Plasmodium vivax is the most geographically widespread species among human malaria parasites. Immunopathological studies have shown that platelets are an important component of the host innate immune response against malaria infections. The objectives of this study were to quantify thrombocytopaenia in P. vivax malaria patients and to determine the associated risks of severe thrombocytopaenia in patients with vivax malaria compared to patients with P. falciparum malaria.P. vivax malaria patients was undertaken. Relevant studies in health-related electronic databases were identified and reviewed. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Fifty-eight observational studies (n = 29 664) were included in the current review. Severe thrombocytopaenia (< 50 000/mm3) to very severe thrombocytopaenia (< 20 000/mm3) was observed in 10.1% of patients with P. vivax infection. A meta-analysis of 11 observational studies showed an equal risk of developing severe/very severe thrombocytopaenia between the patients with P. vivax malaria and those with P. falciparum malaria . This indicates that thrombocytopaenia is as equally a common manifestation in P. vivax and P. falciparum malaria patients. One study showed a higher risk of developing very severe thrombocytopaenia in children with severe P. vivax malaria than with severe P. falciparum malaria . However, a pooled analysis of two studies showed an equal risk among adult severe cases . This indicates that the risk of developing thrombocytopaenia in P. vivax malaria can vary with immune status in both children and adults. One study reported higher levels of urea and serum bilirubin in patients with P. vivax malaria and severe thrombocytopaenia compared with patients mild thrombocytopaenia or no thrombocytopaenia, . A pooled analysis of two other studies showed a similar proportion of bleeding episodes with thrombocytopaenia in severe P. vivax patients and severe P. falciparum patients (P = 0.09). This implied that both P. vivax and P. falciparum infections could present with bleeding episodes, if there had been a change in platelet counts in the infected patients. A pooled analysis of another two studies showed an equal risk of mortality with severe thrombocytopaenia in both P. vivax and P. falciparum malaria patients . However, due to the low number of studies with small sample sizes within the subset of studies that provided clinically relevant information, our confidence in the estimates is limited.A systematic review and meta-analysis of the available literature on thrombocytopaenia in P. vivax malaria. To substantiate these findings, there is a need for well designed, large-scale, prospective studies among patients infected with P. vivax. These should include patients from different countries and epidemiological settings with various age and gender groups represented.The current review has provided some evidence of the clinical relevance of severe thrombocytopaenia in The online version of this article (10.1186/s40249-018-0392-9) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium vivax is the most geographically widespread species of human malaria parasites, affecting almost 40% of the world’s population [P. vivax to any diagnostic method may lead to an underestimation of the true prevalence of P. vivax far more than P. falciparum. This means P. vivax parasites could be highly prevalent in many endemic settings [P. vivax malaria cases originate from South East Asia and the Western Pacific, and a significant number also occur in Africa and South America [3) appears to be a very frequent haematological alteration in acute malaria infections [P. falciparum malaria [P. vivax malaria is limited. Since the life cycle and pathophysiology of P. falciparum and P. vivax infections are different, it is important to understand the role of platelets in P. vivax malaria.pulation –3. The i America –4. Immun America , 6. Throfections , 5–7. Sefections . In briefections . Althoug malaria , its cli malaria . HoweverP. vivax infection. These studies varied with respect to geographical location, thrombocytopaenia grading, study design and other variables. Understanding the occurrence and role of thrombocytopaenia in P. vivax malaria may improve clinical practice guidelines. The aim of this study was to undertake a critical analysis of the available literature on thrombocytopaenia in P. vivax malaria. The overall objectives of this study were to quantify thrombocytopaenia in P. vivax malaria patients and to establish the associated relative risks of severe thrombocytopaenia in patients with P. vivax malaria compared to patients with P. falciparum malaria.There has been a recent increase in studies in the literature which have assessed the frequency of thrombocytopaenia in The current review was done, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline  Additio.P. vivax malaria. A literature search in electronic database of MEDLINE (1946 to October 2017), EMBASE (1947 to October 2017), Google Scholar, the Latin American and Caribbean Health Sciences Literature (LILACS) and African Journals Online (AJOL). The following Medical Subject Heading (MeSH) terms were used: “” combined with “(platelets OR platelet counts OR thrombocytopaenia OR thrombocytopenia).” The search strategy was slightly adjusted according to the requirements of different databases. The search was limited to studies published in English until October 2017. We also checked the references of retrieved articles and relevant reviews for any additional studies.One investigator searched for the original studies, addressing thrombocytopaenia in human P. vivax, residing in malaria endemic countries; (ii) were observational studies, (iii) provided data on frequency of thrombocytopaenia in P. vivax infection, compared frequency/risk of thrombocytopaenia between P. vivax and P. falciparum infection or between severe vivax malaria and uncomplicated vivax malaria/healthy controls, and (iv) provided relative risk (RR) or odds ratio (OR) estimates with its 95% confidence interval (CI) (or raw data to compute the estimates) of the association between P. vivax and P. falciparum or between severe vivax malaria and uncomplicated vivax malaria/healthy controls. If more than one study presented data from the same participants, the study with more complete information was used. Studies not meeting the above criteria were excluded. Studies assessing a special group of the population such as imported malaria or pregnant women were also excluded. This is because acquisition of immunity to malaria in people with imported malaria is different from those residing in endemic countries. Also, physiological changes in pregnant women are different from non-pregnant population.Studies were selected if they (i) had ≥10 participants infected with 3), moderate (50–100 000/mm3) [3) and very severe thrombocytopaenia (< 20 000/mm3) [P. vivax malaria and uncomplicated/non-severe P. vivax malaria in the current analysis were as described by the authors in the primary studies [Thrombocytopaenia was further classified into mild (100–150 000/mm000/mm3) , severe 000/mm3) . The pla studies , 46. TheTwo investigators individually screened the titles and abstracts, and then selected full-text articles, according to the inclusion criteria. The two investigators independently extracted information from each included study using a data extraction form prepared for this meta-analysis. This data extraction form had been pre-tested by the investigators on a sample of papers to check its utility, comprehensiveness and ease of use. Information collected was author, year of publication, country, patient characteristics , study characteristics  and the reported clinical outcomes. Disagreements between the two investigators were resolved by consensus.P. vivax malaria, proportion data was extracted from the included studies. Raw cell counts were extracted from the studies and Freeman-Tukey double arcsine transformation and exact CI were employed with the individual studies for that purpose. Meta-analysis of observational studies, which assessed the risk associated with severe thrombocytopaenia in P. vivax malaria, was performed. The odds ratio (OR) of the pooled analysis was used to compare dichotomous data [CI for the pooled analysis were used. When an individual study provided mean value and range, the range was transformed to SD through the formula SD = ((maximum-minimum)/4). If the studies provided median and interquartile ranges, the data was transformed into mean and SD, using a formula developed by Hozo and associates [I2 test was used for the assessment of statistical heterogeneity between studies. A value of I2 > 50% indicated substantial heterogeneity [To assess the pooled proportions of thrombocytopaenia in ous data . For thesociates . The I2 ogeneity . The ranFigure P. vivax  were from a single large study conducted in Indonesia [All studies included in the present analysis used observational designs. Twenty-nine studies (50% of the included 58 studies) were prospective studies. Of them, the vast majority of participants infected with ndonesia . Half ofCI: 17.0–20.2%) of patients with P. vivax malaria had platelet counts < 150 000/mm3. Of these, 10.1%  of the patients infected with P. vivax had either severe thrombocytopaenia (9%) or very severe thrombocytopaenia (13%) , suggesting a high level of homogeneity between studies in each gradient of thrombocytopaenia.Overall, a pooled analysis showed that 18.7%  and those with P. falciparum malaria (5895/ 38 450) had an equal risk of developing severe/very severe thrombocytopaenia,   had a higher risk of developing very severe thrombocytopaenia than in those with severe P. falciparum malaria (13/145)  [P. vivax malaria and those with P. falciparum malaria  [A subgroup analysis of 11 studies  showed patients with P. vivax malaria compared to the healthy controls  . However, one study on severe malaria [P. falciparum malaria than in those with severe P. vivax malaria . This implied that the platelet counts could vary with severity of malaria.A subset of 4 studies , 41, 58  malaria  showed aCI: 9–21%) of P. vivax malaria patients with thrombocytopaenia developed minor bleeding episodes  and with P. falciparum malaria  . This implied that both P. vivax and P. falciparum infections could present with bleeding episodes should there be a change in platelet counts in the infected patients.A pooled analysis of another 4 studies , 59, 63 P. vivax malaria reported parasite density. Of these, one study showed a higher parasite density among patients with severe thrombocytopaenia in P. vivax malaria than in those with either mild thrombocytopaenia or no thrombocytopaenia (P = 0.524) [P. vivax parasite density in patients with thrombocytopaenia and those without thrombocytopaenia. This was indirectly supported by another study with 456 non-PCR confirmed P. vivax, showing no statistically significant relationship between parasite density and thrombocytopaenia (R2 = 0). This implied that parasite load was not a predictor of change in platelet counts in these patients [Three studies with PCR confirmed = 0.524) . The rem= 0.524) , 38 qualpatients .n = 79) compared to those with moderate thrombocytopaenia  (P = 0.002) or the healthy  participants  (P = 0.05) [P. vivax malaria. However, a small study reported that there was no significant difference in Hb levels between P. vivax malaria with severe thrombocytopaenia (n = 10) and those with mild