Leading malaria vaccine, RTS,S, is based on the circumsporozoite protein (CSP) of sporozoites. RTS,S confers partial protection against malaria in children, but efficacy wanes relatively quickly after primary immunization. Vaccine efficacy has some association with anti-CSP IgG; however, it is unclear how these antibodies function, and how functional antibodies are induced and maintained over time. Recent studies identified antibody-complement interactions as a potentially important immune mechanism against sporozoites. Here, we investigated whether RTS,S vaccine-induced antibodies could function by interacting with complement.A conducted at two study sites of high and low malaria transmission intensity in Manhiça, Mozambique. Samples following primary immunization and 5-year post-immunization follow-up time points were included. Vaccine-induced antibodies were characterized by isotype, subclass, and epitope specificity, and tested for the ability to fix and activate complement. We additionally developed statistical methods to model the decay and determinants of functional antibodies after vaccination.Serum samples were selected from children in a phase IIb trial of RTS,S/AS02RTS,S vaccination induced anti-CSP antibodies that were mostly IgG1, with some IgG3, IgG2, and IgM. Complement-fixing antibodies were effectively induced by vaccination, and targeted the central repeat and C-terminal regions of CSP. Higher levels of complement-fixing antibodies were associated with IgG that equally recognized both the central repeat and C-terminal regions of CSP. Older age and higher malaria exposure were significantly associated with a poorer induction of functional antibodies. There was a marked decay in functional complement-fixing antibodies within months after vaccination, as well as decays in IgG subclasses and IgM. Statistical modeling suggested the decay in complement-fixing antibodies was mostly attributed to the waning of anti-CSP IgG1, and to a lesser extent IgG3.We demonstrate for the first time that RTS,S can induce complement-fixing antibodies in young malaria-exposed children. The short-lived nature of functional responses mirrors the declining vaccine efficacy of RTS,S over time. The negative influence of age and malaria exposure on functional antibodies has implications for understanding vaccine efficacy in different settings. These findings provide insights into the mechanisms and longevity of vaccine-induced immunity that will help inform the future development of highly efficacious and long-lasting malaria vaccines.The online version of this article (10.1186/s12916-019-1277-x) contains supplementary material, which is available to authorized users. B adjuvant (liposome-based adjuvant). Thousands of children and infants were enrolled from multiple study sites across sub-Saharan Africa, and RTS,S was moderately efficacious against clinical malaria at 38–48 months follow-up  , and M3 = 2.231 [0.390], p < 0.001; Ilha Josina N = 49, M0 = 0.030 [0.039], and M3 = 2.457 [0.425], p < 0.001). In contrast, anti-CSP IgG was low at both time points in children who received the comparator non-malaria vaccine   and 30 days after receiving the final RTS,S vaccination (M3) were tested for total IgG levels to full-length CSP. Reactivity was low at baseline but significantly increased after vaccination with RTS,S in both study cohorts   were then tested for IgG subclass responses to CSP. Seropositivity was high for IgG1 (94% and 98%) and IgG3 (90% and 96%), followed by IgG2 (70% and 86%) and IgG4 (22% and 51%) in Manhiça and Ilha Josina cohorts, respectively  but not with comparator vaccine  . In all RTS,S-vaccinated children (N = 99), anti-CSP IgG and IgM responses at M3 moderately correlated, but many demonstrated IgG-high/IgM-low reactivity. Notably, IgM reactivity at M0 and M3 was weakly correlated  . However, this induction of complement-fixing antibodies was not seen with the comparator vaccine  , whereas IgM was mostly NANP-specific .To investigate the targets of functional antibodies induced by RTS,S, we first measured antibody reactivity to NANP and CT antigens, which represented the central repeat and C-terminal regions of CSP, respectively Fig. a, b. Comp < 0.001 for both tests)   Fig. c. Indeedts) Fig. d.N = 50, OD range 2.03 to 4.00; low N = 49, OD range 0.02 to 1.97). Notably, children with high C1q-fixing antibodies had substantially higher levels of IgG to both NANP and CT and higher levels of IgM to NANP  . For the Ilha Josina cohort, which had a substantially higher level of malaria transmission than Manhiça, younger children had significantly higher levels of C1q-fixing antibodies , and age negatively correlated with C1q-fixation . Similar age associations were also observed for C1q-fixation to NANP  in younger and older age groups  and between our two different study sites  but strong for the Ilha Josina cohort . Furthermore, in the Ilha Josina cohort, C1q-fixation to CSP negatively correlated with IgG to MSP2 and AMA1 . Collectively, these data suggest that increased malaria exposure negatively impacts the induction of functional immunity by RTS,S immunization.The Ilha Josina study site had a higher level of malaria transmission intensity than Manhiça, indicating that age associations may be related to malaria exposure. To investigate, we measured antibody responses to well-characterized blood-stage antigens, MSP2 and AMA1, which are established biomarkers of malaria exposure  Fig. 5bb5b. The n = 30) with samples available at months 3, 8.5, 21, 33, 45, and 63 (5-year follow-up). Maximal C1q-fixation responses were observed at M3, which then significantly declined by M8.5   = 55.3; p < .001) and there was also a significant correlation with IgG3  = 29.5; p < .001). There was no significant association with IgG2, IgG4, or IgM.To better understand the factors contributing to the decline in complement-fixing antibodies over time, we developed a model to determine which antibodies most strongly correlated with declining complement-fixing activity using the repeated measures data. We undertook nested multivariable mixed modeling to estimate the unique variance of complement-fixing antibodies by each antibody response type Table . The anaKnowledge of the induction and maintenance of functional immune responses by RTS,S is valuable for strategically improving vaccine design and delivery to generate a highly efficacious and longer-lasting malaria vaccine. Here, we investigated RTS,S-induced antibody responses in children and present new evidence of a functional mechanism that may contribute to vaccine-induced immunity. We show that RTS,S-induced anti-CSP antibodies were predominantly IgG1, and there was also a substantial induction of IgG3, IgG2, and IgM. Antibodies among vaccinated children could strongly fix complement on the whole, but functional responses did vary among individuals. Our data suggest that antibodies targeting both the central repeat and C-terminal regions of CSP are important for complement fixation. Furthermore, functional antibodies were poorly induced in older children with greater malaria exposure, and functional antibodies were poorly maintained after vaccination. This decline in complement-fixing antibodies was largely due to a decline in IgG1 and to a lesser extent IgG3. These findings highlight the diversity of RTS,S-induced immune responses in malaria-exposed populations, demonstrate a new functional mechanism of vaccine-induced antibodies, and shed some light on the maintenance and decay of vaccine immunity. Our findings suggest that generating higher levels of complement-fixing antibodies among a greater proportion of children, and inducing responses that are durable over time, may contribute to greater vaccine efficacy and durability.A strongly induced anti-CSP IgG1 followed by IgG3 and IgG2 in children aged 1–4 years. Similar findings showing a predominant IgG1 and IgG3 response were recently reported for a subset (n = 195) of subjects from the phase III RTS,S/AS01B vaccine trial of infants and young children (6–12 weeks and 5–17 months). A higher ratio of cytophilic antibodies (IgG1 and IgG3) to non-cytophilic antibodies (IgG2 and IgG4) was associated with vaccine-induced protection [B and AS02A adjuvants both contain TLR4 agonist MPL and the saponin QS-21, but AS01B is a liposome-based adjuvant whereas AS02A is an oil-in-water emulsion [IgG1 and IgG3 subclasses have the highest complement-fixing activity, whereas IgG2 and IgG4 have little or no activity . RTS,S/Aotection . Therefoemulsion ; RTS,S aemulsion . It is nemulsion –29, whicComplement activity can also be mediated by IgM antibodies. Interestingly, anti-CSP IgM seropositivity was moderate at baseline, although functional C1q-fixation responses were only apparent after RTS,S vaccination when IgM (and IgG) had significantly increased. Modeling the decay of antibodies over time suggests IgM is not a major mediator of complement-fixing activity, and this was instead largely mediated by IgG1 and IgG3. The role of IgM in immunological memory is not well established, but a recent study demonstrated that IgM memory B cells were long-lived and involved in secondary responses using a murine model of malaria , and IgMB [Complement activity is also influenced by epitope-specificity –34. We fB . AdditioB , 41, andB . HoweverB –47. TakeRTS,S-induced antibodies strongly fixed complement to CSP, but overall functional activity among vaccinated children markedly declined by M8.5. Although total IgG remained moderate at this time, reactivity may have dropped below the threshold required