Plasmodium falciparum infection associated with high mortality even when highly effective antiparasitic therapy is used. Adjunctive therapies that modify the pathophysiological processes caused by malaria are a possible way to improve outcome. This review focuses on the utility of PPARγ agonists as an adjunctive therapy for the treatment of cerebral malaria. The current knowledge of PPARγ agonist use in malaria is summarized. Findings from experimental CNS injury and disease models that demonstrate the potential for PPARγ agonists as an adjunctive therapy for cerebral malaria are also discussed.Cerebral malaria is a severe complication of  Plasmodium. Five species infect humans, Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and most recently, P. knowlesi [P. falciparum infection, with the highest burden born by children and pregnant women. In the absence of prompt and effective treatment, P. falciparum infection can progress quickly, rapidly becoming severe and fatal. The rise in drug-resistant parasites complicates the administration of effective treatment. Few diseases have the global health and economic impact of malaria . In 2009knowlesi . The majSevere malaria has multiple manifestations that can occur singly or in combination. They include hyperparasitemia, high fever, haemoglobinuria, acute renal failure, acute pulmonary edema, metabolic acidosis and respiratory distress, hypoglycemia, anemia, and cerebral malaria, which is characterized by coma and convulsions. Cerebral malaria has the highest mortality rate of all the severe complications and is associated with long-term cognitive and neurological deficits in surviving children –6.P. falciparum cerebral and severe malaria so far, unfortunately with-out much success  binding to receptors expressed on microvascular endothelial cells. Postmortem, in vitro, and genetic studies support that ICAM-1 is the major sequestration receptor for PEs in the brain, while the scavenger receptor CD36 is the major receptor outside the brain [Sequestration of parasitized erythrocytes (PEs) in the microvasculature of the brain (and other organs), resulting in vascular occlusion and local tissue hypoxia and ischemia, is the hallmark feature of cerebral malaria . Sequesthe brain –21.γ, IL-6, IL-1β, IL-1ra, IL-10, MIP-1α and MIP-1β, MCP-1, and IP-10 have been observed in cerebral malaria patients [Parasites produce a variety of bioactive molecules that can elicit innate immune responses in the host . An excepatients , 30–34.Parasite sequestration and inflammation can lead to endothelial activation and dysfunction. Activated endothelium can lead to monocyte and platelet recruitment further impeding vessel flow and contributing to tissue hypoxia and ischemia . WidesprSequestration, inflammation, and endothelial dysfunction can lead to a breakdown of the blood-brain barrier (BBB). Hemorrhages are common autopsy findings in cerebral malaria , 42, 43,Metabolic perturbations are also common in children with cerebral malaria and may contribute to pathology. Vascular obstruction leading to hypoxia, or TNF-induced cytopathic hypoxia, have been proposed as possible causes –49.Recent investigations using fluorescein angiography and fundoscopy have permitted a view of the brain microvasculature in living patients with cerebral malaria, by imaging the retina  vasculature that is available for direct observation). Pediatric cerebral malaria patients had evidence of PE sequestration and thrombi (containing both fibrin and platelets) in their vasculature that were associated with perfusion abnormalities and areas of ischemia and tissue damage . Focal disruptions of the BBB were observed most often, but not always, in association with hemorrhages , 50, 51.γ appears to play an important role in recovery in several models of CNS injury and disease, by limiting inflammation and cytotoxicity and promoting reparative mechanisms. These mechanisms may also be protective in the context of cerebral malaria as well. Interestingly, PPARγ was one of only two genes in a malaria-resistance locus identified using a genome-wide analysis of inbred mouse lines [γ in malaria.Cerebral malaria is a complex disorder that is as yet not fully understood. Multiple processes likely contribute to its development including peripheral and CNS inflammation, PE sequestration, vascular endothelial activation, prothrombotic activation, blood flow obstruction, tissue hypoxia and ischemia, metabolic changes, and BBB dysfunction, leading to neurodegeneration. These processes can contribute to the seizures and coma seen in cerebral malaria patients and the neurologic and cognitive deficits which persist in a portion of cerebral malaria survivors , 29. These lines , supportγ is a member of the family of nuclear hormone receptors which function as ligand-activated transcription factors [γ endogenous ligands include oxidized fatty acids and prostanoids, and synthetic ligands include the thiazolidinedione (TZD) class of antidiabetic drugs . Upon ligand activation, PPARγ heterodimerizes with the retinoid X receptor (RXR), a nuclear receptor for 9-cis-retinoic acid. The ligand-bound PPARγ-RXR heterodimer regulates gene transcription by binding to conserved DNA sequences called PPRE (PPAR response elements) on target genes. PPARγ can also regulate other transcription factors, through nongenomic trans-repression, where the inhibition of transcription occurs by preventing the dissociation of corepressors or by sequestering the coactivators necessary for the binding of the transcription factor to DNA [PPAR factors . PPARγ er to DNA .γ is present in most cell types  and mediates multiple functions in both physiological and pathological conditions [Originally characterized in adipocytes as a regulator of lipid and glucose metabolism, current evidence indicates that PPARnditions , 56. γ agonists have been extensively studied in many inflammatory settings, in vitro, in animal models, and in humans, and in most cases they have demonstrated anti-inflammatory properties [γ agonists can inhibit proinflammatory responses from a variety of cells including macrophages, dendritic cells, T cells, endothelial cells, vascular smooth muscle cells, microglia, and astrocytes [γ on transcription factors including activator protein-1 (AP-1), signal transducers and activators of transcription 1 (STAT-1), nuclear factor kB (NF-κB), and nuclear factor of activated T cells (NFAT). PPARγ agonists can also suppress inflammation by PPARγ-independent mechanisms, for example, the suppression of JAK-STAT-dependent inflammatory responses in activated microglia and astrocytes via the induction of members of the suppressor of cytokine signaling (SOCS) family [PPARoperties . These aoperties . PPARγ atrocytes –74. The ) family , 76. γ agonists in models of CNS injury, ischemic stroke, and diseases of the CNS including multiple sclerosis, ALS, and Parkinson's disease [γ may be involved in coordinating cellular responses to CNS injury and disease. The potential benefit of the anti-inflammatory and neuroprotective properties of PPARγ agonists in cerebral malaria will be discussed below.Data have also been accruing on the neuroprotective properties of PPAR disease –80. ThesPlasmodium falciparum may itself activate PPARγ, perhaps as part of a strategy aimed at enhancing symbiotic survival between the parasite and the host. Hemozoin, a pigment produced by Plasmodium to detoxify free heme generated by the degradation of haemoglobin [γ, and 4-HNE is an inducer of PPARγ [γ mRNA, while the inhibition of PPARγ reversed some of the hemozoin-mediated effects, suggesting that the immunomodulatory effects of hemozoin may be, at least partly, mediated by PPARγ activation [moglobin , can promoglobin . 15-HETEof PPARγ . Hemozoiof PPARγ –89. Hemotivation .γ agonists to modulate immune responses to malaria was initially motivated by reports demonstrating that PPARγ regulates CD36 transcription and that PPARγ agonists have anti-inflammatory properties [The use of PPARoperties , 63. P. chabaudi AS infection) [At that time, the scavenger receptor CD36 was revealed to be a major, noninflammatory, phagocytic receptor for nonopsonised mature-stage PEs . It was fection) . In thisfection) . γ agonists including the natural ligands 15d-PGJ2 and 9-cis-retinoic acid (which binds RXR to activate the PPARγ-RXR heterodimer), and the synthetic TZDs, ciglitazone, troglitazone, and rosiglitazone, were shown to upregulate the CD36 expression on monocytes and enhance the CD36-mediated phagocytosis of PEs [γ agonists also dramatically upregulated the uptake of ring-stage PEs and gametocytes [Various PPARs of PEs , 96, 98.s of PEs , 99. This of PEs , 101. PPetocytes , 96. Theetocytes . This reγ activation to polarize macrophages towards an alternatively activated phenotype [These data are consistent with the reported ability of PPARhenotype . Alternahenotype . α-thalassemia and β-thalassemia) and glucose-6-phosphate dehydrogenase and pyruvate kinase deficiencies [γ agonists may be their most important quality when it comes to the treatment of cerebral malaria.Although reducing the parasite burden by enhancing phagocytic clearance of parasites  will undoubtedly be beneficial to the outcome of infection and may be a contributing mechanism to the genetic resistance offered by hemoglobinopathies , a malaria toxin that interacts with TLR2 [κB and MAPK signaling [γ is known to inhibit the NF-κB signaling [γ-mediated