thrombocytopaenia (n = 45) (13.00 ± 3.25 vs 12.90 ± 3.25) (P = 0.9) or no thrombocytopaenia  (n = 35) (13.00 ± 3.25 vs 12.10 ± 3.25) (P = 0.4) [P. vivax malaria reported that there were higher urea levels in patients with severe thrombocytopaenia (n = 10) compared to patients with mild thrombocytopaenia (n = 45) (41.50 ± 10.37 vs 30.00 ± 7.20) (P < 0.001) or no thrombocytopaenia (n = 35) (41.50 ± 10.37 vs 28.00 ± 7.00) (P < 0.001) [n = 10) (1.70 ± 0.42) compared to those with mild thrombocytopaenia (n = 45) (1.30 ± 0.32) (P = 0.001) or no thrombocytopaenia (n = 35) (0.90 ± 0.25) (P < 0.001) [P. vivax infection. A study on 51 children in India had reported that the association of severe P. vivax malaria in children with severe thrombocytopaenia was 2.6 times significantly greater than those with non-severe thrombocytopaenia  [One study showed a significantly lower haemoglobin (Hb) level with severe thrombocytopaenia  . This inP = 0.4) . The sam< 0.001) . Also, t< 0.001) . TherefoP. falciparum malaria  compared to those with P. vivax malaria  (P = 0.004) [One study has reported that anaemia with thrombocytopaenia was more common in patients with = 0.004) .P. vivax malaria patients  and P. falciparum malaria patients    [A pooled analysis of 2 studies with PCR confirmed parasite species , 64 showents 14%,/57 (OR: 3–18.73) .P. vivax malaria patients. The observational design in these studies was appropriate as it quantified the frequency and associated risks in regular clinical practice, reflecting real life situations. The findings suggest that thrombocytopaenia is a common manifestation equally observed in both human P. vivax and P. falciparum infections. A review of studies restricted to Brazil reported that the mortality rate in P. vivax malaria with severe thrombocytopaenia alone was comparable to that of P. falciparum malaria [P. falciparum and P. vivax malaria. Dating back to the era of induced malaria therapy, the fatality rate with the Madagascar strain of P. vivax was 10–15% when used to treat patients with neurosyphilis in the United Kingdom [Based on available data from 58 individual observational studies across 12 endemic countries, the current study has provided some evidence of the increasing risks of developing thrombocytopaenia in  malaria . Our fin Kingdom . Details Kingdom .P. vivax [P. vivax malaria patient with a platelet count 16 000/mm3 [Plasmodium and platelets has been postulated as P. vivax has been seen within the platelets of patients when using electron microscopy [Although the mechanism of thrombocytopaenia in malaria is not fully understood, numerous studies have hypothesied possible patho-immunological mechanisms of thrombocytopaenia in malaria. Oxidative stress , macrophP. vivax  and platP. vivax  are poss 000/mm3 . Moreovecroscopy . It is pcroscopy .P. vivax infected patients had severe to very severe thrombocytopaenia, regardless of confirmatory tests used for P. vivax malaria. Of note, a large-scale study in Indonesia [CI: 4.2–10.8%) amongst outpatients with P. vivax malaria [In our review, we found that 10.1% of ndonesia  had cont malaria . This di malaria , 79, 89  malaria , 78. The malaria .P. vivax malaria were associated with severe thrombocytopaenia. These findings implied that severe anaemia, and impaired liver and renal functions would be expected in patients with severe thrombocytopaenia and P. vivax malaria. This also indicates that MOD may occur in malaria patients with thrombocytopaenia. This is a similar phenomenon to that seen in P. falciparum infections. This provides further evidence to refute the long-held view that P. vivax infection is benign [P. vivax malaria. This collective evidence supports the need for increased efforts to actively control and then eliminate both P. vivax [P. falciparum infections.A limited number of studies included in this review documented that low Hb, and high serum bilirubin and urea levels in s benign , 39, 80.s benign  of P. viP. vivax  and P. fP. vivax malaria patients found in this review highlights the clinical relevance of this parameter in diagnosis and treatment. However, in the absence of (severe) bleeding manifestations in malaria patients, thrombocytopaenia alone is not regarded as a WHO criterion used to define severe malaria [P. vivax malaria and its complications. Although it was not included in the current analysis, a case study in India had reported retinal haemorrhage in a patient of P. vivax malaria who had a platelet count of 40 000/mm3 [This review reported minor bleeding episodes. Thrombocytopaenia, even in severe cases, was not commonly accompanied with severe bleeding , 24 or c malaria . Additio malaria  and Braz malaria  and furt 000/mm3 .The present study has several limitations. Not all studies included in this review used the same methods for platelet ‘labelling’. Hence, misclassification bias could potentially affect the interpretation of the results. For instance, the haemolysis necessary to count platelets in the chamber creates pseudo-platelets , resultiP. vivax malaria was not confirmed by PCR, the current review attempted to minimize this problem by including studies which focused on specific groups in endemic areas where P. vivax is prevalent. This reinforces our confidence in the effect estimates. Nevertheless, the current work focused exclusively on thrombocytopaenia and also reported comparisons between P. vivax and P. falciparum malaria other than solely giving frequencies of each disease thereby yielding more reliable and comprehensive information.We did not undertake segregated analysis of thrombocytopaenia by use or not of PCR confirmed speciation. This is because microscopy by technicians at the malaria clinics in the endemic countries may contribute to the relatively minor possible errors. Moreover, PCR in the studies included in the current review might have excluded errors made by microscopists in the same studies. Although the quality of the studies included was variable if P. vivax in the Western Brazilian Amazon [We were unable to rule out association of co-infections such as intestinal parasitic infections , acute pn Amazon . AnotherP. vivax malaria between children and adults might be due to a difference in immune status between the age groups. For instance, young children exhibit an “antidisease immunity” that affects the risk and extent of morbidity associated with a given parasite density [The manifestations of thrombocytopaenia could also be influenced by context-dependent factors such as the level of malaria endemicity, accessibility to effective malaria treatment and the age-specific and gender-specific host immunity. We have found that a variation in the risk of developing thrombocytopaenia in  density . InadequOR: 31.8, 95% CI: 25.1–39.7, P ≤ 0.001) [P. vivax infections. Since severe thrombocytopaenia may be associated with several infectious diseases such as dengue and leptospirosis, patients with persistent thrombocytopaenia in malaria endemic areas should also be thoroughly investigated for co-infections or other identifiable causes of thrombocytopaenia.Although further substantiation is required, one study reported that patients with mild thrombocytopaenia were about 32 times more likely to have malaria infection than those with normal platelet count (≤ 0.001) . An esti≤ 0.001) , as it iThe association of severe malaria was found to be stronger with severe thrombocytopaenia in children with a specificity of 88.3% and a positive predictive value of 85% . TherefoP. falciparum and P. vivax malaria [Based on the findings, patients with severe thrombocytopaenia in malaria endemic settings and without other definitive causes for the thrombocytopaenia should be treated as severe malaria  and the  malaria . The pla malaria  and addrP. vivax malaria. To substantiate these findings, there is a need for future well designed, large-scale, prospective studies among patients infected with P. vivax from different countries and epidemiological settings with various age and gender groups represented.The current review has provided some evidence of the clinical relevance of severe thrombocytopaenia in Additional file 1:Multilingual abstracts in the six official working languages of the United Nations (PDF 739 kb)Additional file 2:PRISMA checklist (DOC 58 kb)Additional file 3:The excluded studies (DOC 37 kb)Additional file 4:Characteristics of the included studies (DOC 119 kb)Additional file 5:Proportions of mild and moderate thrombocytopaenia in vivax malaria (DOC 62 kb)Additional file 6:Distribution of mean platelet counts (DOC 29 kb)Additional file 7:Proportion of bleeding manifestation in patients with vivax malaria (DOC 32 kb)Additional file 8:Comparison of mortality with severe thrombocytopaenia between severe vivax malaria and severe falciparum malaria (PDF 99 kb)


Malaria is a life-threatening parasitic infection occurring in the endemic areas, primarily in children under the age of five, pregnant women, and patients with human immunodeficiency virus and acquired immunodeficiency syndrome (HIV)/(AIDS) as well as non-immune individuals. The cytoadherence of infected erythrocytes (IEs) to the host endothelial surface receptor is a known factor that contributes to the increased prevalence of severe malaria cases due to the accumulation of IEs, mainly in the brain and other vital organs. Therefore, further study is needed to discover a new potential anti-adhesive drug to treat severe malaria thus reducing its mortality rate. In this review, we discuss how the aptamer technology could be applied in the development of a new adjunct therapy for current malaria treatment. Anopheles mosquitoes. The disease mostly affects children, pregnant women, non-immune persons, and individuals with chronic diseases such as human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS). Malaria causes complications such as severe anemia, metabolic acidosis, and cerebral malaria, often leading to death if not treated within 24 h [Malaria is a life-threatening disease caused by parasites transmitted to humans through the bite of infected hin 24 h ,2.Plasmodium species, namely P. falciparum, P. vivax, P. malariae, P. knowlesi, and two sub-species of Plasmodium ovale  [P. falciparum causes the most severe disease due to higher parasitemia, and it is responsible for the massive burden of global mortality and morbidity [The World Health Organization (WHO) reported there were 438,000 deaths caused by malaria worldwide, especially in the endemic areas such as Africa [llikeri) ,6. Of thorbidity ,8. Despiorbidity . Other forbidity . HoweverP. falciparum, P. vivax and P. malariae in most parts of the world [Several drugs are available for malaria treatment including chloroquine, sulfadoxine/pyrimethamine (SP), and quinine, which are working well in many parts of the world. Unfortunately, there is a grave concern that the malaria parasites have developed a widespread resistance to anti-malarial drugs, especially in the endemic regions ,12. Antihe world . The SP he world ,15. Thouhe world . The arthe world ,18,19,20As the threat of anti-malarial drug resistance grows, there is increasing pressure to develop alternative treatments. Therefore, taking into consideration the ability of the parasites to infect erythrocytes, the development of an anti-adhesive drug as an adjunct therapy to treat severe malaria could be considered. In this review, we shall focus on how the aptamer technology can be explored as a potential anti-adhesive therapy for