to detect functional activity using our methods, particularly because antibody density is known to influence complement activity, as multiple C1q monomers  bind neighboring IgG molecules to initiate the classical complement pathway . Our staThe present study was conducted in a malaria-endemic setting, and therefore, children will have varying degrees of malaria exposure and naturally acquired immunity. Older children in the higher malaria transmission site, Ilha Josina, had significantly lower levels of complement-fixing antibodies. This was also observed for total IgG to CSP, but the effect was not as pronounced. Older children also had higher levels of antibodies to blood-stage antigens, and this inversely correlated with vaccine-induced anti-CSP antibodies, which has been previously reported , 50. IntWe have provided a detailed characterization of RTS,S-induced antibody responses in malaria-exposed children and identified a new functional antibody activity induced by RTS,S. This is an important finding as recent studies demonstrated that complement fixation by antibodies could inhibit sporozoite traversal and lead to sporozoite cell death , 17, sugAdditional file 1:Figure S1.). Supplementary methods for C1q-fixation assay . Figure S3. RTS,S predominately induces anti-CSP IgG1 and some IgG3, IgG2, and IgM antibodies. Figure S4. Epitope specificity of children with high or low C1q-fixing antibodies. Figure S5. Younger and older children in Manhiça and Ilha Josina cohorts. Figure S6. Functional complement-fixing antibodies decline over time. Figure S7. Antibody concentration and functional C1q-fixation responses. Table S1. Linear regression between epitope-specific IgG and C1q-fixation to CSP, induced by vaccination with RTS,S (N = 99). (DOCX 2573 kb)Induction and decay of functional complement-fixing antibodies by the RTS,S vaccine in children and a negative impact of malaria exposure supplementary material. Supplementary equation 1. Supplementary methods for recombinant C-terminal region of CSP (


Even though malaria is generally on the decline due extensive control and elimination efforts, it still remains a public health problem for over 40% of the world’s population. During the course of malaria infection, parasites and red blood cells come under oxidative stress and there is host immune response in an attempt to protect the red blood cells. The frequency of monocytes and lymphocytes in peripheral blood might, therefore, be expected to reflect the state of an individual’s immune response to the infection. Circulating monocytes and lymphocytes could therefore serve as an index in relation to malaria parasitaemia. The purpose of this study was to determine whether the relative count of monocytes to lymphocytes in peripheral blood (M:L ratio) can predict parasitaemia and, therefore, the severity of malaria infection.Two millilitre of venous blood sample were taken from participants by venisection into anticoagulant tubes. Thick and thin blood films were made and stained with Giemsa and examined for malaria parasites. Whole blood specimen were analysed for full blood count using ABX Pentra 60 C+ automated haematological analyzer. Data was entered into Microsoft Word and analysed using Statistical Package for Social Sciences  and Graphpad prism. Spearman’s correlation was used to determine correlation between occurrences of clinical malaria and the monocytes and lymphocytes ratio. Statistical significance was taken as p ≤ 0.05 with 95% confidence interval.The study comprised of 1629  children up to 5 years presenting with clinical malaria as cases and 445  apparently healthy children as controls. The results indicated that there was a significant positive correlation between the monocytes to lymphocytes ratio and the presence of parasites (p = 0.04) and the level of parasitaemia within the age group of 0–3 years (p = 0.02) and 4–5 years (p = 0.03).The monocyte to lymphocyte ratio obtained correlated positively with the presence of malaria as well as the level of parasitaemia. The outcome of this work implies that monocyte to lymphocyte ratio can be used to predict the level of parasitaemia and together with other factors, the development of severe malaria. In spite of a declining trend in the number of cases and deaths over the last few years, malaria still causes significant mortality and morbidity and the bulk of the burden is observed in children under 5 years of age . The sevPlasmodium species have been known to cause malaria infection in humans namely; Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. However, more recently, Plasmodium knowlesi, which use to infect long-tailed and pig-tailed macaque monkeys, has been implicated as a cause of human malaria in Southeast Asia . For the thin smears, a minimum of 200 fields under oil immersion objectives was examined for species identification.Results of the full blood count obtained from the automated haematological analyzer were used to estimate the monocytes, lymphocytes and neutrophil levels of each sample. The absolute count of the monocytes and lymphocytes level of each sample was entered into a calculator and the ratio was estimated by dividing the monocytes by the lymphocyte counts. The other ratios were calculated using the same formula.Data was collected using notebooks and transferred to a computer and kept confidential. They were later entered into Microsoft Word and analysed using Statistical Package for Social Sciences  and Graphpad prism. Normally distributed data were analysed using independent sample t-test expressed and as Mean ± SD. Skewed data were analysed using Mann–Whitney u test and expressed in Median and interquartile range. Spearman’s rank correlation was used to determine the correlation between occurrence of clinical malaria and the monocytes and lymphocytes ratio (M:L). Statistical significance was taken as p ≤ 0.05 with 95% confidence interval.A total of 1629 and 445 children aged 6 months to 5 years 4 months whose parents or guardians consented participated in the study as cases and controls, respectively. Out of the total number of cases, 35.3% were recruited from the Korle-bu Teaching hospital, 42.6% from the Sunyani Regional Hospital and 22.1% from the Wa Regional Hospital. Again out of the total number, 55% were males and 45% were females. Out of the total number of controls, 44.5% of them were recruited from the Korle-bu municipality, 35.5% from Sunyani municipality and 20% from Wa municipality. Here also, 57.8% were males and 42.2% were females  were within the age group of 0–3 years with an average age of 1.76 ± 0.32 and 700 (43%) were within the age group of 4–5 years with an average age of 4.13 ± 0.82. Also, out of the 445 children recruited as control group, 210 (47.2%) were within the age group of 0–3 years with an average age of 1.95 ± 0.75 and 235 (52.8%) were within the age group of 4–5 years with an average age of 3.81 ± 1.06  and the monocytes to lymphocytes ratios (p = 0.0001 and p = 0.0008). Monocytes (p = 0.0175) and neutrophil to lymphocytes ratio (0.0087) showed significance among age 0–3 only whilst haemoglobin p = 0.02) showed significant difference among age 4–5 years .Again, from Table Also as seen in Table P. falciparum infections in children up to 5 years and 4 months. A total of 1629 children with an average age of 2.14 ± 1.57 participated in the study with parental/guardian consent. The number distribution from the study sites were as follows: Korle-bu Teaching hospital (35.3%), The Sunyani Regional Hospital (42.6%) and the Wa Regional Hospital (22.1%). Also, 55% of the participants were males and 45% were females. Four hundred and forty-five control subjects were also recruited for the study with an average age of 2.45 ± 1.62 years distributed as follows: Korle-bu Teaching Hospital (44.5%), Sunyani Regional Hospital (35.5%) and Wa Regional Hospital (20%) with 57.8% being males and 42.2% females  even though there was no significant difference in the total white cell count between the groups . Monocytes are essential component of the innate immune response as they act as a link to the adaptive immune system through the presentation of antigen to lymphocytes. Subsequently, during an infection, there is variation or derangement in the monocytes and lymphocytes numbers which also affect their functions . The stuP. falciparum. This antiparasitic immune response though requires tempering by the adaptive immune response if effective immunity to clinical malaria is to be achieved [Furthermore, the relative frequency of monocytes to lymphocytes in blood circulation reflects one’s capacity to initiate an effective immune response against an ongoing infection. It can therefore be postulated that the monocytes to lymphocytes ratio can be a marker of an individual’s capacity to mount an effective immune response against malaria infection . Boströmachieved .Warimwe et al.  have shoA high monocyte to lymphocyte ratio might indicate a predominantly pro-inflammatory immune response that renders an individual susceptible to clinical malaria and increased parasitaemia. The main conclusion as far as this work is concern is that increased monocyte to lymphocyte ratio correlates positively with the presence and increased parasitaemia among children up to 5 years of age. As parasitaemia increases, development of severe malaria is almost always the consequence. The outcome of this work therefore indicates that when M:L ratio is used with other clinical and haematological parameters, it can significantly improve malaria diagnosis and further assist with treatment choices.


