trial. The majority of cases presented to 2 hospitals in eastern Uganda that are both situated within an area of very high malaria transmission. BWF has a high hidden burden of morbidity and mortality, with 90% requiring transfusions and >30% requiring multiple transfusions. Moreover, 28-day mortality was 12%, and 3% of survivors remained severely anemic at this time point. We conclude that malaria and its treatment are important factors in the etiology of BWF in the study population and that this may be linked to the recent switch toward orally administered ACTs as first-line treatment for malaria within the region. Further studies investigating the incidence and etiology of childhood BWF in the context of ACTs are indicated.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 Supplement_FigureClick here for additional data file.