inhibitory effect on MAPK signaling has recently been described [γ activation was not directly examined. Human monocytes and murine macrophages treated with PPARith TLR2 , 108. Thignaling . PPARγ iignaling , and a Pescribed . Howeverγ agonists in vivo have been tested in a mouse model of experimental cerebral malaria (P. berghei ANKA). Cerebral pathology in this model is the result of an uncontrolled proinflammatory response to infection [β ratio, and higher IL-10 levels . Patients receiving rosiglitazone also had significantly lower levels of IL-6 and MCP-1 and trended towards significantly lower levels of TNF at 24 and 48 hours posttreatment [Given the encouraging data in the mouse models, a phase I/IIa randomized double-blind placebo-controlled trial was undertaken to test the safety, tolerability, and efficacy of rosiglitazone adjunctive therapy in 140 Thai adults with uncomplicated  malaria . Rosigli malaria . A nonstreatment . Both thγ (via RXR ligation), and like rosiglitazone, has been shown to enhance CD36-mediated PE uptake and reduce malaria-induced TNF production in vitro [The findings in the rosiglitazone trial share some similarities to those of a randomized trial of vitamin A supplementation in children from Papua New Guinea . 9-cis-rin vitro . Childrein vitro , suggestγ agonists in models of neuroinflammatory and neurodegenerative disease states may give us an insight into how PPARγ agonist could function in cerebral malaria [Data on the anti-inflammatory and neuroprotective properties of PPAR malaria –80.γ is expressed not only in immune cells and in peripheral organs, but also in the CNS  and in human brain microvascular endothelial cells [γ agonists such as rosiglitazone and pioglitazone can cross the BBB [Relevant to cerebral malaria pathology, PPARal cells –116. Fur the BBB , and thuγ agonists. PPARγ agonists have been shown to inhibit the following: the expression of inflammatory mediators, such as TNF, IL-6, IL-1b, and COX-2, from activated monocytes and microglial [As mentioned above cerebral malaria is an inflammatory disease , 49. Procroglial , 119; thcroglial , 121; thcroglial , 122; thcroglial , 124. Inγ agonists also have antioxidant properties. PPARγ agonists enhance the endothelial and neuronal expression and activity of superoxide dismutase-1 (SOD-1) and catalase  [γ can also suppress superoxide generation by decreasing the expression of components of the NAD(P)H oxidase complex [γ activation [Malaria is associated not only with inflammation, but also with oxidative stress, conditions that together can lead to increased cytotoxicity. Elevated levels of TNF in addition to oxidants such as superoxide and free heme can lead to neuronal damage , 126. TNromoter) –132. SOD complex , 133. Ro complex , 134. Hetivation . HO-1 istivation . HO-1 intivation . HO-1 antivation . γ agonist activity may enhance NO bioavailability [Oxidative stress can also result in decreased NO bioavailability, via scavenging by cell-free hemoglobin and/or superoxide-mediated formation of the toxic peroxynitrite . Low NO lability . A trialγ agonists is their ability to regulate the expression of the glutamate receptor GLT1/EAAT2 (GLT1/EAAT2 has six putative PPREs in its promoter region) [γ agonists increased astrocytic expression of GLT1/EAAT2 mRNA and protein in vitro [An additional neuroprotective property of PPAR region) . Glutama region) . Glutama region) , 148. Thin vitro  and protin vitro , 150. Inin vitro .γ agonists via the attenuation of inflammation, oxidative stress, and cytotoxicity [γ agonist use in models of ischemic and hemorrhagic stroke [γ agonist use was associated with reduced brain injury and with improved neurological outcomes [γ agonists attenuated neuron loss, prevented motor dysfunction, improved motor performance, and reversed memory decline [Collectively these data support a neuroprotective role for PPARtoxicity . Such prc stroke –158, andc stroke . In the outcomes , 159–162 decline –166. Sup decline , 168. In decline . Diabeti decline . Clinicaγ activation may enhance the tolerance of the host to malaria infection by immunoregulatory mechanisms (modulation of the inflammatory response to infection), and by mechanisms that render tissues more resistant to inflammatory damage. Such immunomodulatory effects are likely to be protective in the context of cerebral malaria. However, whether PPARγ activation following the onset of cerebral malaria (once the inflammatory cascade has begun) will be protective is an open question. Other immunomodulatory therapies tested in cerebral malaria in the past  have failed [γ activation impacts several pathways and may have not only neuroprotective but also neuroregenerative effects improves the