The global malaria burden has decreased over the last decade and many nations are attempting elimination. Asymptomatic malaria infections are not normally diagnosed or treated, posing a major hurdle for elimination efforts. One solution to this problem is mass drug administration (MDA), with success depending on adequate population participation. Here, we present a detailed spatial and temporal analysis of malaria episodes and asymptomatic infections in four villages undergoing MDA in Myanmar. In this study, individuals from neighborhoods with low MDA adherence had 2.85 times the odds of having a malaria episode post-MDA in comparison to those from high adherence neighborhoods, regardless of individual participation, suggesting a herd effect. High mosquito biting rates, living in a house with someone else with malaria, or having an asymptomatic malaria infection were also predictors of clinical episodes. Spatial clustering of non-adherence to MDA, even in villages with high overall participation, may frustrate elimination efforts. The global burden of malaria has decreased over the last decade. Many countries now aim to banish malaria. One obstacle to elimination is people who carry malaria parasites without showing symptoms. These asymptomatic people are unlikely to be diagnosed and treated and may contribute to further spread of malaria. One way to clear all malaria infections would be to ask everyone in a community to take antimalarial drugs at the same time, even if they do not feel ill. This tactic is most likely to work in communities that are already reducing malaria infections by other means. For example, by treating symptomatic people and using bed nets to prevent bites from malaria-infected mosquitos.Several studies have shown that mass drug administration is a promising approach to reduce malaria infections. But its success depends on enough people participating. If enough community members take antimalarial drugs, then even those who cannot participate, such as young children or pregnant women, should be less likely to get malaria. This is called the herd effect.Plasmodium falciparum. The analysis looked at malaria infections among residents of four villages in the Kayin State of Myanmar that used mass antimalarial drug administration. People who lived in neighborhoods with high participation in mass drug administration were almost three times less likely to get malaria than people who lived in communities with low participation. Even people who did not take part benefited.Now, Parker et al. demonstrate that mass antimalarial drug administration reduces infections with malaria caused by the parasite The analysis suggests that mass antimalaria drug administration benefits individuals and their communities if enough people take part. To be successful, malaria elimination programs that wish to use mass drug administration should approach communities in a way that encourages high levels of participation. P. falciparum malaria (Mass drug administration (MDA) is the provision of medications to entire target populations and the approach has been used for many infectious diseases, including lymphatic filariasis, soil-transmitted helminths, onchocerciasis, schistosomiasis, and trachoma . MDA has malaria  and has  malaria  and Asia malaria . It is b malaria , and has malaria .P. falciparum malaria  provides a survival advantage for resistant parasites, there has been some hesitance in using MDA for malaria. One historical malaria eradication campaign relied on the inclusion of sub-therapeutic levels of antimalarials distributed in table salt across large populations . This pr malaria .While antimalarials are usually administered following diagnosis (confirmed or presumed) or used as a prophylactic, MDA is used because of an intended population- or community-level effect. The rationale is that the transmission potential or reproductive rate of malaria is so high that a sufficient amount of the parasite reservoir needs to be removed in order to disrupt transmission. This group-level effect is also referred to as a ‘herd effect’  and the P. falciparum malaria in villages undergoing MDA. We investigate associations between individual- and group-level participation in MDA ; subclinical infections; and clinical episodes of P. falciparum post-MDA. Such empirical research is important for providing an evidence base for further research, intervention, and policy work.Drawing from detailed micro-epidemiological and spatial data from an MDA trial in Kayin State, Myanmar , we descP. falciparum. 201 participants were found to have P. falciparum by uPCR. 325 uPCR-positive participants had Plasmodium infections not identifiable at the species level and thus were not included in these analyses. Total numbers of clinical episodes and uPCR-detected infections were higher than the total number of infected individuals because some participants had multiple infections.3229 villagers  were included in this study. During the study period, 80 study participants were diagnosed with clinical P. falciparum episodes occurred in only one of the study villages (TOT). 66 out of the 80 participants who had a clinical P. falciparum episode were from TOT village .After MDA, the vast majority of clinical P. falciparum episode during the study period had repeated clinical episodes and 19 of the 80 (24%) participants who had a clinical episode were found to have a uPCR-detected P. falciparum infection in at least one of the surveys. uPCR-detected P. falciparum infections were more prevalent in males than females .Eleven of the 80 participants (14%) who had a clinical P. falciparum infections were widespread in all villages at baseline  prior to MDA.uPCR-detected baseline . These iP. falciparum infections in each village at baseline but subsequently no significant clusters were detected . The cluster in KNH occurred from M5 through M7 but included only four episodes. There were two separate clusters in village TOT. A cluster in the western portion of the village began in M12 and lasted until M18 . A single-house cluster occurred in the eastern portion of the village (M15 through M18) with five episodes among four house members .There were statistically significant clusters of uPCR-detected detected . ClusterThere were significant clusters of non-participation in the MDAs in three of the study villages . The noP. falciparum episodes occurred in village TOT following MDA (MDA was completed by M3), followed by a small outbreak beginning in M12  and individual participation in MDA  and participants who lived in a house with someone else who had a clinical P. falciparum episode during the same month , after adjusting for other covariates .After MDA, clinical variates . Individvariates . The humP. falciparum malaria. There was an apparent group level effect from MDA adherence, suggesting herd protection and evident from three lines of evidence.The primary objective of this research was to look for a potential herd effect and at the impact of non-adherence with regard to MDA for P. falciparum outbreak began (M12), there was a lag of approximately 1 month between the onset of clinical episodes in neighborhoods with mid and low MDA adherence and then occurred in neighborhoods with high MDA adherence  of the administered antimalarials. This group level protective effect from MDA was also evident in the rainy season following MDA  which codherence .P. falciparum episode  at the same time as in village TOT  when searching within houses and occasionally in neighboring houses, but these would have only been a small proportion of all infections within the villages , a neighborhood-level effect was not included in this analysis. It is possible that the confidence intervals around the neighborhood MDA adherence variable are therefore too small and would not have been statistically significant had a neighborhood effect been included. This work would benefit from analyses with larger datasets.There are several limitations to this work. Individuals who did not participate in MDA also did not participate in blood screenings immediately after MDA (i.e. M3 in village TOT). uPCR-detected infections are therefore likely to be underdiagnosed for these individuals, and it is likely that such infections clustered and overlapped with the clusters of non-adherence to MDA. While no genetic analyses were done with samples from the village, it is likely that these underdiagnosed infections, combined with the high HBR, led to a resurgence of clinical episodes. There is also evidence of a poorly functioning MP in this village, which could have led to undiagnosed clinical episodes, especially during the beginning of the study. Some infections are likely to be acquired outside of the village, leading to complex spatial patterns in infections that are mapped at the house level. Within-household clustering can be the result of within-household transmission, or shared exposure outside of the household or village among household members. Finally, these data come from a limited number of villages , with analysis of P. falciparum remaining after MDA (TOT). Likewise, this work points toward the need for considering spatial scale in MDA and in MDA adherence. Most current MDA trials and programs in the GMS and elsewhere consider a single village or community as the target unit. In some cases, especially when high prevalence villages are spatially clustered across a landscape, it may be necessary to target units above the village level (i.e. groups of villages).This work has relevance with regard to further research and practice concerning MDA. While participation is obviously crucial to success, there is unlikely to be a single adherence proportion that can be applied in all situations. In this study, the elimination efforts were successful in three out of four villages even though one of those villages (HKT) had a similar overall adherence to the village with P. falciparum malaria prevalence surveys using ultrasensitive quantitative PCR (uPCR)  along the Myanmar-Thailand border, in Kayin (Karen) State, Myanmar . The vilR (uPCR)  and wereR (uPCR) . The norR (uPCR) . The stuA full population census was completed in each of the four study villages at baseline May – June 2013. Everyone enumerated in the census was given a unique identification code. Geographic coordinates were collected for all houses in the four study villages and a unique identification code was assigned to each house. All individuals were then linked to their respective houses.Blood surveys were conducted at baseline in each village, aiming to screen all individuals above an age of 9 months. Venous blood (3 mL) was drawn from each participant, transported to a central laboratory and analyzed using a highly sensitive quantitative PCR (uPCR) assay with a limit of detection of 22 parasites per mL. . InfectiP. falciparum or P. vivax. Malaria episodes were treated with dihydroartemisinin-piperaquine (DHA + P) for P. falciparum and chloroquine for P. vivax. Radical cure for P. vivax was not provided because the absence of G6PD (Glucose-6-phosphate dehydrogenase) tests required to prevent hemolysis in G6PD individuals  was established in each village at the beginning of the project, as part of the malaria intervention. Village health workers were trained to diagnose malaria using rapid diagnostic tests (RDTs) and to treat RDT positive infections with dose based on weight and age. The ID code of each participant who self-presented at the MP was recorded, along with RDT results, and these cases are hereafter referred to as clinical malaria episodes of either ividuals .MDAs were conducted in two villages at the beginning of the study  and extended to the two control villages beginning in month 9 (M9). Restricted randomization was used to decide which villages received early or deferred MDA. MDA consisted of 3 days of DHA + P, with a single low dose of primaquine on the third day, repeated over three months . Follow-up blood surveys were conducted in each village every third month after M0 until M18. A final full blood survey was completed in each village at M24.Mosquitoes were collected monthly using human landing catches to estimate the human biting rate (HBR). Mosquito catching teams were based at five sites (both indoors and outdoors) within each of the four study villages  for five consecutive nights during the study period M0 through M20. Mosquitoes were caught using glass tubes and later identified morphologically .The locations of study villages, MPs, catch sites, and village houses are indicated in All individuals recorded in the census with a house address in the four study villages were included in this analysis.P. falciparum. Likewise, individuals who did not have a clinical episode within a given month were coded with a ‘0’ for that respective month. Individuals who were ever diagnosed with a clinical episode or uPCR-detected P. falciparum infections were likewise coded as a ‘1’ for analyses of having ever been detected by uPCR for an infection or having