malaria.Anopheles mosquito injects a sporozoite of P. falciparum into the human host, which invades the host’s hepatocytes. The asexual life cycle of the parasite begins within 8 to 10 days, when merozoites are formed. The merozoites are then released into the bloodstream and rapidly invade normal erythrocytes. During the asexual blood stage, the merozoites develop into the ring-stage, the pigmented-trophozoite stage, and the schizont-stage inside the infected erythrocytes (IEs) within 48 h. The cycle is repeated through the replication and the release of new merozoites to invade other uninfected erythrocytes. Repeated cycles of IEs invasion, replication, and merozoite release result in an exponential growth of the parasite leading to the progression of the disease.Malarial parasites have a complex life cycle involving sexual and asexual reproductive stages. The sexual stage takes place inside the mosquito vector. An infected female P. falciparum erythrocyte membrane protein 1 (PfEMP1). The transported protein on the surface of the red blood cell is known to play an important role in the adhesion mechanism to host endothelial tissues and stimulate immune recognition [Most of the symptoms and clinical complications of malaria are observed during the asexual stage. It starts with a high fever and is associated with “flu-like” symptoms followed by a headache, chills and vomiting . Howeverognition ,24,25. Uognition . Figure Cytoadherence is believed to be one of the factors that cause severe malaria. In cytoadherence, IEs acquire novel adhesive properties which are sequestration , rosseting (interaction of IEs with uninfected erythrocytes), and platelet clumping (interaction of IEs with platelets causing them to bind to other IEs) as shown in CD36 is known as a multiligand scavenger receptor that mediates the binding and uptake of a wide variety of particulate ligands such as oxidized low-density lipoproteins, bacteria, β-amyloid plaque, and apoptotic cells by macrophages ,33. In mIn cerebral malaria (CM), it has been shown that there is an association between IE binding to ICAM-1, although there is no explanation on how the mechanism of IEs cytoadherence in the brain leads to CM ,38. A reSeveral studies have demonstrated that endothelial dysfunction and resulting inflammation preferentially affected the brain, but the mechanism behind it is still unclear . Post-moThe association between the loss of local EPCR and TM with CM are caused by the sequestration of IEs, which disturbs the coagulation and inflammatory mechanism . In an eFurthermore, Lavsteen et al. recently revealed that PfEMP1 domain cassettes 8 and 13 can bind to EPCR endothelial receptor near or at the same region as APC, which can inhibit APC-mediated EPCR-dependent cytoprotective effects on endothelial cells ,45,56. TSince 1990, many studies have been conducted using the RNA molecules that specifically bind to many targets, including small molecules, proteins, enzymes, nucleic acids, protein receptors, viruses, and considerably more targets . Single-SELEX  is a method used to isolate aptamers that specifically bind to a target with high affinity through a repetitive amplification and selection process. Previous studies revealed that the SELEX method has been used extensively to isolate high affinity ligands that bind at picomolar and low nanomolar affinities to a wide variety of proteins and cell surface epitopes . For ins14 to 1016 unique randomized sequences flanked by primer binding regions [In the SELEX technology, there are several crucial steps which must be followed until the aptamer with high specificity and strong affinity to a target can be isolated. (i) The SELEX process starts with a chemical synthesis of a randomized oligonucleotide containing 10 regions ,66. Usua regions . For DNA regions ,70,71. H regions ,69,70.After that, (ii) the oligonucleotide library is incubated with the target molecules to allow the binding to occur. The selection incubation is commonly performed at room temperature or on ice (4 °C). Other conditions depend on the target aptamer assay development. After the incubation, (iii) the aptamer that bound to the target is separated from unbound aptamer. Plentiful methods have been established, from simple methods  to using high-technology instruments , Capillary Electrophoresis (CE) and flow cytometry) to partition the unbound aptamer from target-bound aptamer ,73,74,75Most of the current therapeutic and diagnostic applications use a monoclonal or polyclonal antibody against a specific antigen or protein. Since aptamer technology was discovered in the early 1990s, numerous studies have been conducted to isolate specific aptamers to specific molecules, especially intercellular molecules, which are unreachable by an antibody. Aptamers have more advantages than antibodies in terms of production, stability, binding affinity, small size, and less immunogenicity ,80.Production of monoclonal antibodies (mAb) normally uses a vertebrate organism such as a mouse, goat, or rabbit, and injecting the animals with a specific antigen to produce a specific antibody. Aptamer production does not require organisms for oligonucleotide synthesis . InsteadMoreover, the in vitro production of monoclonal antibodies (mAb) using mammalian cell culture is laborious and very expensive. The process involves a large number of colonies and requires confirmation of antibody activity by immunoassay in each new batch produced, as the same antibodies tend to change in different batches ,79,82. SAs mentioned earlier, an aptamer can reach target molecules which are difficult to reach by antibodies such as those inside tissues or intercellular molecules due to their size (8–25 kDa) compared to the antibody (approximately 250 kDa). The intercellular molecules allow the aptamer to penetrate the tissue more efficiently to reach their target in vivo in comparison to the larger size of the protein antibody.Since aptamers are a single stranded nucleic acid, theoretically they should not be recognised by the human immune system as foreign molecules. However, a study by Eyetech Study Group showed a slight immunogenicity of a VEGF-specific aptamer when given to a monkey at a 1000-fold higher dose than the therapeutic dose . This isFurthermore, the antibody is well known for its irreversible denaturation that can cause it to lose its binding ability to a specific target, as its tertiary structure is lost when exposed to high temperatures. Aptamers are more thermally stable even after 95 °C denaturation ,88. ThusO-methoxy pyrimidine and purine. This modification not only increases nuclease resistance but also allows stronger aptamer binding with the target and increased binding specificity [A limitation of aptamers compared to antibodies is that the RNA aptamers are susceptible to nuclease degradation at the 2′-OH on the ribose sugar of nucleic acid. Nevertheless, it still can be improved through chemical modification by substitution of the 2′-OH group with 2′-fluoro pyrimidine, 2′-amino pyrimidine, or 2′-cificity .S-transferase, and streptavidin for protein purification and surface immobilization purposes [In terms of aptamer generation for anti-adherence of the malaria parasite to the microvascular endothelial surface receptor, the target should be identified. In other words, it is crucial to determine whether a purified recombinant protein or a whole cell surface should be used. Similar to protein-based antibody development, the recombinant protein of a selected surface receptor such as ICAM-1, CD36, and VCAM-1 can be expressed in the prokaryotic or eukaryotic system by tagging them with certain affinity molecules such as histidine, immunoglobulin, glutathione-purposes ,90,91. Hpurposes . The newpurposes .However, a previous study revealed that Cell-SELEX caused a low aptamer enrichment efficiency because a number of proteins or molecules co-expressed on the cells . This miTherefore, a potential anti-cytoadherence could be developed using Cross-Over SELEX by combining the purified recombinant protein  and human umbilical vein endothelial cells (HUVEC). HUVEC is a cell line that expresses small levels of ICAM-1 but is CD36-deficent. However, tumour necrosis factor (TNF) will promote the up-regulation of expression of ICAM-1 ,97. AfteStudy on inhibiting the IEs binding property to isolated aptamers can be performed by incubating the mature stage of Plasmodium IEs culture with immobilized recombinant ICAM-1 protein or HUVEC which previously blocked the isolated aptamer. After incubating and washing steps, the remaining parasite will be calculated and compared with control (untreated with aptamer). Aptamer that shows significant reduction in the number of bound IEs might possess the cytoadherence inhibition property. Study on reversing the IEs binding property can be performed by introducing the aptamer candidate into the assay containing IEs culture bound to immobilized protein or cells. Any significant reduction in the number of bound IEs might indicate that the aptamer has the ability to reverse the IEs binding to the receptor protein.The potential aptamer that is able to inhibit or reverse the binding of IEs from endothelial protein receptor can allow the clearance by the spleen and relieve microvascular obstruction in severe malaria patients . This ad14 unique sequences [P. falciparum FCR3S1.2 [Even though the mechanism on how parasite cytoadherence to microvasculature can result in the severity of the malaria disease is yet to be comprehended, post-mortem studies have shown that the presence of IE in the microvessels is associated with the disease’s pathology . The cytequences . They haFCR3S1.2 ,100.2+) oxidize to form hemozoin (Fe3+) which precipitates the inside of the parasite vacuoles, also known as a malaria pigment [P. falciparum [Current treatment of malaria involves drug usage such as chloroquine, quinine, sulfadoxine/pyrimethamine, and artemisinin to disturb the biological mechanism of the live parasite in the human blood cell from surviving due to the heme toxicity. The drugs prevent the heme from crystallizing to hemozoin through the formation of deprotonated drug, which caps the hemozoin to prevent further crystallization of the heme. This heme molecule is highly toxic to the cell and leads to the dysfunction of the membrane resulting in cell lysis and parasite cell autodigestion. Therefore, to avoid this toxic molecule, the heme molecules  study, Dondrop and colleagues revealed that there is correlation between high parasite burdens in the blood vessel and the severity of malaria where 15% of patients died even after they were given an artesunate treatment . The stuSeveral mAb were discovered to be capable of blocking or inhibiting IE cytoadherence such as mAb OKM5 which can bind to an epitope of the ectodomain region of CD36 to block the cytoadherence of IEs to CD36 . MeanwhiTherefore, discovering a new technology such as aptamer development is vital to inhibit and reverse parasite binding on endothelial cells. Current aptamer development for malaria targets PfEMP1 which is associated with rosette formation and heme metabolism. Unfortunately, an aptamer targeting the host endothelial surface receptors to block or reverse the binding of IEs is yet to be explored. In this review, a number of endothelial receptors were mentioned and could be potentially used for future studies to isolate the aptamer to select the receptor protein, a potential for the malaria anti-adherence.