likelihood of efficacy. However, it is unknown whether the regenerative effects seen with long-term PPARγ agonist use in chronic CNS disease will also be obvious with a short treatment course, as would be administered in cerebral malaria.PPARe failed . That PPγ agonists could have an impact on the acquisition of adaptive immunity to malaria via modulatory effects on dendritic cells, T cells, and B cells [Rosiglitazone (4 mg twice daily for 4 days administered in combination with atovaquone-proguanil) was found to be safe and well tolerated in uncomplicated malaria. Mean serum glucose, alanine aminotransferase, and aspartate aminotransferase levels did not differ between patients receiving placebo and those receiving rosiglitazone . In addi B cells . γ agonists in malaria are encouraging, with rosiglitazone being safe, well tolerated, and efficacious in uncomplicated malaria patients. Given the anti-inflammatory, neuroprotective, and neuroregenerative properties reported for PPARγ agonists in models of CNS injury, ischemic stroke, and diseases of the CNS, we can hypothesize that PPARγ activation in cerebral malaria may lead to improved outcome and possibly less long-term cognitive and neurological deficits. However, a randomized double-blind placebo-controlled trial in patients with cerebral malaria will be required to determine if these hypotheses are correct.The existing data on the use of PPAR


Plasmodium falciparum infection and contributes to the highest number of deaths by falciparum malaria. This study aims to evaluate the relationship between ABO blood group and severe malaria in an adult population at the tertiary care centre in Odisha.Erythrocyte-associated antigenic polymorphisms or their absence have perhaps evolved in the human population to protect against malarial infection. Studies in various populations consistently demonstrate that blood group 'O' confers resistance against severe falciparum infection. In India, Odisha state has one of the highest incidences of P. falciparum infected subjects and 174 healthy controls were screened for ABO blood group. Falciparum-infected individuals were categorized as severe malaria and uncomplicated malaria. Severe malaria was further clinically phenotyped into cerebral malaria, non-cerebral severe malaria and multi-organ dysfunction. A meta-analysis was performed to assess the role of ABO blood group in severe malaria.A total of 353 Frequency of blood group 'B' was significantly higher in patients with severe malaria compared to the uncomplicated cases  and healthy controls . Irrespective of the level of clinical severity, blood group 'B' was significantly associated with cerebral malaria , multi-organ dysfunction  and non-cerebral severe malaria patients  compared to the uncomplicated category. Prevalence of 'O' group in uncomplicated malaria  and healthy controls  was significantly high compared to severe malaria. Meta-analysis of previous studies, including the current one, highlighted the protective nature of blood group 'O' to severe malaria (P = 0.01). On the other hand, carriers of blood group 'A' (P = 0.04) and 'AB' (P = 0.04) were susceptible to malaria severity.Results of the current study indicate that blood group 'O' is associated with reduced and 'B' blood group with increased risk of development of severe malaria in Odisha, India. Meta-analysis also supports the protective nature of blood group 'O' from severe falciparum infection. Plasmodium and transmitted by the bite of infected Anopheles mosquitoes. Out of the four species that infect humans, Plasmodium falciparum is the principal cause of severe clinical manifestations [P. falciparum malaria [Malaria is an infection caused by protozoan parasites of the genus stations . Cyto-adstations . An assostations . Howeverstations -7. On thstations . An enzystations . Variati malaria  and this malaria . Tri-sac malaria ,7,11.There are limited reports in literature on association of ABO blood group and susceptibility to severe falciparum malaria.. The association of blood group 'AB' and severe malaria has been demonstrated in various populations, viz. Sri Lanka , Mali 11, and EtP. falciparum malaria [Malaria remains a major health problem in India. The National Vector Borne Disease Control Programme (NVBDCP), India, has reported that 1.8 million cases of malaria and 1,000 malaria-related deaths occur annually . However malaria  and cont malaria , no studP. falciparum infection by Giemsa-stained thick and thin blood smears and immune chromatography test (SD Bio Standard Diagnostics India). Detection of P. falciparum was also performed by nested polymerase chain reaction (PCR). Individuals infected only with P. falciparum were included. Clinical