ever had a clinical episode during the study period.The data were aggregated into 1 month time steps and individuals were coded with a ‘1’ for any month in which they presented at the village MP and were diagnosed with Predictor variables (covariates) are listed in Neighborhood MDA non-adherence was calculated as the proportion of people who took no rounds of MDA within 100 m radius of each house in the study population. This proportion was calculated for each house in the study villages and non-adherence proportions were then attributed to individuals based on the house to which they were attributed.Anopheles minimus s.l., An. maculatus s.l., and An. dirus s.l.) was calculated for each month.The human biting rate (HBR) for primary vectors  were calculated for binary predictors and Wilcox rank sum tests were calculated for continuous variables. Cumulative hazards curves were used to analyze temporal patterns in clinical episodes. uPCR-detected infections (from surveys) and clinical episodes (from the MPs) were mapped at the house level across time. Maps were created for each village and each survey time point (months 0 through 24: M0 – M24), with clinical episodes aggregated to align with surveys .P. falciparum episodes for each survey time point in the one village with sufficient P. falciparum episodes (TOT) post-MDA. Clinical episodes were aggregated to align with survey time points (i.e. months 1 through 3 were aggregated and plotted in month 3). One SDD was calculated, corresponding to approximately 68% of all points falling inside of the resulting circle.The weighted standard distance deviation (SDD) was used to visually analyze the distribution of clinical P. falciparum infections across survey months; clinical P. falciparum episodes across all months of the study period; and MDA non-participation , the analysis of clinical P. falciparum episodes focuses on this village alone.A multivariable mixed effects logistic regression was used to estimate model adjusted odds ratios (AOR) and confidence intervals for predictor covariates with regard to the individual odds of having a clinical P. falciparum infections are listed in Appendix 1. While this study focuses on P. falciparum, P. vivax data were also collected and are included in secondary analyses in the Appendix.Covariates for these models are listed in https://cran.r-project.org/) and the ‘epiR’, ‘lme4’, and ‘survival’ packages. All maps were created using ArcGIS 10.5 (https://www.arcgis.com/). Exploratory spatial data analysis was conducted using ArcGIS 10.5 and SatScan v9.5 (https://www.satscan.org/). The neighborhood participation variable was created using ArcGIS and the Python programming language .Exploratory statistics and regressions were calculated using R , the Tak Province Community Ethics Advisory Board (T-CAB), and by village committees in each of the four study villages. Survey and mass drug administration participation were voluntary and all participants provided written informed consent. Participants < 18 yo provided assent to participate, along with consent from their guardian(s). Potential participants received study information in their mother language (Karen or Burmese) both through community engagement activities on through one-on-one meetings. The ethics committees require consent to publish when individuals are identifiable. Since no individuals are identifiable through this publication, no consent to publish was obtained. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Plasmodium falciparum infections conferred by mass antimalarial drug administrations" for consideration by eLife. Your article has been reviewed by Prabhat Jha as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal her identity: Gillian Stresman (Reviewer #3).Thank you for submitting your article "Herd protection against The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper presents an interesting dataset on the spatial and temporal patterns of malaria before and after a mass drug administration intervention in 4 villages. Understanding the effect of community-level protection that results from mass drug administration campaigns is currently a key gap in planning for malaria elimination activities. The data are impressively detailed.Essential revisions:This paper presents a very interesting dataset on the spatial and temporal patterns of malaria before and after a mass drug administration intervention in 4 villages. Understanding the effect of community-level protection that results from mass drug administration campaigns is currently a key gap in planning for malaria elimination activities. The data are impressively detailed.However, we have concerns around the presentation of ideas, results, and conclusions with the overall narrative lacking cohesion/focus. For example, the aim of the work is to look at the presence of herd immunity as a result of MDA but the majority of the introduction section focuses on justifying the use of MDA instead of the literature around adherence being a critical factor in the success of MDA, the concept of herd immunity, how critical thresholds for coverage do not exist but potential ways of estimating them etc. Also, the subsequent sections jump between outcome of temporal and spatial analysis, between Pf vs. Pv and infection vs. clinical case making it hard to follow. Overall, the aims, results, and discussion do not match the title and conclusions of paper. We suggest that the authors take time to carefully frame the story they are trying to tell. One suggestion would be to include the Pv work as supplemental information. The outcome of the MDA on Pv is essentially a null result with the MDA implementation not being designed to have an impact on transmission . The findings are important but not surprising and therefore could be included as supplemental so it doesn't detract from the main and more interesting outcome of the impact on Pf.A second major concern is that the work does not appear to be placed within the larger research and programmatic context. A lot of the research from the GMS region come from this institution, but degree of self-citation is concerning with ~50% of references coming from the research group. Furthermore, the authors appear to be drawing conclusions achieved in other papers without referencing instead of focusing on the aims of this specific piece of work. For example, the Discussion section comments on individual-level results, that were not presented in this paper but have been highlighted in other papers, on the same data without referencing .Third, the approach taken to estimate HBR, a key factor suggested to contribute to reintroduction of Pf transmission is questionable. Although a pragmatic approach, what level of spatial bias has been introduced by attributing household-level HBR values based on the nearest trapping site? Given the known high-degree of spatial heterogeneity at small scales in mosquito abundance, would this add an additional level of bias when just selecting the nearest sampling point? Would it be more appropriate to aggregate HBR according to neighborhood as has been defined by the adherence variable to ensure spatial congruence? This can be achieved by creating prediction surfaces using model based geostatistical methods with the HBR variable as outcome and then taking the mean predicted HBR using the same window size as for adherence.That said, there are many strong points about the analysis e.g. the possibility to look at both individual level and area-level covariates, and the measurement of both symptomatic and asymptomatic infections and relationship between the two.An interesting result was the apparent clustering of resurgent cases in the area with lower adherence to MDA in one village . But the evidence for this was rather weak – it was based mainly on one cluster of non-adherence in one village, which also happened to experience a higher number of cases. This seems potentially explained by the higher biting rate of mosquitoes in this part of the village rather than the non-adherence, but it was not clear whether there was multivariate adjustment to check this? Also, the asymptomatic infections did not seem to follow the same pattern. Including this finding in the title and as a key result elsewhere in the paper places too much confidence in this result, and the conclusions should be more cautious. Perhaps generally the limitations of spatial analysis within only 4 villages could be further acknowledged.P. falciparum and P. vivax episodes”). If a critical cutoff value for adherence levels to achieve a 'herd immunity' during MDA campaigns could be estimated using these data, it would be a major finding and significant improvement to this work. This would be possible to do with these data using ROC analysis with the continuous adherence variables.We also suggest the authors explore further the effect of individual level adherence vs area-level average adherence, specifically are non-adherent individuals living in high-adherence neighbourhoods protected, compared with non-adherent individuals in low-adherence neighbourhoods? (and vice versa) It is not really clear at the moment whether this is a spatial effect, or whether it's just perhaps that non-adherent people are both more likely to be infected and to live in non-adherent neighbourhoods , a Reviewing Editor, and three reviewers.Thank you for resubmitting your work entitled "Herd protection against The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Reviewer #1:The rewritten paper is much clearer and easier to read than the initial version. Most of the points raised in the previous reviews have been well addressed.Reviewer #2:The authors have made substantial revisions based on the previous reviews and most points are thoroughly addressed. I like the new Figure 4.The idea of a herd effect is interesting and certainly worth reporting, but my only remaining concern is that the conclusions and title are too strong based on the limited data from one village and also analytical problems. The authors have clarified in response to another reviewer that their multivariate model (Table 2) does not include random neighbourhood effects, which is not an appropriate analysis with which to test neighbourhood variables since it will overestimate the statistical significance of the herd effect result.Ideally an alternative analysis needs to be tried, for example separating the village into discrete spatial units and adding random effects at this level. At the very least the authors should consider wording the Abstract and Title more cautiously – to clarify that this herd effect was only seen in one village and more investigation is needed to verify whether this is a real phenomenon.Reviewer #3:This paper is an interesting and very timely assessment of the community effect of a mass drug administration trial. The revised version of the paper is much improved but could be further strengthened by focusing the Introduction and Discussion section around the objective of the paper, instead of the more general role of MDA as is currently written. Specifically, I suggest that the authors should address the following comments.Introduction: Are the authors trying to say that MDA should supplement existing malaria programs/used when the full package doesn't eliminate transmission ? There is an argument that this intervention could bypass weak health systems to interrupt transmission and it has been used under alternative scenarios (e.g. Ebola epidemic response). Should this be 'should ideally be used in settings…'The potential risks associated of using MDA are missing from the introduction (i.e. developing drug resistance). Why has the WHO avoided recommending it for so long? What's changed their stance?Introduction: The justification that MDA is used to achieve a population level effect is missing important nuances (and offers some repetition). The rationale of MDA is typically argued from the biological perspective whereby the transmission potential/reproductive rate of malaria is so high, to achieve interruption, enough of the parasitic reservoir needs to be removed before it has the chance to fuel subsequent infections. The concept of a "herd effect" applies in most communicable disease systems: If enough of the population is protected, transmission chains cannot be sustained and transmission with die out. What is 'enough' is unknown as stated and is affected by the propensity for diseases to cluster in populations . If the aim of MDA for malaria is to interrupt transmission chains, the notion of a herd effect providing additional levels of population protection is plausible but has never been examined empirically.Subsection “Analysis”: what was the rationale for using a 100m radius. Are the results affected according to size of buffer?Subsection “Analysis”: is there an impact when considering individual level  vs household level (e.g. households with </> 80%? compliance) non-compliance?Discussion section: MDA adherents in low-adherence areas had highest risk? Even