The World Health Organization has selected Malawi as one of three sites to pilot the roll-out of RTS,S/AS01 in phase 4 trials. As policy discussions for the expanded use of RTS,S/AS01 continue, it will be critical to determine the performance of the vaccine according to seasonal patterns of malaria transmission in regions of Africa. Given waning vaccine efficacy over time, this secondary analysis demonstrates that administering the vaccine to children in the months prior to malaria season could maximize impact of the vaccine. We followed children (5–17 months) and infants (6–12 weeks) assigned to one of three groups: (1) vaccine with four doses; (2) vaccine with three doses; (3) control. The primary endpoint was defined as episodes of clinical malaria. During the 4-years of follow-up, 658 of 1544 (42.6%) children and infants had at least one episode of clinical malaria. With each 1-inch increase in rainfall per month there was an associated increase in the rate of malaria by 12.6%  among children and 15.9%  among infants. There was no evidence of effect modification of vaccine efficacy by precipitation (89% power). Despite substantial financial and developmental resources directed toward the reduction of malaria incidence in Africa, Malawi has not seen a significant decrease in malaria transmission. The entomological inoculation rate (EIR) in Malawi varies depending on the method of sampling and calculation, but previous researchers have estimated a weighted median (25–75 percentile) EIR of 9.1 . This value is similar to Ghana’s weighted median EIR of 7.6  but is much higher than Kenya’s EIR of 0.2 2. With regard to long-lasting insecticidal-treated nets (LLINs), Malawi has seen an increase in the percentage of households with at least one LLIN from 27% in 2002 to 58% in 2012, with over 18 million LLINs distributed in the decade. Still, this rate is far below the Global Fund’s target of 80% of households with at least one LLIN3. Though Malawi’s population is at mesoendemic transmission risk for malaria4, it was one of only two (out of 44) malaria-endemic countries that saw an increase in a population-adjusted Plasmodium falciparum parasite rate among children aged 2–10 years5.Globally, malaria incidence among at-risk populations fell by 37% between 2000 and 2015, with a corresponding 65% reduction in malaria deaths among children under 5 years of age6. The European Medicines Agency has adopted a positive scientific opinion of the vaccine, leading to a recommendation by the WHO’s Strategic Advisory Group of Experts on Immunization (SAGE) and the Malaria Policy Advisory Committee (MPAC) for the implementation of large-scale pilot projects1.The multi-site, phase 3 trial of RTS,S/AS01, which is the most advanced candidate malaria vaccine, is complete. Across 11 study sites in seven countries in Africa, administration of the vaccine in four doses was shown to reduce clinical malaria by 36.3%  in children 5–17 months old and by 25.9%  in infants 6–12 weeks old, although efficacy varied across sites7. The expected four-year program will provide insights on the feasibility of delivering the vaccine in real-life settings, safety in the context of routine use, and impact on childhood survival rates. Results from the MVIP will be useful before full-scale deployment across the continent. To inform these policy discussions, it will be critical to understand the variation in vaccine efficacy over space and time and within different environmental contexts. It is widely understood that RTS,S/AS01 will not serve as a panacea and that the vaccine must be used in conjunction with other control measures, such as LLINs and prompt and appropriate artemisinin combination therapies. However, implementation of the vaccine may be particularly effective in certain environments, such as areas where malaria transmission is high or seasonal8.In April 2017, the WHO announced that Ghana, Kenya, and Malawi had been selected to participate in the Malaria Vaccine Implementation Program (MVIP) of RTS,S/AS01 beginning in 2018. The three countries were chosen for their high coverage of LLINs, a high malaria burden even after LLIN scale-up, promising malaria and immunization programs, and participation in the Phase 3 trial of RTS,S/AS019. Mathematical models have also shown that while absolute vaccine impact would increase with increasing levels of malaria transmission, a higher proportion of averted episodes and high cost-effectiveness may be possible in lower transmission regions10. Precipitation is particularly interesting because greater exposure to infected mosquito bites during the peak rainy season may overwhelm vaccine efficacy. We undertook an analysis of the phase 3 trial of RTS,S/AS01 in Lilongwe, Malawi, to evaluate its efficacy against episodes of clinical Plasmodium falciparum malaria and its effect modification by seasonal variation in precipitation.Site-specific data that considers the seasonality of vaccine efficacy have been limited. Apart from other programmatic considerations, an understanding of the seasonality of transmission and temporal variations in each pilot location is critical in deploying effective pilot interventions. For example, seasonal malaria chemoprevention is optimally targeted at regions with shorter, more intense malaria transmission seasons and requires accurate timing within that season13. The primary endpoint was defined as development of clinical malaria  for both age groups13. Participants were followed passively but parents or legal representatives were told to seek medical care for participants at specified health settings for any illness. Parents or legal representatives of all participants witnessed and provided informed consent at enrollment to the study. Non-literate parents indicated consent by giving a thumbprint in place of a signature. Precipitation data were obtained from the Chitedze Agricultural Center in Lilongwe, Malawi . The trial protocol was approved by both the institutional review board (IRB) at UNC Chapel Hill and by the Malawi National Health Sciences Research Committee.The details of the study design have been described previouslyFrom June 2009 until January 2011, children aged 5–17 months and infants aged 6–12 weeks were recruited in Lilongwe and randomly assigned (1:1:1) by block randomization to one of three groups: (1) a group that received 3 doses of RTS,S/AS01 and a fourth dose 18 months after the third dose (V + 1 group); (2) a group that received the same schedule with a control as fourth dose (V group); (3) a group that received a rabies vaccine  for children 5–17 months of age and a meningococcal serogroup C conjugate vaccine  for infants 6–12 weeks of age (C group).Malaria treatment during the study was done in accordance with Malawi national guidelines and cases were detected by passive surveillance. The Lilongwe vaccine trial catchment area was identified using neighborhood location information from patients who came to the Area 18 Health Center in the previous year. Three different geographic data sources were examined to identify the catchment area. To recruit participants, community-based information sessions were conducted for families with infants and children who met the enrollment age criteria. Flyers were also distributed in the identified communities.Analyses of vaccine efficacy against clinical malaria were explored in an intention-to-treat cohort and included all episodes from the time of enrolment until the date on which the last enrolled infant or child completed follow-up.14. Monthly precipitation was considered in the proportional-hazards model as a time-varying covariate with a time lag of two months. Interaction terms for vaccination status and precipitation were included to assess effect modification. Cumulative risk of malaria was estimated using the Kaplan-Meier method, and log-rank tests were conducted to compare malaria incidence between randomized groups.Vaccine efficacy was defined as the percent reduction in the incidence or hazard rate. Cox proportional-hazards models were used to compare rates of first or only malaria episodes between groups15. In the negative binomial regression model, the logarithm of follow-up time was used as an offset. Waning of vaccine efficacy was assessed in the negative binomial regression by examining efficacy in years one through four of the study follow-up period. The AG model considers time at-risk since study enrolment, assumes a common baseline hazard function for all episodes, and includes a single parameter for the vaccine effect16. The PWP models are stratified proportional hazards models where all children and infants are included in the first stratum, children and infants with at least one malaria episode are included in the second stratum, and so forth. The PWP-TT model considers time at-risk since study enrolment, whereas the PWP-GT model resets the time index to zero after each malaria episode16. PWP models that allow for vaccine effects to vary across malaria episodes were used to estimate vaccine efficacy in each malaria episode , the Prentice Williams Peterson total time (PWP-TT), and the Prentice Williams Peterson gap time (PWP-GT) modelsPower to detect effect modification of vaccine efficacy by precipitation was assessed by a simulation study. R software, version 3.3, was used to conduct the power calculation.The study protocol was approved by the UNC Institutional Review Board and the GlaxoSmithKline Investigational Site at Lilongwe, Malawi. The trial was overseen by a Data Safety Monitoring Board operating under a charter assisted by a Local Safety Monitor. The study was registered with ClinicalTrials.gov, number NCT00866619. The study was first received on March 19, 2009 and last updated on April 24, 2017.The data that supports the findings of this study are available from GlaxoSmithKline but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of GlaxoSmithKline.A total of 1986 participants were screened. Of the 1055 children who were screened, 220 did not meet eligibility criteria, 8 withdrew consent, 9 migrated, and 18 were lost to follow-up; 800 children received the first dose of treatment. Of the 931 infants who were screened, 92 did not meet eligibility criteria, 4 withdrew consent, 7 migrated, and 2 were lost to follow-up; 826 infants received the first dose of treatment. A total of 1544 participants in Lilongwe were in the intention-to-treat cohort and had surveillance data available after the third dose. Among the 760 children, 247 were randomly assigned to the V + 1 group, 263 to the V group, and 250 to the C group. Among the 784 infants, 255 were randomly assigned to the V + 1 group, 257 to the V group, and 272 to the C group.LLIN household use among study participants was higher than in the general population, with LLIN household use among children at 87.8% in the V + 1 group, 82.6% in the V group, and 85.4% in the C group at study month 31. Among infants, LLIN household use was 90.4% in the V + 1 group, 87.1% in the V group, and 90.9% in the C group  children and infants had at least one episode of clinical malaria. Among children, 299 of 760 (39.3%) developed malaria: 81 of 247 (32.8%) in the V + 1 group, 95 of 263 (36.1%) in the V group, 123 of 250 (49.2%) in the C group. Among infants, 359 of 784 (45.8%) developed malaria: 102 of 255 (40.0%) in the V + 1 group, 112 of 257 (43.6%) in the V group, 145 of 272 (53.3%) in the C group (Table .6% childPrecipitation in the rainy season (November through April) averaged 5.4 inches per month from 2009 through 2014 with a peak mean of 8.2 inches per month in January. Over each study year, malaria incidence was lowest during the period from August through October of the dry season Fig. . PrecipiPower to detect effect modification by precipitation was assessed by a simulation study. Two thousand data sets were simulated as follows: The sample sizes for infants and children allocated to the V + 1 group, V group, and C group matched the sample sizes in the trial. Annual loss to follow-up was 15% and maximum follow-up was four years. Incidence of first malaria episode was 0.27 per person-year for infants in the C group when precipitation was 2.75 inches per month . Vaccine efficacy was 65% when precipitation was less than 2.75 inches per month and vaccine efficacy was 25% otherwise. The average number of malaria episodes in the 2,000 simulated data sets was 654 . Effect modification was assessed for each data set by fitting a Cox model and testing for an interaction between vaccination and precipitation. The empirical power to detect effect modification was 89%.Multiple malaria