categorization was done based on WHO guidelines [P. falciparum infection. The risk of exposure to malaria was similar for both HC and patients. Criteria for analysis of mortality: Since death occurred in the CM and MOD groups' only patients from these groups were included for analysis. The study was approved by the Institutional Ethics Committee of the Medical College and blood samples were collected after written consent of the patients or accompanying person, depending on the clinical scenario.The study was conducted at S.C.B. Medical College, Cuttack, Odisha, India between 2008-2009. Patients (age ≥15 years) admitted to the Department of Medicine with a short history of fever were clinically examined in detail and screened for idelines . UncomplP. falciparum is the major cause of malaria (>80%) followed by P. vivax (10-15%) [P. falcipaurm and/or P. vivax infections [Plasmodium genus) was performed in a 20 μl PCR reaction containing 3 μL genomic DNA, 1× Taq buffer containg MgCl2 (SIGMA), 250 μM dNTP (SIGMA)), 200 nM of two genus-specific primer rPLU1 (5'-TCA AAG ATT AAG CCA TGA AAG TGA-3') and rPLU5 (5'-CCT GTT GTT GCC TTA AAC TCC-3') (Integrated DNA technologies) and 1 U of DNA polymerase (SIGMA). The cycle conditions were as follows: an initial de-naturation step at 95°C for 7 minutes, followed by 38 cycles of 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, and a final extension at 72°C for 7 minutes. For secondary PCR , 3 ul of PCR product of previous reaction was used as template and species-specific primers set were used {rFAL1(5'-TTA AAC TGG TTT GGG AAA ACC AAA ACC AAATAT ATT)/rFAL2(5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3') and rVIV1(5'-CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC-3')/rVIV2(5'-ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA-3')} in different tubes. Reaction mixture and thermo-cycler condition was similar to primary PCR. PCR products were analysed in agarose gel electrophoresis.Genomic DNA was extracted by GenElute™ Blood Genomic DNA Kit (SIGMA) from whole blood according to the manufacturer's instructions. Polymerase chain reaction (PCR), has been used to detect up to species level - falciparum, vivax, ovale and malariae ,21. In t(10-15%) . With slfections . In brieBlood samples of both falciparum-infected individuals and healthy controls were typed by commercial haemagglutination kit . In brief, about 20 ul of whole blood was taken on a clean slide and 20 ul of antisera A, B and Rhesus blood group applied, mixed by means of an applicator stick and results were noted.P. falciparum malaria. Cross-references were checked including studies not located in PubMed. The following key words and subject terms were searched: ABO blood group, Plasmodium falciparum, severe malaria and uncomplicated malaria.An extensive PubMed search was performed for articles published on the association of ABO blood group in severe Statistical analyses were performed by using GraphPad Prism (version 5.01). Difference between means was analysed by ANOVA. The Fisher exact test was used to analyse the association between blood groups and severe malaria or different clinical manifestation. Odds ratio (OR) were calculated with 95% confidence interval. The 'O' blood group was taken as a reference to compare the prevalence of other blood groups, 'A', 'B', 'AB' and non 'O', defined as A+B+AB combined, in severe and uncomplicated malaria. A probability value of ≤ 0.01 (0.05/4) was considered statistically significant after Bonferroni correction. Meta-analysis and all related statistics were obtained by Comprehensive Meta-analysis V2 software. Variation within and among different study (heterogeneity) was assessed by Q-test with the null hypothesis that all studies have the same effects. The random effects model was used for meta-analysis if the Q-statistic was significant (P < 0.05) which indicate heterogeneity across studies. On the other hand, lineage of all studies to null hypothesis, the fixed effect model, was used for necessary meta-analysis.P. falciparum malaria patients were further subdivided into cerebral malaria (CM) (n = 59), multi-organ dysfunction (MOD) (n = 80) and non-cerebral severe malaria (NCSM) (n = 108). 