compared to those in the same neighborhoods with low-adherence? This is an interesting finding consistent with what has been observed with bednets: those who have the most malaria are more likely to adhere to interventions, potentially related to perceptions of risk.Discussion section: I would encourage authors to split this paragraph into 3 sections to ensure that these key observations can be discussed in depth.Discussion section: do the authors have evidence to support this counterfactual? Or do they mean would have likely/may have resulted in… ?Discussion section: I'm not sure of the relevant of this paragraph to the objectives of the work. The focus is on the herd protection or am I missing a connection between adherence and the more general rational for MDA? What are the potential limitations to MDA when considering the potential impacts of herd protection that may be specific to this setting?Discussion section: Similar to point above, the objective is the work is related to herd immunity and the concluding remarks seem to focus on the general impact of MDA. Here, I would encourage the authors to include some insight revolving around the potential that comes with an improved understanding of the herd effect for malaria control and elimination activities. Next steps for further exploring this phenomenon?Discussion section: The objective of the study was to look at the herd effect and the impact of non-adherence. I would encourage the authors to re-write this first paragraph of the discussion to focus on this instead of what has been reported in other publications.Plasmodium falciparum infections conferred by mass antimalarial drug administrations" for further consideration at eLife. Your revised article has been favorably evaluated by Neil Ferguson (Senior Editor), a Reviewing Editor, and 3 reviewers.Thank you for resubmitting your work entitled "Potential herd protection against The manuscript has been improved but there are some minor remaining issues that need to be addressed before acceptance, as outlined below in the individual reports.Reviewer #1:I had no substantive concerns with the previous version and feel that the manuscript is essentially suitable for publication.However, in the course of the review process the authors have made incremental amendments that understate the very real concerns that exist around the potential for selection of resistance by MDA. In particular, reference 18  is cited to support the contention that there is "no evidence of resistance emerging as a result of controlled, targeted MDA with the use of therapeutic drug levels". Arguable, such evidence is exactly what was reported by Trigg et al. for pyrimethamine/sulfadoxine in Tanzania . Even if there were no evidence, the absence of evidence is not good evidence for absence of an effect, and the case for no effect seems to be summarised in the Abstract of von Seidlein and Dondorp 2015 by the statement "it is difficult to conceptualize how targeted malaria elimination could contribute to artemisinin resistance…". The wording here is critical: it is rather easy to conceptualise how failed attempts at elimination using MDA could lead to resistance, especially if drug use is not well controlled. Since this paper is not about drug resistance, I think that the claims about resistance in the Introduction should be removed.Reviewer #2:I copy here my previous review and the authors' response, with my current response below:“The authors have made substantial revisions based on the previous reviews and most points are thoroughly addressed. I like the new Figure 4.The idea of a herd effect is interesting and certainly worth reporting, but my only remaining concern is that the conclusions and title are too strong based on the limited data from one village and also analytical problems. The authors have clarified in response to another reviewer that their multivariate model (Table 2) does not include random neighbourhood effects, which is not an appropriate analysis with which to test neighbourhood variables since it will overestimate the statistical significance of the herd effect result.Ideally an alternative analysis needs to be tried, for example separating the village into discrete spatial units and adding random effects at this level. At the very least the authors should consider wording the abstract and title more cautiously – to clarify that this herd effect was only seen in one village and more investigation is needed to verify whether this is a real phenomenon.”“Yes, this is an approach used, for example, by Ali et al.  and in some ways it would be ideal. With our data some problems would remain though because any discrete spatial units ("neighborhoods") that are imposed on the villages would be arbitrary and since the villages are quite small, there would only be 4-5 each . For the one village with Pf after MDA, we'd only have 4 or 5 neighborhoods and we would question if a random intercept would actually be useful, given that there are assumptions about the mean and variance of the random intercept.There is also a spatial argument to be made in that an arbitrary boundary or line that would split such areas within the village would be creating a boundary between houses that are extremely close to each other. If geographic proximity is important here, and we believe it is, then we would actually lose a lot of information by using this approach.Regardless, the reviewer is correct that we need to be cautious in our wording here. We have taken the suggestion to word the Title and Abstract more cautiously, and we discuss some of these issues in the Discussion section.”Thank you for addressing these points very well in terms of altering the abstract and title. For my last suggestion, please can the authors either (a) explicitly state that the confidence interval reported for the paper's main result is likely to be much too narrow, because it tests for the effect of a neighbourhood level variable (coverage within 100m of a household) using individual level data without allowing for neighbourhood effects. Or (b) remove the confidence interval entirely and explain why it could not be calculated. Or (c) try an alternative analysis.P. falciparum episodes” as it stands must therefore be incorrect . It would be unlikely to be a significant result if neighbourhood effects were included. It is still an interesting result, which could be explored further in other larger datasets.I appreciate the difficulties of doing an analysis which incorporates neighbourhood effects, but the confidence interval of the main result in the Abstract, shown in subsection “Longitudinal multivariable analysis of clinical Otherwise the manuscript is very interesting and useful, and I have no further comments.Reviewer #3:This work is much improved. I have no additional comments and recommend the work for publication. Essential revisions:[…] 1) However, we have concerns around the presentation of ideas, results, and conclusions with the overall narrative lacking cohesion/focus. For example, the aim of the work is to look at the presence of herd immunity as a result of MDA but the majority of the introduction section focuses on justifying the use of MDA instead of the literature around adherence being a critical factor in the success of MDA, the concept of herd immunity, how critical thresholds for coverage do not exist but potential ways of estimating them etc. Also, the subsequent sections jump between outcome of temporal and spatial analysis, between Pf vs. Pv and infection vs. clinical case making it hard to follow. Overall, the aims, results, and discussion do not match the title and conclusions of paper. We suggest that the authors take time to carefully frame the story they are trying to tell. One suggestion would be to include the Pv work as supplemental information. The outcome of the MDA on Pv is essentially a null result with the MDA implementation not being designed to have an impact on transmission . The findings are important but not surprising and therefore could be included as supplemental so it doesn't detract from the main and more interesting outcome of the impact on Pf.P. falciparum malaria after MDA had been administered, looking at both individual and group-level effects. Since the work was focused on clinical P. falciparum we’ve now removed all analysis of P. vivax and uPCR-detected P. falciparum and offer these analyses in the supplementary material. We’ve rewritten the Introduction to focus more generally on MDA for P. falciparum malaria and have revised much of the rest of the manuscript in order to improve readability. We believe that these changes make the manuscript much more clear and concise.We have now refocused and rewritten the manuscript in order to address this valid critique. The main goal of this work was to analyze clinical 2) A second major concern is that the work does not appear to be placed within the larger research and programmatic context. A lot of the research from the GMS region come from this institution, but degree of self-citation is concerning with ~50% of references coming from the research group. Furthermore, the authors appear to be drawing conclusions achieved in other papers without referencing instead of focusing on the aims of this specific piece of work. For example, the Discussion section comments on individual-level results, that were not presented in this paper but have been highlighted in other papers, on the same data without referencing .In our refocusing of this work, we now begin by discussing MDA in general rather than the GMS context. We’ve now attempted to appropriately reference other research teams in this revision.P. falciparum episode after MDA (results in Table 2) uses a multivariable regression which includes both individual and group (“neighborhood”) level variables for MDA adherence. We hope that this is now made clear through our revision of the manuscript.Regarding results from other manuscripts – these data have been previously analyzed . However, the effects that we mention  are from this analysis. The analysis of risk factors for acquiring a clinical 3) Third, the approach taken to estimate HBR, a key factor suggested to contribute to reintroduction of Pf transmission is questionable. Although a pragmatic approach, what level of spatial bias has been introduced by attributing household-level HBR values based on the nearest trapping site? Given the known high-degree of spatial heterogeneity at small scales in mosquito abundance, would this add an additional level of bias when just selecting the nearest sampling point? Would it be more appropriate to aggregate HBR according to neighborhood as has been defined by the adherence variable to ensure spatial congruence? This can be achieved by creating prediction surfaces using model based geostatistical methods with the HBR variable as outcome and then taking the mean predicted HBR using the same window size as for adherence.The reviewer is correct, both about micro-heterogeneities in HBR and that our specification of this variable is overly simplistic. We agree that a smoothed surface would be better, however we don’t believe that these data are appropriate for making prediction surfaces. There were only 5 catch sites per village and most villages are not in geographic proximity to each other . The catch sites are not systematically distributed through the villages and their geographic extent is not as broad as the geographic extent of houses in each village. We would therefore have to create interpolations from only 5 points and then extrapolate outside of the 5 points to meet the geographic range of the villages. To address this problem in our analysis we have therefore opted to use HBR calculations at the village level in the regressions. This is still simplistic and doesn’t address micro-scale heterogeneities. In the absence of detailed human movement data and HBR outside of the village, we believe that this specification for HBR is the best possible approach with these data.4) An interesting result was the apparent clustering of resurgent cases in the area with lower adherence to MDA in one village . But the evidence for this was rather weak – it was based mainly on one cluster of non-adherence in one village, which also happened to experience a higher number of cases. This seems potentially explained by the higher biting rate of mosquitoes in this part of the village rather than the non-adherence, but it was not clear whether there was multivariate adjustment to check this? Also, the asymptomatic infections did not seem to follow the same pattern. Including this finding in the title and as a key result elsewhere in the paper places too much confidence in this result, and the conclusions should be more cautious. Perhaps generally the limitations of spatial analysis within only 4 villages could be further acknowledged.Please see our response to point 2 above. We hope that the revised manuscript now clarifies that the models were multivariable and are looking at both individual and group level MDA adherence as well as HBR.The reviewer is correct that these are a limited set of data and that we should be cautious in over-interpreting the results (which mostly come from a single village that had remaining Pf after MDA). We have added the following statement to the Discussion section:P. falciparum episodes coming from the sole village that continued to have P. falciparum after MDA.”