episodes were common. From study initiation until study end, there were 143 episodes of clinical malaria that met the primary case definition in children in the V + 1 group, 219 episodes in the V group, and 300 episodes in the C group. In the infant group, there were 263 episodes in the V + 1 group, 304 episodes in the V group, and 420 episodes in the C group  in the V + 1 group and 29.0%  in the V group compared to the C group. Among infants, vaccine efficacies against multiple episodes were 31.2%  in the V + 1 group and 20.9%  in the V group. By the Andersen-Gill model, among children, efficacies against multiple episodes were 49.7%  in the V + 1 group and 29.1%  in the V group compared to the C group. Among infants, vaccine efficacies against multiple episodes were 31.1%  in the V + 1 group and 27.5%  in the V group. By the PWP-TT model with common effects, among children, efficacies against multiple episodes were 33.1%  in the V + 1 group and 14.3%  in the V group. Among infants, vaccine efficacies against multiple episodes were 13.9%  in the V + 1 group and 8.4%  in the V group. By the PWP-GT model with common effects, among children, efficacies against multiple episodes were 36.2%  in the V + 1 group and 17.1%  in the V group. Among infants, efficacies against multiple episodes were 17.0%  in the V + 1 group and 11.8%  in the V group , PWP-TT model , and PWP-GT model . Neither did vaccine efficacy change for infants during the rainy season, as indicated by the non-significant interaction between treatment group and precipitation in the AG model , PWP-TT model , and PWP-GT model .Waning of vaccine efficacy was assessed by estimating efficacy separately for each year of study follow-up. Among children, vaccine efficacy was 70.1%  in the V + 1 group and 55.1%  were still protected with a vaccine efficacy of 42.1% , but those who did not (V group) were no longer protected, with a vaccine efficacy of 25.9% . Among infants, vaccine efficacy declined from 63.0%  in the V + 1 group and 41.2%  in the first year to 17.5%  in the V + 1 group and 11.0%  in the V group in the fourth year. By the third year, as in the case of children, infants in the V + 1 group were protected, with a vaccine efficacy of 37.0%  while infants in the V group were no longer protected, with a vaccine efficacy of 8.5%  . This time lag is consistent with previous time series analyses in a holo-endemic area of Ghana and across the East African highlands, which showed that high-resolution precipitation data could directly predict malaria incidence among children less than 15 years of age21. This period of time also closely matches the theoretical vector-parasite-host cycle, which includes the growth of the Anopheles mosquito from egg to reproductive age, the development of the Plasmodium falciparum parasite in the vector from gametocytes to sporozoites, and the incubation period in the human host from infection to onset of malaria symptoms22.Precipitation was significantly associated with increased malaria incidence after a time lag of two months. This finding suggests that there is a strong, direct association between rainfall and malaria incidence. The intensity of transmission is dependent on the density, biting, and survival rates of the mosquito vector, which are heavily influenced by seasonal temperature and precipitation patterns6. This observed difference may be explained in part by differences in naturally acquired immunity and physiological effects between the two age groups23. Due to the lower efficacy rates observed among infants, the WHO Strategic Advisory Group of Experts on Immunization and the Malaria Policy Advisory Committee jointly decided that it would not recommend RTS,S vaccination among this younger age group24. However, it is likely that continued reductions in malaria exposure will lead to an increased burden among children over five-years old. In a comparison of four mathematical models, enhanced prevention measures and RTS,S vaccination was predicted to cause such an age shift in malaria incidence, disproportionately affecting children of older ages10. Pilot implementations should assess the magnitude of the predicted age shift and its impact on severe disease and mortality. Future studies would be needed to establish safety and immunogenicity of the vaccine against older age groups, including adults.Age played an important role in the number of clinical malaria episodes observed, with infants significantly more likely to experience multiple episodes of malaria. Consistent with findings from the phase 3 trial, vaccine efficacies were higher among children compared to infants in the negative binomial model, AG model, and PWP models15. The negative binomial model is a reasonable choice for estimating an overall effect in disease reduction with time as an offset26, but the added value of the PWP models is the ability to accommodate time-dependent variables like precipitation and a more efficient use of data because timing of malaria episodes is considered16. The PWP models are especially useful when the effects of covariates such as vaccination and precipitation are different in subsequent episodes, which is likely the case because RTS,S efficacy has been shown to wane over time16. However, the PWP models also have limitations. For example, the conditional nature of the models leads to very small, nonrandom risk sets for later episodes16, making vaccine efficacy estimates for subsequent episodes imprecise  indicated lower vaccine efficacies compared to the negative binomial and Andersen-Gill models Table . This obIn conclusion, precipitation was significantly associated with increased malaria incidence among children and infants, with a surge in new cases following a time lag of two months. In assessing rates of first malaria episode or all malaria episodes, there was no evidence of effect modification of vaccine efficacy by precipitation. These findings suggest that despite increased malaria incidence in the rainy season, efficacy of RTS,S/AS01 does not change based on time of year. Thus considering waning vaccine efficacy, vaccination prior to peak transmission seasons is likely to maximize impact.Supplementary Tables and Code


Plasmodium. Complex inter-relationships between Mycoplasma species and Plasmodium parasites have been previously noted in vitro. This is the first report of Plasmodium falciparum and Mycoplasma pneumoniae co-infection in a human host presenting with cerebral malaria manifesting orofacial dyskinesias and haemophagocytic lymphohistiocytosis.Malaria is a mosquito-borne infectious disease with diverse clinical manifestations caused by a parasitic protozoan of the genus Mycoplasma infection. He subsequently developed encephalopathy with orofacial dyskinesia. A diagnosis of severe P. falciparum infection with significant parasitaemia was established. Peripheral blood cytopaenia occurred due to haemophagocytic lymphohistiocytosis in the bone marrow. Complete clinical and haematological recovery was achieved with intravenous artesunate.A 55-year-old Sri Lankan man with a recent visit to South Africa presented with an acute febrile illness, cough and worsening dyspnoea with alveolar-interstitial infiltrates on chest radiography. Serological evaluation confirmed a diagnosis of Plasmodium falciparum and Mycoplasma pneumoniae co-infection occurring in vivo manifests clinical features that are plausibly a result of the interaction between the two microorganisms. This is the first report of orofacial dyskinesia in either infection.The online version of this article (doi:10.1186/s12936-016-1517-x) contains supplementary material, which is available to authorized users. Plasmodium, of which five species are known to cause human disease; it is transmitted by Anopheles spp. mosquitoes. The clinical manifestations range from uncomplicated infection to severe multisystem affliction with significant mortality. Plasmodium falciparum infections have a high predilection of multisystem manifestations. This case report highlights the unusual combination of Mycoplasma and P. falciparum co-infection presenting with cerebral malaria manifesting orofacial dyskinesias and haemophagocytic lymphohistiocytosis.Malaria is caused by protozoa of the genus A 55-year-old Sri Lankan man employed as a chef in Dubai, United Arab Emirates, presented to the National Hospital of Sri Lanka with fever for five days and associated headache, arthralgia, myalgia and severe prostration approximately 3 weeks after spending 2 months in South Africa as part of his employment. He had been previously well and had not been on chemoprophylaxis for malaria while in South Africa. He has had a worsening non-productive cough and shortness of breath 1 day prior to presentation. On examination, he was pale and had posterior cervical lymphadenopathy. His respiratory rate was 30 cycles/min and he had bilateral diffuse scattered crepitations and rhonchi in both lung bases. Rest of the systems examination was normal.3) and the chest radiograph showed alveolar-interstitial shadowing. The patient was empirically treated with clarithromycin for 7 days, which resulted in the resolution of the chest symptoms and signs, but with poor resolution of fever. His serum Mycoplasma IgM ELISA (Sigma Aldrich) antibody titre was strongly positive at a dilution of 1/1260 with a repeat titre noted at 1/7560, four weeks after the initial presentation. Given the recent travel history, a blood film was examined for evidence of malaria, but was noted to be negative. Despite initial improvement, progressive thrombocytopenia and worsening anaemia were noted from day 7 of his illness. By day 10, his level of consciousness deteriorated demonstrating abnormal behaviour and worsening drowsiness. Frequent, stereotyped, intermittent orofacial dyskinetic movements, not attributable to any of the drugs administered, were noted . Although severe malaria  malaria , 8. AlthP. falciparum and Mycoplasma pneumoniae co-infection manifesting clinical features that are plausibly a result of the interaction between the two microorganisms. It is also the first report of orofacial dyskinesia in either disease.This is the first report of human


P. falciparum infection. One hundred and twenty healthy male volunteers, living in a malaria endemic area of Senegal were randomised to receive either the Chimpanzee adenovirus (ChAd63) ME-TRAP as prime vaccination, followed eight weeks later by modified vaccinia Ankara (MVA) also encoding ME-TRAP as booster, or two doses of anti-rabies vaccine as a comparator. Prior to follow-up, antimalarials were administered to clear parasitaemia and then participants were monitored by PCR for malaria infection for eight weeks. The primary endpoint was time-to-infection with P. falciparum malaria, determined by two consecutive positive PCR results. Secondary endpoints included adverse event reporting, measures of cellular and humoral immunogenicity and a meta-analysis of combined vaccine efficacy with the parallel study in Kenyan adults.We show that this pre-erythrocytic malaria vaccine is safe and induces significant immunogenicity, with a peak T-cell response at seven days after boosting of 932 Spot Forming Cells (SFC)/106 Peripheral Blood Mononuclear Cells(PBMC) compared to 57 SFC/ 106 PBMCs in the control group. However, a vaccine efficacy was not observed: 12 of 57 ME-TRAP vaccinees became PCR positive during the intensive monitoring period as compared to 13 of the 58 controls (P = 0.80). This trial confirms that vaccine efficacy against malaria infection in adults may be rapidly assessed using this efficient and cost-effective clinical trial design. Further efficacy evaluation of this vectored candidate vaccine approach in other malaria transmission settings and age-de-escalation into the main target age groups for a malaria vaccine is in progress.Malaria transmission is in decline in some parts of Africa, partly due to the scaling up of control measures. If the goal of elimination is to be achieved, additional control measures including an effective and durable vaccine will be required. Studies utilising the prime-boost approach to deliver viral vectors encoding the pre-erythrocytic antigen ME-TRAP (multiple epitope thrombospondin-related adhesion protein) have shown promising safety, immunogenicity and efficacy in sporozoite challenge studies. More recently, a study in Kenyan adults, similar to that reported here, showed substantial efficacy against  Malaria transmission has been on the decline in many parts of Africa in association with scaling up of effective control measures –3. ElimiP. falciparum