“Finally, these data come from a limited number of villages , with analysis of 5) We also suggest the authors explore further the effect of individual level adherence vs area-level average adherence, specifically are non-adherent individuals living in high-adherence neighbourhoods protected, compared with non-adherent individuals in low-adherence neighbourhoods? (and vice versa) It is not really clear at the moment whether this is a spatial effect, or whether it's just perhaps that non-adherent people are both more likely to be infected and to live in non-adherent neighbourhoods .Please see our response to point 2 above. We hope that the revised manuscript now clarifies that the models were multivariable and are looking at both individual and group /area level adherence and effects.6) If a critical cutoff value for adherence levels to achieve a 'herd immunity' during MDA campaigns could be estimated using these data, it would be a major finding and significant improvement to this work. This would be possible to do with these data using ROC analysis with the continuous adherence variables.Our goal in this paper is to show that there is a measurable herd effect, but we believe that it is drastically complicated by context and that a single proportion of adherence won’t be applicable in all target populations. This analysis will soon be followed by a mathematical modelling paper using these and other data in order to further explore this topic.7) Last, in general it was not clear whether multivariate analysis was done – if so, it would be important to see the effect of each variable after adjustment. If not, the authors need to explain why multivariate analysis was not performed.Please see our response to point 2 above. We hope that the revised manuscript now clarifies that the models were multivariable and are looking at both individual and group /area level adherence and effects.Reviewer #2:[…] The idea of a herd effect is interesting and certainly worth reporting, but my only remaining concern is that the conclusions and title are too strong based on the limited data from one village and also analytical problems. The authors have clarified in response to another reviewer that their multivariate model (Table 2) does not include random neighbourhood effects, which is not an appropriate analysis with which to test neighbourhood variables since it will overestimate the statistical significance of the herd effect result.Ideally an alternative analysis needs to be tried, for example separating the village into discrete spatial units and adding random effects at this level. At the very least the authors should consider wording the Abstract and Title more cautiously – to clarify that this herd effect was only seen in one village and more investigation is needed to verify whether this is a real phenomenon.Yes, this is an approach used, for example, by Ali et al., (2005) and in some ways it would be ideal. With our data some problems would remain though because any discrete spatial units (“neighborhoods”) that are imposed on the villages would be arbitrary and since the villages are quite small, there would only be 4-5 each . For the one village with Pf after MDA, we’d only have 4 or 5 neighborhoods and we would question if a random intercept would actually be useful, given that there are assumptions about the mean and variance of the random intercept.There is also a spatial argument to be made in that an arbitrary boundary or line that would split such areas within the village would be creating a boundary between houses that are extremely close to each other. If geographic proximity is important here, and we believe it is, then we would actually lose a lot of information by using this approach.Regardless, the reviewer is correct that we need to be cautious in our wording here. We have taken the suggestion to word the title and abstract more cautiously, and we discuss some of these issues in the Discussion section.Reviewer #3:[…] Introduction: Are the authors trying to say that MDA should supplement existing malaria programs/used when the full package doesn't eliminate transmission ? There is an argument that this intervention could bypass weak health systems to interrupt transmission and it has been used under alternative scenarios (e.g. Ebola epidemic response). Should this be 'should ideally be used in settings…'Yes. We have now changed the wording of this statement (Introduction).The potential risks associated of using MDA are missing from the introduction (i.e. developing drug resistance). Why has the WHO avoided recommending it for so long? What's changed their stance?We have now added a few sentences discussing historical problems and concerns regarding MDA:Introduction“Given that drug pressure  provides a survival advantage for resistant parasites, there has been some hesitance in using MDA for malaria. One historical malaria eradication campaign relied on the inclusion of sub-therapeutic levels of antimalarials distributed in table salt across large populations [15]. This program likely led to the emergence of parasite resistance in the same regions [16] and this has in part led to hesitance among some institutions  to implement MDA for malaria [17]. There is, however, no evidence of resistance emerging as a result of controlled, targeted MDA with the use of therapeutic drug levels [18].”Introduction: The justification that MDA is used to achieve a population level effect is missing important nuances (and offers some repetition). The rationale of MDA is typically argued from the biological perspective whereby the transmission potential/reproductive rate of malaria is so high, to achieve interruption, enough of the parasitic reservoir needs to be removed before it has the chance to fuel subsequent infections. The concept of a "herd effect" applies in most communicable disease systems: If enough of the population is protected, transmission chains cannot be sustained and transmission with die out. What is 'enough' is unknown as stated and is affected by the propensity for diseases to cluster in populations . If the aim of MDA for malaria is to interrupt transmission chains, the notion of a herd effect providing additional levels of population protection is plausible but has never been examined empirically.We have now attempted to incorporate these nuances and some of this text (reworded) into this part of the manuscript:Introduction“While antimalarials are usually administered following diagnosis (confirmed or presumed) or used as a prophylactic, MDA is used because of an intended population- or community-level effect. […] If the aim for of antimalarial MDA is to interrupt transmission, the notion of a herd effect providing additional levels of population protection is plausible but has not been examined empirically [22].”Subsection “Analysis”: what was the rationale for using a 100m radius. Are the results affected according to size of buffer?The 100m radius was relatively arbitrary, but we did do a sensitivity analysis on the buffer size. We’ve added this detail in the Appendix now:> 180m) most houses were included within a buffer (furthest distance between any two houses was approximately 1km) leading to little spatial heterogeneity in participation across the village.”“The sensitivity of the model results with regard to the neighborhood MDA non-adherence buffer size was also tested. Neighborhood MDA-non-adherence was tested with varying buffer sizes, beginning with a radius of 20m and going up to a radius of 220m, by 40m intervals. Neighborhood buffers from 60m to 140m were statistically significant and had comparable model adjusted odds ratios . Small buffers (i.e. 20m) included only one or very few households. At larger buffer sizes  vs household level (e.g. households with </> 80%? compliance) non-compliance?For this analysis we’ve only considered the individual level. We prefer to not do the analysis using the household level because of the variation in household size (histogram attached here). For example, a house with 10 members and 80% adherence could still have 2 members with parasites in their blood while a house with 5 members and only 60% adherence would still have 2 members with parasites in their blood. Arguably, these 2 individuals are equally infective for others in the neighborhood, but would be treated as though they were completely different if we analyzed based on the house level.Discussion section: MDA adherents in low-adherence areas had highest risk? Even compared to those in the same neighborhoods with low-adherence? This is an interesting finding consistent with what has been observed with bednets: those who have the most malaria are more likely to adhere to interventions, potentially related to perceptions of risk.We have now added the following statement along these lines:Discussion section“As has been described in other settings, this individual-level finding may be related to relative perceptions of risk; with potential complacency among individuals living in areas with lower levels of malaria [23].”Discussion section: I would encourage authors to split this paragraph into 3 sections to ensure that these key observations can be discussed in depth.Done.Discussion section: do the authors have evidence to support this counterfactual? Or do they mean would have likely/may have resulted in….We have softened the statement as requested (Discussion section).Discussion section: I'm not sure of the relevant of this paragraph to the objectives of the work. The focus is on the herd protection or am I missing a connection between adherence and the more general rational for MDA? What are the potential limitations to MDA when considering the potential impacts of herd protection that may be specific to this setting?We have now dropped this paragraph.Discussion section: Similar to point above, the objective is the work is related to herd immunity and the concluding remarks seem to focus on the general impact of MDA. Here, I would encourage the authors to include some insight revolving around the potential that comes with an improved understanding of the herd effect for malaria control and elimination activities. Next steps for further exploring this phenomenon?P. falciparum, general implications of this work, and future directions.We have now rewritten the conclusion to focus more specifically on herd effects from MDA for Discussion section: The objective of the study was to look at the herd effect and the impact of non-adherence. I would encourage the authors to re-write this first paragraph of the discussion to focus on this instead of what has been reported in other publications.Done.Reviewer #1:[…] However, in the course of the review process the authors have made incremental amendments that understate the very real concerns that exist around the potential for selection of resistance by MDA. In particular, reference 18  is cited to support the contention that there is "no evidence of resistance emerging as a result of controlled, targeted MDA with the use of therapeutic drug levels". Arguable, such evidence is exactly what was reported by Trigg et al. for pyrimethamine/sulfadoxine in Tanzania . Even if there were no evidence, the absence of evidence is not good evidence for absence of an effect, and the case for no effect seems to be summarised in the Abstract of von Seidlein and Dondorp 2015 by the statement "it is difficult to conceptualize how targeted malaria elimination could contribute to artemisinin resistance…". The wording here is critical: it is rather easy to conceptualise how failed attempts at elimination using MDA could lead to resistance, especially if drug use is not well controlled. Since this paper is not about drug resistance, I think that the claims about resistance in the Introduction should be removed.We were unsure of whether or not the reviewer wanted us to discard all discussion/claims about resistance in the paragraph – but we agree with a previous suggestion that the hesitancy for using MDA should at least be mentioned. We felt that the most contentious statement was the one referred to above  and we have therefore removed this statement from the manuscript.Reviewer #2:[…] For my last suggestion, please can the authors either (a) explicitly state that the confidence interval reported for the paper's main result is likely to be much too narrow, because it tests for the effect of a neighbourhood level variable (coverage within 100m of a household) using individual level data without allowing for neighbourhood effects. Or (b) remove the confidence interval entirely and explain why it could not be calculated. Or (c) try an alternative analysis.I appreciate the difficulties of doing an analysis which incorporates neighbourhood effects, but the confidence interval of the main result in the Abstract, shown in subsection “Longitudinal multivariable analysis of clinical P. falciparum episodes” as it stands must therefore be incorrect . It would be unlikely to be a significant result if neighbourhood effects were included. It is still an interesting result, which could be explored further in other larger datasets.Done. We have included the following statement at the end of the Discussion section, immediately before stating our conclusions:P. falciparum episodes post-MDA were only possible to analyze in a single village, and that neighborhoods were not discrete and overlapped (100m buffer around each house), a neighborhood-level effect was not included in this analysis. It is possible that the confidence intervals around the neighborhood MDA adherence variable are therefore too small and would not have been statistically significant had a neighborhood effect been included. This work would benefit from analyses with larger datasets.”“Given that clinical