malaria antigens [P. falciparum pre-erythrocytic antigen ME-TRAP (multiple epitope string thrombospondin-related adhesion protein) is one of the most advanced malaria vaccine candidates, capable of inducing sterile protection in 21% of malaria naïve adults following controlled human malaria infection (CHMI)[The pre-erythocytic malaria vaccine RTS, S targeting the circumsporozoite protein, is the most advanced vaccine in clinical development. In the recently published Phase III clinical trial in over 15,000 infants and children in several African countries, the efficacy of RTS, S, with AS01 as adjuvant, in young children was about 50% during the first year –11, but antigens –15. ChAdon (CHMI).Recently in Sukuta, The Gambia and Kilifi, Kenya, two phase Ib dose-escalation clinical trials were undertaken to assess the safety and immunogenicity of this approach in 46 healthy malaria-exposed adults as previously, immunogenicity had only been described in predominantly Caucasian, malaria-naïve adults. These two studies showed encouraging results, with both vaccines shown to be safe and well tolerated, with high-level T cell responses induced  vaccine and boosting with MVA ME-TRAP (2 x 108 pfu), 8 weeks apart in healthy adult volunteers in Senegal.Using malaria infection detected by PCR as an endpoint for efficacy, we assessed the safety, immunogenicity and efficacy of priming with the same vaccines and doses, ChAd63 ME-TRAP ; http://www.pactr.org2. Its population is estimated at 452,168 inhabitants and the average annual rainfall amounts to approximately 600 mm. Over the past few years, malaria transmission has reached record levels in all 5 municipal districts within the Départment, namely Médina Gounas, Whakhinane Nimzath, Golf Sud, Sam Notaire and Ndiareme Limamoulaye. There are several reasons that account for the resurgence of malaria in the Dakar suburbs: (i) a high population density, (ii) cyclic flooding in lowland areas since the year 2000, (iii) the lack of a drainage system, and (iv) the existence of several wetland areas The study was conducted in the peri-urban area of Dakar in Senegal, West Africa, in one of the Université Cheikh Anta Diop’s (UCAD) sites located in the Roi Baudoin Hospital in Guédiawaye from June 2012 to March 2013. Guédiawaye is a small town situated on the coastal shoreline in the Dakar region, bordered to the East and the South by the town of Pikine and to the West by the city of Dakar, specifically by the municipal district of Parcelles Assainies [Conseil National d’Ethique pour la Recherche en Santé” (CNERS) of Senegal as well as the “Direction de la Pharmacie et des Médicaments” (DPM), the Senegalese regulatory authority, and Oxford Tropical Research Ethics Committee (OXTREC). An independent Data Safety Monitoring Board (DSMB) provided oversight for the study. An on-site Local Safety Monitor (LSM) acted as liaison between the site and the DSMB. The study was conducted in accordance with the Helsinki Declaration of 1964 (revised in 1996) and in accordance with Good Clinical Practice . The University of Oxford acted as study sponsor. The trial was registered at the Pan-African Clinical Trial Registry (Ref No: PACTR 201-303-000-499-409); http://www.pactr.org/ and ClinicalTrials.gov (Ref No: NCT01658696); http://clinicaltrials.gov.The study was reviewed and approved by the “5 vp) as prime vaccination, followed eight weeks later by MVA ME-TRAP (2x108 pfu) as booster or two doses of anti-rabies vaccine (0.5ml) as comparator at the same interval. All vaccines were administered intramuscularly.We conducted a randomised, controlled, single-blinded phase IIb efficacy trial. An independent statistician based at the University of Oxford provided sealed randomisation envelopes to the study site. Participants, fieldworker and laboratory analysts were blinded to study allocation of participants and remained blinded until after analysis was completed. The recombinant vectors have been previously described in detail . EligiblHealthy men aged 18–50 years old were screened and 120 eligible volunteers were selected to participate in this trial. Prior to the commencement of the study, local community sensitisation  meetings were held to introduce to members of the community the purpose and nature of the clinical trial. Screening of eligible volunteers commenced after signed informed consent was obtained. Volunteers enrolled into the study were required to be in good health, within the age range, and residing in the study area for the duration of the study. Exclusion of volunteers occurred if there was any physical, haematological or biochemical evidence of ill health. In order to maintain confidentiality, anonymised HIV test were conducted using the standard rapid diagnostic kits and testing algorithm used by the Senegalese Ministry of Health and Prevention. A trained counsellor provided pre-test counseling. Eligible volunteers who participated in the trial met all of the inclusion criteria and none of the exclusion criteria.Volunteers were observed for at least 30 minutes after each vaccination. A number of volunteers were assigned to each of the 12 field workers who visited them at home on day one post-vaccination and were assessed in the clinic on day 14 and 7 after the first and second vaccinations respectively. The local solicited adverse events (AEs) were; pain, discoloration, swelling, warmth, itch, scaling or blistering at the injection site. Systemic solicited AEs were; headache, malaise, arthralgia, reported fever, nausea, myalgia and documented fever (temperature ≥37.5°C). Using pre-determined criteria, the intensity of AEs was graded as mild, moderate or severe. Serious adverse events (SAEs) were recorded during the whole study period. Laboratory abnormalities, recorded as adverse events were assessed by full blood count, creatinine and alanine aminotransferase.P. falciparum infections.We administered a three-day course of directly observed atovaquone/proguanil (250mg/100mg) and artesunate (250mg) 7 days after the final vaccination (Days 63–65) in order to clear parasitaemia prior to PCR monitoring for new ex-vivo interferon-gamma (IFNγ) ELISpot. The immunogenicity was determined at D0, D14 (2 weeks post vaccination with ChAd63 ME-TRAP), D63 (7 days post vaccination with MVA-ME-TRAP) and D161. While ME-TRAP ELISpots were performed on samples from all volunteers at D63 (the peak time-point for vaccine-induced cellular immunity), due to the high cost and staff requirement of the immunology analysis, we did a second randomization to allocate measurement of ELISpot responses at D0, D14 and D161 to 30 volunteers only. Fresh PBMCs were isolated from 40 ml of heparinized blood using Lymphoprep (Axis-Shield Diagnostics Ltd). ELISA and ELISpot assays were performed as previously described [Immunogenicity to the TRAP antigen was assessed by IgG ELISA and escribed . An AID Neutralising antibodies to the ChAd63 vector were assayed as previously described , on a seEfficacy was assessed by time to detection of malaria parasites in blood by quantitative PCR (qPCR) assays. Volunteers were followed up initially for 4 weeks by three times weekly blood sampling (0.5mls) for qPCR monitoring. Subsequently blood sampling was done once weekly for a further 4 weeks. Prior to the onset of the qPCR monitoring period, drug treatment was administered to volunteers to clear parasitaemia. Follow-up began 2 weeks after the second and final vaccination and 7 days after drug treatment. Volunteers diagnosed with malaria infection during follow-up were treated according to national guidelines.qPCR monitoring and assays were conducted as previously described ,18,19,22The DSMB, sponsor and investigators agreed an analysis plan before unblinding. The primary efficacy analysis was the hazard ratio for vaccination as determined by Cox regression model for first PCR positive blood sample post-vaccination (1972. Regression models and life tables. J. R. Stat. Soc. 34: 187–220). The secondary efficacy analysis was the hazard ratio for vaccination, again by Cox regression for first PCR positive blood sample above a threshold of 10 parasites per ml. The time at risk was considered to begin one week after the final vaccination with MVA ME-TRAP. All analyses are presented unadjusted and then adjusted for potential confounders (age and bednet use). Safety data is presented for the ITT cohort , and immunogenicity and efficacy data is presented for the ATP cohort (i.e. excluding 3 subjects who did not complete their vaccination course).Analysis was done using Stata version 11 .Differences between immunological responses were assessed by Kruskal Wallis tests.We expected about 70% of adults to be PCR positive during follow up, thus providing 90% power to detect an efficacy of 50% and 80% power to detect an efficacy of 40% with 100 volunteers. The sample size was increased to 120 to allow for loss to follow up.One hundred and seventy one people were screened prior to randomisation and enrollment of 120 participants. The most common reasons for exclusion were positive hepatitis B (HBsAg) serology (n = 21), isolated hematology and or biochemistry abnormalities (n = 16) and abnormalities at the clinical examination (n = 13). One participant travelled to outside the study area before treatment allocation and was loss to follow up. No positivity to HIV was found. The participant flow is summarised in No serious adverse event (SAE) related to vaccination was reported during the study period. One SAE was observed in the control group. The volunteer reported to the outpatient ward with fever, headache, chills, articulations pains, asthenia and dizziness and typhoid fever was diagnosed following examination and investigation. The volunteer was withdrawn from the study on September 2012.Pain, itching, redness, swelling and warmth were the most common local adverse events associated with the administration of the ChAd63 ME-TRAP vaccine With regard to malaria incidence, 5 cases of clinical malaria occurred before the boost vaccination mostly in the malaria vaccine arm and 6 clinical malaria episodes after the boost vaccination, 4 of which were in the control group. A very low number of clinical malaria cases were recorded probably due to the unusually low malaria transmission in the year of the study. In previous years, higher seasonal transmission had been observed .10 geometric mean titre (LGMT) 1.76 CI (1.5–2.0) and boosting with MVA in the ME-TRAP vaccinated group  165–412) compared with 48 SFC (CI 30–79 SFC) among control vaccinees , with 56% of responses above the clinically relevant threshold of 200. In contrast, in a smaller cohort of UK adults [Kaplan Meier plots of the distribution of time to PCR positivity are shown in Although the study in Senegal was underpowered to detect vaccine efficacy due to the unexpectedly low number of infections during the follow-up period, a protocol-specified meta-analysis after pooling the data of the Kenyan and Senegalese trials showed significant protective efficacy of 50%  in both groups, with baseline responses comparable to those observed in previous studies in Kenyan, Gambian and UK adults.  