The World Health Organization (WHO) recommendation of treating uncomplicated malaria during the second and third trimester of pregnancy with an artemisinin-based combination therapy (ACT) has already been implemented by all sub-Saharan African countries. However, there is limited knowledge on the effect of ACT on pregnancy outcomes, and on newborn and infant’s health.Pregnant women with malaria in four countries  were treated with either artemether–lumefantrine (AL), amodiaquine–artesunate (ASAQ), mefloquine-artesunate (MQAS), or dihydroartemisinin–piperaquine (DHA–PQ); 3127 live new-borns  were followed-up until their first birthday.Prevalence of placental malaria and low birth weight were 28.0% (738/2646) and 16.0% (480/2999), respectively, with no significant differences between treatment arms. No differences in congenital malformations (p = 0.35), perinatal mortality (p = 0.77), neonatal mortality (p = 0.21), and infant mortality (p = 0.96) were found.Outcome of pregnancy and infant survival were similar between treatment arms indicating that any of the four artemisinin-based combinations could be safely used during the second and third trimester of pregnancy without any adverse effect on the baby. Nevertheless, smaller safety differences between artemisinin-based combinations cannot be excluded; country-wide post-marketing surveillance would be very helpful to confirm such findings.Trial registration ClinicalTrials.gov, NCT00852423, Registered on 27 February 2009, https://clinicaltrials.gov/ct2/show/NCT00852423 Plasmodium falciparum infection  while the population-attributable fraction was the percentage of LBW cases that were due to malaria infection .Infant mortality and hospital admissions rates were calculated as number of subjects with the event over the time at risk. The rate was compared between treatment arms using Poisson regression or negative binomial regression model  adjustedA total 3258 out of the 3428 mothers enrolled had a recorded delivery outcome. Maternal characteristics such as gestational age , and maternal malaria infection status at delivery were not significantly different between treatment groups Table . Fifty-tPrevalence of LBW was 16.0% 480/2999), and was significantly more frequent among women with acute and chronic placenta malaria  than in those with past or no infection  (p < 0.01). Nevertheless, LBW (p = 0.54) and mean birth weight were similar between treatment arms . Attributable fraction analysis suggests placenta malaria (acute and chronic) was responsible for 35% of the LBW among women with placental malaria infection (infected attributable fraction). At the prevalence found in this trial, the percentage of all LBW babies due to malaria (population-attributable fraction) was estimated at 13%  polydactyly), the most commonly reported SAEs among infants were infections and infestations  were similar between treatment arms: AL: 90, ASAQ: 104, MQAS: 56, DHAPQ: 85 (p = 0.91)  (p = 0.04) or with a congenital abnormality  were more likely to be admitted in hospital. Infants sleeping under bed nets  (p < 0.01) were less likely to be admitted in hospital than those who were not  of all infant deaths, with no difference between study arms  (p = 0.47). The large majority of neonatal deaths  occurred in the first week of life, at the mean age of 1.3 (SD 1.9) days, with no statistically significant difference between treatment arms  (p = 0.19). Neonatal mortality (early and late) rate was 22.4 deaths per 1000 live births  (p = 0.21); perinatal mortality rate was 21.9 deaths per 1000 total births  (p = 0.77), with no significant difference between the treatment groups . Babies born with birth asphyxia  (p < 0.01) and congenital abnormality  (p < 0.01) had significantly higher risk of dying than other infants (Table The PREGACT study aimed at evaluating the safety and efficacy of four artemisinin-based combinations when administered to pregnant women with malaria . The folMore than two-third of all neonatal deaths occurred in the first week of life. In this age group, malaria is usually an indirect cause of death as it causes LBW which in turn increases the risk of dying in the first year of life , 24. ThiP. falciparum isolates during gestation in Cameroonian pregnant women showed that 77% of placental parasites were acquired from 30 weeks’ gestation onwards [Placenta sequestration of malaria parasites can lead to inflammatory response, particularly in first-time mothers who often have high density infections, and also affect development of the fetal circulation. This decreases the supply of nutrients and oxygen to the fetus, resulting in intrauterine growth retardation and LBW, which is a risk factor for higher infant mortality . More th onwards .Fetal anaemia plays a role in neonatal survival , 39. CorThe longer recall period between the post-partum visits, at 4–6 weeks, and then at 6 and 12 months could have affected the accuracy of information on morbidity. Nevertheless, it is unlikely the study team missed any infant death. Moreover, such recall bias would have equally affected the 4 study arms.In summary, there were no major differences between treatment arms in terms of perinatal, neonatal and infant mortality, nor on the overall occurrence of SAEs in babies, indicating that any of the tested artemisinin-based combinations can be used for the treatment of malaria during the second and third trimester of pregnancy without any adverse effect on the baby. As women were treated only once, repeated treatments may have resulted in a different outcome. It is reassuring that repeated administration of DHAPQ as IPTp did not increase the occurrence of adverse birth outcomes, indicating that at least this ACT could be safely administered 2–3 times during the second and third trimester of pregnancy . Althoug


Malaria is still a public health problem in Africa. Seasonal Malaria Chemoprevention (SMC) is an efficient control strategy recommended by WHO that targets children under five year old living in areas of seasonal malaria transmission. SMC uses the combination amodiaquine (AQ) – sulfadoxine-pyrimethamine (SP). However SP selects rapidly drug resistant parasites. And malaria burden may increase in older children where SMC is implemented. We initiated a pilot study to assess an alternative approach to SMC in older children in Mali.A randomized open-label clinical trial was conducted to test the efficacy and safety of SMC using artesunate – amodiaquine in school aged children in Mali. Two hundred pupils aged 6–15 years old were enrolled and randomized into two arms of 100 each, to receive either artesunate–amodiaquine (ASAQ) monthly or no intervention. Both arms were followed and clinical malaria were diagnosed and treated with arthemeter-lumefanthrine as recommended by Mali National Malaria Control Program. ASAQ was administered 3 days under study team direct observation and during 4 consecutive months starting in October 2013. Follow up was continued until April 2014.Overall, 20 cases of uncomplicated clinical malaria were encountered in the Control arm and three cases in the ASAQ arm, showing a protective efficacy of 85% 95% CI [80.1–89.9] against clinical malaria. Protective efficacy against malaria infection was 69.6% 95% CI [58.6–21.4]. No effect on anemia was observed. ASAQ was well tolerated. Most common solicited adverse events were abdominal pain and headaches of mild intensity in respectively 64% and 44% of children that swallowed ASAQ.peri urban setting in Mali. Its administration at schools is a feasible and accepted strategy to deliver the intervention.ASAQ is effective and well tolerated as SMC targeting older children in a  While in = 171) . The vil2.2Participants were children aged 6 to 15 years attending the fundamental school in Sirakoro-Meguetana. After a screening for eligibility, they were included if they agreed to comply with study follow up procedures, had no clinical signs and symptoms of danger, had no known allergic reaction to amodiaquine and artesunate, and if their parents or legal guardians gave a written informed consent. Exclusion criteria were the presence of a low hemoglobin level (<10 g/dL), a low blood sugar level (<70 mg/dL) or a positive malaria smear at screening, the presence of any concurrent acute illness, the presence of chronic illnesses such as diabetes or hypertension, the intake of any antimalarial drugs within 15 days preceding the screening.2.3The Institutional Ethics Committee from the faculty of Medicine, Pharmacy and Odonto-Stomatology approved the study protocol and informed consent forms, approval letter 2013/76/CE/FMPOS dated on 31 July 2013. Community permission was sought as described  from loc2.4The study drugs are marketed in Mali and freely available. Artemisinin-based combination therapy (ACT), composed of artesunate (AS) and amodiaquine (AQ), so-called ASAQ is one the of first line drug recommended by World Health Organization and the Mali National Malaria Control Program (MNMCP) to treat uncomplicated malaria. Artemether-Lumefantrine, abbreviated as ARLU is the other ACT recommended by MNMCP. These drugs are widely available in Mali and distributed free of charge in the public health care system to children aged under 5 years. ASAQ used in this study was manufactured under GMP conditions by Guilin Pharmaceutical Co., Ltd., and donated solely to the purpose of the study.Children in the study arm under SMC received tablets of ASAQ that they swallowed under direct supervision by study team on study day 0, 1 and 2. After drugs intake, children remained 1 h under study team surveillance. Adverse reactions after drugs intake as well as concomitant medications were recorded. If a child vomited <30 min after his oral intake, a full dose was given. When vomiting occurred after 30 mn and up to 1 h after drug administration, half dose was given. The first dose of ASAQ was given to all eligible children in October 1st, 2013. Subsequent doses were given one month apart; the second dose in November 2013, the third dose in December 2013 and the fourth dose in January 2014.In the control arm, no product was administered. Children were followed as recommended by MNMCP and whenever they had signs or symptoms they were fully examined for diagnosis and treatment. In both arms, clinical episodes of malaria were diagnosed using a Rapid Diagnostic Test (RDT), the SD Bioline Malaria Ag Pf/Pan . Malaria2.5The primary outcome was the efficacy of the intervention. This was determined by comparing the incidence of clinical malaria between the ASAQ arm and the control arm. It was also determined by comparing the incidence of malaria infection and anemia between the two study arms. The secondary outcome was the safety of ASAQ determined as incidence of adverse events after oral intake of ASAQ tablets.2.5.1P. falciparum on malaria smear, in the absence of signs of severity or complications. School teachers were trained to detect children presenting signs or symptoms and to refer them the study team. In addition, children were also informed regularly to self-refer to the study team whenever they would feel ill. Malaria infection was defined as the presence of asexual forms of P. falciparum on malaria smear without associated clinical signs or symptoms. These cases were identified after the end of the follow up period when smears collected at schedule visits were read. Hemoglobin rate was determined with Hemocue® on capillary blood. Moderate anemia was defined as a hemoglobin rate equal or below 10 g/dL and severe anemia was defined as hemoglobin rate equal or below 05 g/dL. A questionnaire asking for use of bed nets impregnated with insecticides and status of others socio-economic variables was used to interview enrolled children at baseline.Clinical malaria was the primary endpoint and was defined as the presence of clinical symptoms consistent