As expecThe surprising difference in immunogenicity found between Kenya and Senegal may partially be explained by several reasons including the lower immunogenicity at baseline in Senegal (60 SFC in Guédiawaye compared with 112 SFC in Kilifi) The ChAd63 ME-TRAP priming in Kilifi may have acted as a booster dose to naturally acquired immunity. The absence of malaria exposure in the first weeks of follow up in Senegal may have pushed the rate ratio towards 1 and obscured any genuine protective efficacy. In other words, with the absence of malaria risk in both study groups in the first two weeks of the trial, the rate ratio at day 14 was still 1 and vaccine efficacy (1-RR) = 0. This period of zero vaccine efficacy pulls down the overall efficacy for all eight weeks. Malaria transmission is nearly perennial in Kenya with two different transmission seasons, while the transmission pattern in Senegal is very different. There is a long dry  season of about 9 months followed by a wet season of 3 months. Malaria is actually transmitted only during part of the wet season. Transmission is therefore intense and short. Outside this transmission window, there may be a total absence of exposure.in vitro vector-neutralizing immunity [Among 205 adults from another Senegalese population , serum samples tested for anti-ChAd63 neutralizing antibodies indicated that all had previous exposure to ChAd63 or a related adenovirus. Such results have been observed in other populations  and are immunity –30 make Vaccine efficacy against the primary endpoint (parasitaemia>0) was significantly different than that observed in the parallel study in Kenya where a vaccine efficacy of 67% was observed. A pre-specified pooled analysis of the data from both studies using Cox regression showed an overall vaccine efficacy in the two populations of 50%  for time to first PCR positivity.Although the unadjusted confidence intervals of the efficacy estimates in the two sites were overlapping, there was a significant interaction between the effects of site and vaccination (p = 0.009) but not with time since vaccination (p = 0.13). This provides statistical evidence from pooled Senegalese and Kenya datasets that there was variation in efficacy between the two studies rather than variation in efficacy over time since vaccination.Several parasitological differences between the two sites that may be relevant to the apparent different vaccine efficacy are noteworthy:PCR positivity was more prevalent before monitoring commenced in Kenya compared to Senegal;After antimalarial treatment, the incidence of PCR positivity was higher in Kenya yielding more power to detect efficacy than in the Senegalese study.The PCR data showed higher density parasitaemia in the trial in Senegal compared with the one in Kilifi, Kenya, representing a potentially more stringent challenge for the vaccine in the presence of less background malaria immunity. However, this likely reflects lower malaria transmission in the Senegalese population where the study was done, so that bites are less frequent and PCR positivity is less frequent; but when an infection occurs because prior exposure has been lower there is less blood stage immunity and therefore a higher peak parasitaemia is reached. The PCRs became positive in Kilifi only for a two-week period after anti-malarial treatment with no positives appearing later. In our Senegal study in contrast, the emergence of infections was evenly distributed throughout the follow-up period so that if vaccine efficacy falls very quickly lower efficacy could be seen in Senegal. However, the lack of significant interaction between site and time since vaccination in the combined model  does not support this possibility.It should be noted that in both sites examples of brief PCR positivity, without sustained parasite growth was observed, likely reflecting blood-stage immunity. Microscopy along with the qPCR was not performed after Atovaquone/Proguanil treatment.An alternative explanation for the lack of vaccine efficacy in Senegal simply relates to lower vaccine immunogenicity. In UK CHMI studies a level of > 2000 SFU / million PBMCs was induced consistently by the ChAd-MVA regime –21 and tIn addition to enhancing cellular immune responses to TRAP, vaccine efficacy might potentially be improved by including additional liver-stage antigens, such as PfLSA1 and PfLSAP2. These antigens have recently been shown to induce sterile efficacy against transgenic parasites in a murine model of malaria and are progressing to clinical evaluation . AnotherOverall, the results of safety, immunogenicity and efficacy of the ChAd63-MVA vaccination strategy in Kenyan and Senegalese young men gave encouraging results and provide, to our knowledge, the first indication of potential field efficacy of any vaccine designed to induce protective cellular immunity. It is now important that protective efficacy be assessed in younger populations, especially young children and infants, and these trials are underway.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.


Seasonal malaria chemoprevention (SMC) is a new strategy recommended by WHO in areas of highly seasonal transmission in March 2012. Although randomized controlled trials (RCTs) have shown SMC to be highly effective, evidence and experience from routine implementation of SMC are limited.A non-randomized pragmatic trial with pre-post design was used, with one intervention district (Kita), where four rounds of SMC with sulfadoxine + amodiaquine (SP + AQ) took place in August–November 2014, and one comparison district (Bafoulabe). The primary aims were to evaluate SMC coverage and reductions in prevalence of malaria and anaemia when SMC is delivered through routine programmes using existing community health workers. Children aged 3–59 months from 15 selected localities per district, sampled with probability proportional to size, were surveyed and blood samples collected for malaria blood smears, haemoglobin (Hb) measurement, and molecular markers of drug resistance in two cross-sectional surveys, one before SMC (July 2014) and one after SMC (December 2014). Difference-in-differences regression models were used to assess and compare changes in malaria and anaemia in the intervention and comparison districts. Adherence and tolerability of SMC were assessed by cross-sectional surveys 4–7 days after each SMC round. Coverage of SMC was assessed in the post-SMC survey.During round 1, 84% of targeted children received at least the first SMC dose, but coverage declined to 67% by round 4. Across the four treatment rounds, 54% of children received four complete SMC courses. Prevalence of parasitaemia was similar in intervention and comparison districts prior to SMC  as was the prevalence of malaria illness . After SMC, parasitaemia prevalence fell to 18% in the intervention district and increased to 46% in the comparison district . Prevalence of malaria illness fell to a greater degree in the intervention district versus the comparison district  and the same for moderate anaemia (Hb < 8 g/dL) . The frequency of the quintuple mutation  remained low (5%) before and after intervention in both districts.Routine implementation of SMC in Mali substantially reduced malaria and anaemia, with reductions of similar magnitude to those seen in previous RCTs. Improving coverage could further strengthen SMC impact.Trial registration clinical trial registration number NCT02894294 Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa. The region is disproportionally affected by malaria, with 88% of the estimated 214 million malaria cases and 90% of 438,000 malaria deaths worldwide occurring in this region in 2015, according to recent World Health Organization (WHO) estimates. More than two-thirds (70%) of all malaria deaths occur in children under 5 years of age [Seasonal malaria chemoprevention (SMC) is a new strategy for malaria control recommended by WHO in March 2012 for sub-Saharan countries in the Sahel region. It involves the administration of therapeutic doses of anti-malarials [amodiaquine (AQ) + sulfadoxine–pyrimethamine (SP)] at monthly intervals during the high malaria transmission season. Specifically, the WHO recommends SMC at monthly intervals for up to 4 months per season for children aged 3–59 months living in highly seasonal transmission areas in the Sahel in West Africa where a high level of resistance to AQ or SP has not been recorded [The Mali National Malaria Control Programme adopted SMC as policy in 2012 with a plan to expand implementation to all districts by 2016. Clinical trials conducted in Mali and Burkina Faso have shown a strong protective effect of SMC , 6. In MCurrent evidence on SMC comes mainly from randomized control trials. Recent modelling studies –10 indicSeasonal malaria chemoprevention was implemented in Kita district in 2014. Kita is located approximately 180 km north of the capital Bamako in the western Kayes Region. The health infrastructure includes a district hospital and 47 community health centres providing primary health, including curative, preventive, social, and promotional services for a catchment area of up 15 km, usually composed of several villages or quartiers. At the village level, community health centres are supported by community health workers who are members of the community chosen by community and who have received shorter training than professional workers to provide basic mother and child health services. In 2014, the district population was estimated at 516,649 inhabitants with about 77,497 children aged 3–59 months in 336 villages/quartiers. Bafoulabe, a neighbouring district in the same Kayes Region, with highly seasonal rainfall and malaria transmission similar to Kita, was chosen as the comparison district because it is adjacent to Kita, with similar geographic, social and demographic characteristics. In 2014 the rainfall was about 854 mm in the area of Bafoulabe and 839 mm in Kita. SMC was not implemented in Bafoulabe in 2014 due to funding constraints. Bafoulabe has a district hospital and 24 community health centres; in 2014 the population was estimated at 152,976 inhabitants with 22,946 children aged 3–59 months in 336 villages/quartiers. In 2013, parasitaemia prevalence among children ages 6–59 months in the Kayes Region, where Bafoulabe and Kita are located, was 36.9% according to the recent Demographic and Health Survey . Both diA non-randomized, pre-post design was used, with an intervention district (Kita) where SMC was implemented through the health system, and a comparison district (Bafoulabe) where SMC was not implemented. SMC implementation consisted of the administration of SP + AQ at monthly intervals in children 3–59 months in August, September, October, and November 2014. During each round, children aged 3–11 months received 75 mg of AQ given once daily for 3 days plus a single dose of 250/12.5 mg of SP, while children aged 12–59 months received 150 mg AQ base given once daily for 3 days and a single dose of 500/25 mg of SP. The single dose of SP was given only on the first day, at the same time as the first dose of AQ. Tablets were crushed into water and sugar added and administered under direct observation by the health care worker. Children were observed for 30 min after drug administration, and drugs were re-administered if vomiting occurred during this period. The first day of treatment (SP + AQ) was administrated by health workers at fixed points in the village . The doses of AQ for the second and third days were given to parents to be administrated at home. The administration of the first day’s treatment was recorded on a special SMC card that was given to all children receiving SMC during the first round. The SMC implementation in Kita was carried out by health staff in the district, including 588 heath workers  organized into 133 teams of 2–6 health workers each; 37 teams were based in health centres and 96 teams were deployed to fixed distribution sites in surrounding villages. The same locations were used at each of the four SMC rounds. Children between 3 and 59 months of age are eligible to receive SMC if they are afebrile, have no history of allergy to SP or AQ, not under co-trimoxazole prophylaxis for HIV infection or exposure.Seasonal malaria chemoprevention activities were funded by the US President’s Malaria Initiative (PMI) through the Maternal and Child Survival Programme (MCSP) of the Save the Children. The National Malaria Control Programme and regional and national health directorates, in collaboration with PMI and Save the Children, led the planning and implementation of SMC. A research team of the Malaria Research and Training Centre of the University of Science Techniques and Technologies of Bamako that was not involved in the implementation conducted the surveys.Baseline and follow-up, cross-sectional, household surveys were carried out in the intervention and comparison districts to assess the impact of SMC on malaria parasitaemia, fever, malaria illness, and anaemia. The baseline survey was performed in July 2014 prior to the start of SMC implementation and the post-intervention (follow-up) survey took place in December 2014. Thirty clusters (villages or quartiers), 15 in each district, were selected using a random sampling with probability proportional to the size of the population. In each cluster, a sample of about 38 children aged 3–59 months were randomly selected and surveyed using the WHO EPI method . HousehoCross-sectional surveys were carried out 4–7 days after each of the four rounds of SMC to assess caregivers’ adherence to the administration of SMC drugs and determine the frequency of adverse events in the intervention district of Kita. Adherence surveys were conducted in 10 clusters  selected randomly among the 15 already selected for the pre- post-intervention surveys. The same localities (clusters) but different households were surveyed after each round. The main indicators assessed through caregiver interview and verification of the SMC card included: the proportion of children who received the second and