with malaria such as fever, headaches, abdominal pain, diarrhea, vomiting, or myalgia, associated with the presence of asexual forms of 2.5.2After each administration of ASAQ, participants were monitored for 1 h, during which, occurrence of solicited adverse events was determined on study days 0, 1 and 3. Solicited adverse events were vomiting, nausea, abdominal pain, fever , headache, anorexia, diarrhea, myalgia, dizziness, and pruritis. All others adverse events were considered as unsolicited adverse events. Adverse events were evaluated for relationship with ASAQ oral intake and graded by intensity into four classes. Adverse events were of: class 1, if they were mild with signs and symptoms easily tolerated; class 2, if they were moderated with intensity high enough to disturb daily activities; class 3 if they were severe, with invalidating intensity impeding daily activities; class 4 if they were life threatening.2.6Sample size was determined based on the primary endpoint, the incidence of clinical malaria. The primary analysis population was the intent-to-treat (ITT) population. Power calculations were based on the inverse sine transform approximation . With a 2.7Participants were randomized in a 1:1 ratio to receive either ASAQ or no intervention. Randomization to study arms was done using a computer-generated randomization list. The randomization list contained sequential codes that linked a study number to an arm assignment. Study numbers were assigned to children in the order in which they were enrolled in the trial. There were no masking procedures of study products assignments.2.8The trial was conducted in compliance with the Bandiagara Malaria Project (BMP) Clinical Trial Quality Assurance Procedures and the MRTC Clinical Laboratory Quality Assurance Plan. The sponsor ensured three monitoring visits of the study. After the pre-study visit, one interim monitoring visit and a close-out visit were done. Data were collected on standard case report forms that were monitored and sent to MRTC Data Management Unit for data entry into a GCP compliant database designed with Microsoft ACCESS 2007. Data was double entered by two independent data entry clerks and then reconciled by a senior data manager. After resolution of discrepancies, the database was frozen and used by the study statistician for analysis.2.9p-values was made for additional analyses.Categorical data are summarized using counts and percentages. For efficacy analysis, incidence of clinical malaria and proportion of malaria infection episodes between study arms were compared using Chi square test and relative risk ratios. Parasite density and anemia were analyzed as binary variables using Chi square test and also as continuous variables using ANOVA after appropriate logarithmic transformation. Survival analysis was done to assess difference in time to first or only clinical malaria episodes between the two arms. The time to first clinical episode of malaria or malaria infection was analyzed using a Kaplan–Meier curve and the Log rank test. All tests were 2-sided, and no correction of 33.1Three hundred fifty-four children were screened and 200 were randomized, 100 in each study arm. Main reasons for exclusions were concurrent illnesses . The mea3.23.2.1−3 per person month in the ASAQ arm and 35.98.10−3 per person month in the control arm. Relative Risk of having a clinical malaria in the control arm when compared to the ASAQ arm was 6.58 CI 95% [1.95–22.15]. Protective efficacy of the SMC ASAQ compared to control was 85% CI 95% [80.1–89.9] against clinical malaria  such as Dihydroartemisinin-Piperaquine (DHA-PQ) have been tested for SMC . DHA-PQ P. falciparum clinical malaria and a reduction of 67% in the prevalence of malaria infection. However no impact on the prevalence of anemia was observed. Several studies have assessed the impact of SMC in school aged children using various drug regimens  in Mali reduce the risk of clinical malaria by 86%, and the prevalence of malaria infection by 67%. ASAQ was a highly effective combination therapy with the potential to replace SP-AQ in case of spread of resistance to one of SP components. The study also showed that SMC delivery at schools with schools teachers as partners is feasible. This is a potential strategic approach to achieve universal coverage in view of malaria elimination in areas of Sahel where malaria transmission is seasonal.None.The study drug ASAQ was donated by Guilin Pharmaceutical Co., Ltd.Centre National de la Recherche Scientifique et Technologique (CNRST), the Malian National Center for Scientific Research and Technology, award number Programme de Recherche CNRST 10. The study was conducted within the African Network for Drugs and Diagnostics (ANDI) Center of Excellence for Clinical Development of Malaria Products.The trial was funded by the The funding source had no role in study design, the collection, analysis and interpretation of data; the writing of the report; and in the decision to submit the article for publication.MAT, AKK, OKD, conceived and designed the study. AKK, BT, ED, SN, BD conducted the study and collected data. MSS analyzed the data. MAT, AKK, MSS, BT and OKD wrote the manuscript. All authors read and approved the final manuscript.


Placental malaria (PM) is an important predictor of infant morbidity and mortality in sub-Saharan Africa. Although placental histology is the gold standard test to diagnose PM, the placenta impression smears remains widely used in epidemiological studies. This study is set to evaluate the performance of placental impression smears to detect PM in pregnant women in southern Benin.Plasmodium falciparum infections have been assessed in placental blood and placental biopsy.A cross-sectional analysis was performed on data collected in the framework a multicenter randomized clinical trial . Samples from 491 pregnant women were examined in the district of Allada, Southern Benin. Placental malaria detected by placenta impression smears and histology were prevalent in 11.4% and 10.8%, respectively. Sensitivity and specificity of placental impression smears were 90.6% and 98.4%. Among 55 pregnant women tested positive by placenta impression smears, 48 were positive by the histology, while 7 were negative . Four hundred and twenty four (424) of the 429 tested negative by the placenta impression smears, were also negative according to histology whereas the rest (5 of 429) of the women were positive .Placenta impression smear is an accurate and easy method for the diagnosis of placental malaria. P. falciparum infection during pregnancy is a serious public health problem in sub-Saharan Africa (SSA). A quarter of women has evidence of placental malaria (PM) at the time of delivery [P. falciparum-infected erythrocytes to syncytiotrophoblast leads to parasite sequestration in the intervillous space. The parasite adheres specifically to chondroitin sulfate A expressed on syncytiotrophoblast [Malaria due to delivery ,2. PM codelivery ,4. It's delivery -8. Durinphoblast . Then, Pphoblast . PM is wphoblast . Malariaphoblast . Placentphoblast , we had Study design: a cross sectional analysis of data collected on four hundred and ninety-one pregnant women between January 2010 and May 2012 has been performed. Pregnant women were followed-up from the first antenatal clinical (ANC1) visit until the time of delivery.Study site, population and procedures: the study site, population and procedure have been described elsewhere [lsewhere ,15. BriePlasmodium falciparum: is the predominant species transmitted (97%). The study population was composed of HIV-negative pregnant women residing in the district of Allada. During the study, socio-demographic , clinical  and biological  data were collected. Two doses of IPTp (1500/75 mg SP per dose or 15 mg/kg MQ per dose) were administered throughout ANC visits. The second dose of IPTp was given at least of 1 month apart from the administration of the first dose. At enrolment, each woman received a LLITN and their use was assessed at each ANC visit. Clinical malaria episodes were treated with oral quinine or artemether-lumefantrine in the first and subsequent trimesters, respectively, for uncomplicated malaria, and with parental quinine for severe malaria. At delivery, women's peripheral blood, cord blood, and placenta (biopsy and impression smears) samples were collected for hematological and parasitological evaluation.Laboratory methods: thick and thin blood films were stained and read for Plasmodium species detection according to standard quality-control procedures [ocedures . Tissue ocedures . Impressocedures ,19.Placental impression smears: a 2.5 x 2.5 cm3 sample from the selected placenta area was cut. The sample included the thickness of tissue from the maternal to the fetal side limited by the fetal membranes. One of the internal faces of sample was carefully put into contact with the slide, on the closest location to the identification area of slide. Then, the placental section was dry with a small piece of filter paper. One of the faces of the dried placental section was put into contact with the slide, on the most distal location to the identification area in the slide. The same procedure was repeated on a second slide.Placental histology: the 2.5 x 2.5 cm3 sample collected for placental impression smears was immediately put in a 50 ml of 10% buffered formalin container. This container was stored in a 4°C fridge until the placental tissue is processed at the department of pathology of the faculty of Medicine of the University of Abomey Calavi. The maximum of fixation was of 5 days. PM was characterized using the classification of Bulmer et al. [r et al. : uninfecEthical considerations: this study was approved by the Ethics Committee of the Faculty of Medicine of Cotonou in Benin. Before each inclusion, all participants involved in the study provided their written informed consent. In case the woman could not read, an impartial witness was involved in the process.Statistical analysis: data were double-entered into Microsoft Access 2003 database and analyzed with Stata 12.0 Software for Windows. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive and negative likelihood ratio (LH ±) were calculated to determine the accuracy diagnosis of placental impression smears vs. placental histology. Sensibility was calculated as true positives / , Sp as true negatives / , PPV as true positives / , NPV as true negatives /  [gatives) .Four hundred and eighty-four placentae have been examined. Women’s mean age was 25.9 years (95% CI: (25.4-26.4). Ninety-one (18.5%) of the women were primigravidae. The mean body mass index (BMI) was 24.4 kg/m² (95% CI: (24.1-24.7). Gestational age at delivery was 39.5 weeks (95% CI: (39.1-39.8) and 7.3% of deliveries were preterm births . Table 2To appreciate the accuracy of placental impression smears to diagnose PM in pregnant women, we compared placental impression smears to placental histology. The prevalence of PM was 11.4% and 10.8% by impression smears and placental histology, respectively. Less than 12% of pregnant women presented a PM. This finding supports similar results reported in a neighborhood area  and in Kenya (11.4%) ,23. Thiset al in Malawi and Anchang-Kimbi et al. in Cameroun reported prevalence rates of 64.6% and 50%, respectively. However, the specificity of 98% that we report is comparable to those reported by Rogerson and Anchang-Kimbi in their respective studies [In this study, the sensitivity of placental impression smear was 90.6%. This performance is much higher than that reported in the literature. Indeed, Rogerson  studies ,28. Some studies , but in  studies . This in studies . Positiv studies . The pla studies ,33. HencPM was relatively common in the study, probably due to the effect of preventives strategies against malaria in pregnancy and appropriate curative treatment during their follow-up. Placental impression smear seems to be a good diagnostic tool to detect the placental malaria infection because of its good sensitivity, specificity, positive and negative predictive values compared to the placental histology. Placental impression smear is easy to perform and less expensive compared to placental histology. Placental impression smear could be considered as a good diagnostic tool to detect PM in low income countries where malaria is endemic. The use of impression smear for the diagnosis of PM in epidemiological studies will allow more characterizing the burden of morbidity and mortality attributable to malaria during pregnancy and defining adequate malaria in pregnancy preventive strategies.Malaria during pregnancy raises a greater diagnostic challenge especially in sub-Saharan Africa;Placental histology is the “gold standard ” for the diagnosis of malaria during pregnancy;Placental histology is relatively expensive, labor intensive and often unavailable.Placenta impression smear is an accurate and easy method for the diagnosis of placental malaria.The authors declare no competing interests.