third dose of AQ at home, the proportion who spat out or vomited the treatment, the nature and frequency of the adverse events, and opinions of parents/caregivers regarding the intervention.During the post-SMC survey in Kita, 15 additional clusters (villages/quartiers) were added to the 15 surveyed at baseline, yielding a total of 30 clusters for the coverage survey in Kita. Parents or guardians of children in the target age range at the time of the SMC administration were questioned using standardized questionnaires about receipt of SMC. In addition, information from SMC cards was recorded for each child, when available. Interviews in each household were conducted by two trained interviewers in the local language. Primary indicators assessed from the coverage survey included administration of each day of the 3-day SMC treatment course during each of the four rounds and the use of insecticide-impregnated bed nets (ITN) during the previous night. Completed questionnaires were verified at the end of each day by a supervisor and corrected if necessary. Coverage of SMC at each round was defined as the proportion of the children aged 3–59 months at the time of SMC who received the 3 days’ treatment of SMC during that specific round. Full SMC coverage was defined as the proportion of children aged 3–59 months who received the complete 3-day treatment course during all four rounds of SMC.The study team initially intended to use routine data reported from health centres in the intervention and the comparison districts to assess trends in malaria cases, but these data were too incomplete. For example, data from several weeks (up to 3 months) were not reported for several health centres. For this reason, the impact on malaria morbidity was assessed using routine data on confirmed malaria cases extracted from the registers by the research team in nine of the 47 community health centres in Kita and 7 of the 24 health centres in Bafoulabe; these health centres were randomly selected from among the 15 health areas of pre- post-intervention surveyed in each of the two districts. These data on confirmed malaria, uncomplicated and severe cases for 2013 and 2014 were collected from registers in February 2015.Plasmodium falciparum). These RDTs can detect infection with Plasmodium by detecting histidine-rich protein 2 antigen specific to P. falciparum (PfHRP2). RDT results were given to caregivers of children who were tested; those testing positive received malaria treatment according to national guidelines in Mali.Malaria RDTs were performed in the field according to the manufacturers’ recommendation using the same RDTs used in the primary health centres  were used to measure haemoglobin (Hb) concentration in children using capillary blood obtained by finger prick.HemoCueThick and thin blood smears were prepared in the field for participating children in the pre-post intervention surveys. Thin smears were fixed in methanol. All slides were air dried and stained using Giemsa stain 3% for 45–60 min the field, and transferred to the Malaria Research and Training Centre (MRTC) laboratory for reading and storage. The stained thick and thin blood films were examined microscopically at a magnification of 1000× to identify the parasite species and to determine the parasite density. Parasite density was assessed by experienced certified microscopists unaware of the intervention and comparison groups, by counting the number of asexual parasites in a set number of white blood cells  with a hand tally counter according to established standard operating procedures, assuming 8000 white blood cells (WBC)/µL.P. falciparum parasitaemia ≥160/uL (4 parasites/200 WBC) by blood smear. Samples were analysed by nested polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymorphism (RFLP) for mutations at codons 51, 59 and 108 of the dhfr gene, 437 and 540 of the dhps gene, mutations at codon 76 in the P. falciparum chloroquine transporter gene (pfcrt), and at codon 86 of the P. falciparum multidrug resistance gene one (pfmdr1) according to published methods [Filter paper samples were air dried and stored individually in small Ziploc bags with a desiccant and transferred to the MRTC laboratory in Bamako for analysis. Molecular analysis was performed on samples from children whose blood smear showed  methods , 14. CasBased on a meta-analysis of SMC that found a reduction of parasitaemia prevalence of 53% in trial settings , and assThe adherence surveys were powered to estimate an adherence rate  of 80%, within a 95% confidence limit of ±10%. Assuming a design effect of 2 and a 10% non-response rate, 156 children were required after each round. To achieve this sample size, 10 clusters were selected in Kita, and 20 households per cluster were visited and surveyed after each SMC round.Assuming a SMC coverage of 50%, with an alpha of 5%, design effect of 2 and non-response rate of up to 10%, a total of 840 children were required for the coverage survey. To achieve this sample size, 30 clusters were randomly selected in Kita and 38 children were surveyed in each cluster.Survey data were collected on standardized forms, entered into a MS Access database, and analysed using Stata (version 12.1). As described above, data from health facility registers were extracted from the registers in selected heath centres in intervention and comparison districts. Analyses from household surveys were adjusted for cluster sampling, using the survey commands in Stata. Logistic regression was used for binary outcomes of anaemia and parasitaemia, and used a difference-in-differences approach, which can be useful in estimating impact from observational study designs . These rThe coverage survey was completed for 1141 children aged 4–63 months in December 2014 (children aged of 3 month at least at the time of the last round of SMC in November and did not reach the fifth anniversary at the time of the first round of SMC in August). The SMC card was available for 805 (70.6%) of 1141 children. Based on information collected by interview, the proportion of children who received at least the first day of treatment administered by the health worker was 84% at the first SMC round, but declined with subsequent rounds and was 67% for the fourth round  of children were reported to have spat out or vomited the first day’s treatment, while 6.2% 50/808) and 6.6% (52/792) did so on days 2 and 3, respectively Table . Overall0/808 andOverall, 99.9% of parents of children surveyed reported that SMC is good or very good, with 68.3% saying it is very good  and in the post-intervention survey (29.4 months in Kita vs 28.6 months in Bafoulabe p = 0.47).The prevalence of malaria infection aged 3–59 months was similar in the two districts at baseline (24.1% in Kita vs 30.5% in Bafoulabe p = 0.32)  was similar across the two districts at baseline . In post-intervention period, the prevalence of fever was twice as less in the intervention district as in the comparison district but the difference was not statistically significant 2.8 vs 1.4%); p = 0.11) . At the end of the intervention period, the prevalence of anaemia in children aged 3–59 months was 68.9% in the comparison district and 49.3% in the intervention district, corresponding to a reduction in odds of anemia of 53% in the intervention area  adjusted for age, gender, ITN use and cluster design  as well as in post-intervention survey (97.1 vs 98.8% p = 0.14%).dhfr, dhps, Pfcrt-76T and Pfmdr1-86Y were similar between the two districts before and after the intervention except for single mutation dhps A437G, which was higher in the intervention district compared to the comparison district at the end of the intervention (p = 0.01) (Table dhps K540E mutation (12.5% at the highest) was well below the 50% limit recommended by WHO for not using the SP in intermittent preventive treatment in infants and did not vary before and after intervention in the two districts. There was also no significant change in prevalence of the quintuple mutants before and after the intervention and between the two districts.The frequencies of individual and multiple mutations in In the localities surveyed in Kita, the number of confirmed malaria cases during the 2014 transmission season (July to December) was reduced by 61% compared to 2013, whereas in the comparison district of Bafoulabe there was an increase of 23%  during the first round. However, this proportion decreased over the consecutive rounds and was only 67% for the fourth round based on interviews and 58% based on SMC cards. This emphasizes the importance of continuing to monitor SMC coverage over subsequent years, as well as undertaking activities, including social mobilization and community engagement, to prevent the decrease in coverage seen in subsequent rounds. Travel was reported as the main reason for missing SMC rounds. At the time of this evaluation SMC in the Kayes Region was implemented only in Kita district. With the extension of the SMC to other districts and the plan to cover the whole country in 2016, it is expected that fewer children will miss their rounds because of travel within country. Nevertheless, information and sensitization of communities about the SMC schedule will remain essential to reduce the proportion of children who miss their SMC rounds. After travel, the next most common reason parents reported for missing a round was that they were not informed; efforts should be undertaken to improve information and reminders about the dates for upcoming SMC rounds, for example by providing information at each SMC round, and through reminders from community health workers/other community channels closer to the next round. It is possible that the impact of SMC on malaria and anemia demonstrated in this study could be even greater if coverage of the intervention is improved.Seasonal malaria chemoprevention was well tolerated with similar or fewer patients reporting side effects than reported in previous clinical trials in Mali , 6. A reNot surprisingly, parents’ opinions about SMC were very positive, with 99.9% of parents reporting they felt the strategy is good or very good, and 99% of them supporting the continuation of the intervention. This strong support and favorable opinion of SMC are major assets for continuation and scale-up, and are reflected in the very high rates of administration of drugs for days 2 and 3 by parents at home 95% of the times. Not giving the second and third days treatment, although a major concern, was not an issue according to data from this study, since these treatments were given at least 95% of the time, and only parents of two children reported that they saved the drugs of future treatment or gave it to another child.dhps K540E mutation, which serves as a marker for the quintuple mutation that is most associated with the resistance to SP in West Africa [Pfmdr1-86Y; these were similar before and after the intervention in the two districts. This is reassuring despite the increase in dhps A437G mutation given the 50% threshold for dhps K540E mutations recommended by WHO for not using SP in intermittent preventive treatment in infants [dhps K540E mutations were detected in samples collected after 1, 2 and 3 years of SMC in Senegal [dhfr mutants was reported after 2 years of implementation of IPTi [The frequency of t Africa , remaine infants . These d Senegal . Pilot i Senegal ; however of IPTi  and an iStrengths of this study include the fact that evaluation was done by a research team not involved with the SMC implementation, the choice of appropriate comparison district, the pre and post design and the assessment of several indicators for SMC implementation. As with any observational study, there is potential for selection and observational bias. To minimize these biases, study participants were selected randomly and technicians who read blood smears or performed the molecular analysis were blinded. Potential for recall bias could not be excluded despite the fact coverage survey was performed only about 4 weeks post SMC rounds and adherence surveys in 4–7 days post SMC rounds.In summary, routine implementation of SMC in Mali substantially reduced prevalence and incidence of malaria, as well as prevalence of anemia, with reductions of similar magnitude to those seen in previous clinical trials. Improving coverage through social mobilization and communication could further strengthen SMC impact. One year of implementation of SMC was not associated with an increase in molecular markers known to be associated with resistance to SP and AQ, but resistance markers should continue to be closely monitored.