174 healthy controls (HC) were included. The mean age between all clinical categories and HC was comparable. Significantly higher levels of haemoglobin were observed in HC than UM and other clinical categories of severe malaria (P < 0.0001)  and 106 uncomplicated patients (UM). Severe P. falciparum infection, prevalence of each ABO blood group was compared in severe malaria patients (n = 247), uncomplicated malaria (n = 106) and healthy controls (n = 174). Of the 174 blood samples examined in healthy controls, 74 (42%), 36 (21%), 50 (29%) and 14 (8%) individuals were from O, A, B, and AB blood group, respectively. As shown in Table To assess possible associations of ABO blood groups with severe course of ents n = 47, uncoments n = 47, uncomThe association of ABO blood group in different clinical phenotypes of severe malaria, categorized as CM, NCSM and MOD, was studied. As highlighted in Table Since the study revealed a significant association of ABO blood group and severe falciparum malaria, possible role of ABO blood groups in disease outcome was explored. Prevalence of blood groups were analysed among subjects who died during treatment and those who survived Out of 247 cases of severe malaria, twenty patients died during course of treatment: five patients from CM category (n = 59), 15 patients from MOD (n = 80) and none from NCSM (n = 108). Therefore, NCSM patients were not included in the analysis. Furthermore, fifteen patients from CM and MOD groups left the hospital against medical advice, which necessitated their exclusion since their survival status was not known. As a result, a total of 124 patients in CM and MOD groups were analysed. Although, frequency of blood group 'B' was higher in patients who died (80%) compared to those who survived (54%), the difference was not statistically significant (P = 0.06). Distribution of other blood groups in patients who died and survived was comparable  study must be case-controlled, 2) patients placed under well defined categories as severe and uncomplicated, 3) the sample size should be more than 50 in each category. Out of nineteen studies only five publications satisfied all inclusion criteria and were considered for meta-analysis. Data from the recent study was also included for analysis. Table s. For 'O', 'B' and 'AB' blood groups the funnel plots were symmetric. However, plot for blood group 'A' was asymmetric. Trim-and-fill technique was used to adjust publication bias of blood group 'A', for recomputing the effect size [A funnel plot has been used to test publication bias in meta-analysis. The funnel plot remains symmetric in absence of publication biaect size .Heterogeneity Q test was performed to evaluate inter- and intra-study variations and based upon significance value; different models were used for the meta-analysis. Blood group 'O' (P < 0.0001), 'B' (P = 0.004) and 'AB' (P = 0.037) showed significant heterogeneity and, therefore, a random-affect model was employed. On the other hand, a fixed-effect model was used for association of blood group 'A' since the Q-test was not significant (P = 0.120).Results of meta-analysis are shown in Figure Results of the current study suggest that patients with blood group 'B' have a four-fold increased risk of developing severe infection. In addition, it also reiterates the observation that 'O' blood group was significantly associated with a decreased risk of severe malaria. Other blood groups ('A' and 'AB') did not show any association.The role of ABO blood group in malaria has been investigated in various populations, but robust data is limited . This stIt is presumed that the prevalence of blood group 'O' would be higher in malaria endemic areas due to its capacity to confer protection. An analysis of blood group in healthy controls revealed a distribution of 'O' (42%), which was much higher compared to 'A' (21%), 'B' 29%) and 'AB' (8%). A community-based study in a tribal population of Odisha, where malaria is endemic, also showed higher prevalence of blood group 'O' . Signifi% and 'ABMeta-analysis combines results of several similar studies to produce a single estimate of the major effect with enhanced precision . CurrentThere are however limitations in the present study. Several RBC polymorphisms, including those linked to glucose-6-phosphate dehydrogenase, pyruvate kinase, complement receptor-1 and haemoglobinopathies, have a role in the clinical outcome of malaria , but werThis study reveals a significant association of blood group 'B' to severe malaria. The association is valid across all grades of severity. Blood group 'O' confers protection to severe disease. The meta-analysis reiterates the observation of protection conferred by blood group 'O' and highlights susceptibility of group 'A and 'AB' to severe malarial infection.The authors declare that they have no competing interests.AKP was involved in detection of malaria by PCR, analysis, interpretation, performed statistics and writing the first draft of the manuscript. SKP was also involved in species-specific PCR and analysis. ANS, RT, BR and BKD made a contribution in the design, data interpretation, work supervision and critically revising the manuscript. All authors read and approved the manuscript.


To guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure.Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years , recent travel to Mozambique (OR 4.4 [95% CI 1.0–19.0]) and residence in southeast Swaziland .Using a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to The prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts. Global progress in malaria control has led to increased interest in and optimism for elimination. Of 99 countries that remain endemic, 32 are moving towards elimination, including four in sub-Saharan Africa The burden of malaria is typically estimated by passive and active surveillance. Passive surveillance generally involves health facility based reporting of malaria cases, which can be limited by incomplete reporting, healthcare seeking in the private sector, and poor diagnostic capacity, particularly in low transmission settings where health workers see few malaria cases. Active surveillance addresses some of these limitations and generally involves cross-sectional surveys of defined sample populations, where the primary malaria indicator is the proportion of persons infected with malaria parasites  Estimating parasite prevalence typically relies on microscopy, which is time and labor intensive, and often inaccurate in operational settings Swaziland is a country in southern Africa that has experienced recent declines in malaria burden 5613 participants living in 1751 households were surveyed. Most participants lived in urban areas (53.3%), and in the eastern region of Lubombo (79.2%). Twelve percent of participants reported international travel in 2010, with South Africa (84.3%) and Mozambique (14.4%) being the most frequent destinations. Most participants resided in their current residence for >3 years (76.1%) .Indoor residual spraying (IRS) in the past 12 months was reported in 44.5% of households. Bed net use was low: 3.6% of participants reported sleeping under an ITN the previous night. Among children and women who sought care for fever in the last two weeks, 0% and 15.4%, respectively, received a finger or heel stick for malaria diagnosis . Among 4P. falciparum . Using PCR as the gold standard, RDTs identified none of the two PCR positive samples resulting in a sensitivity of 0.0%, specificity of 99.9%, positive predictive value of 0%, and negative predictive value of 100%.RDTs were performed in 4330 of 5613 (77%) participants and three were positive for lciparum . By PCR,e border . NeitherUsing a pooled strategy for PCR, a total of 182 assays were performed to test the 4028 dried blood spots . CompareCost and other operational issues were considered in a comparison amongst RDT, microscopy, individual PCR, and pooled PCR . Cost to42 antigens. There was a step increase in seroprevalence at 20 years of age (P<0.001). Exclusion of participants who reported recent travel to Mozambique yielded the same age-specific pattern of seroprevalence (data not shown). Analysis of seroconversion rates suggested a significant change in seroconversion rate 20.1 years ago (95% CI 18.8−20.5), suggesting a temporal change in malaria exposure in the year 1989.1820 of 4031 participants with dried blood spots were sampled for serology. Overall, 82 participants  had antibodies to both AMA-1 and MSP-1s of age . CompareP = 0.048).There were no significant relationships between seropositivity and wealth index, urban vs rural residence, altitude, IRS coverage, or bed net use. Reported travel to Mozambique, but not South Africa, within 2010 was significantly associated with seropositivity . Two secondary clusters of higher relative risk were identified within the primary cluster: one extending from Sithobela to Somntongo constituency  and a smaller one limited to Somntongo  (P = 0.02) and a secondary cluster was located in Somntongo . Analyses examining responses to individual antigens or either antigen showed similar clustering (data not shown). Analysis excluding participants with reported recent travel to Mozambique yielded the same three clusters.Spatial cluster analysis was performed to identify areas with higher than expected seroprevalence. A primary cluster was identified in the southeast region of the country  . To asseP. falciparum and one P. malariae infection were identified among 4028 participants. Compared to RDT, pooled PCR identified infections missed by RDT and provided improved efficiency and affordability excluding capital costs. Serological data identified a potential focus of recent transmission in the southeast and low seroprevalence in younger age groups, suggesting low exposure to malaria in recent years. Report of recent travel to Mozambique was identified as a risk factor for both P. falciparum infection and seropositivity.To guide strategic planning for the transition from malaria control to elimination, we utilized novel malaria surveillance techniques of pooled PCR and serology to perform the first large scale cross-sectional malaria survey in Swaziland and found that prevalence of infection and recent exposure was extremely low. By pooled PCR, only one P. falciparum HRP-2 antigen despite resolution of infection Large-scale prevalence surveys have traditionally relied on microscopy, but considering the significant time and labor required, and potential operational limitations, the Swaziland malaria program decided to use RDTs. As a simple point-of-care test, RDTs are convenient, but can give false positive results with underlying autoimmune conditions, non-malarial infections, or persistence of the P. falciparum-specific RDT missed one P. malariae infection, and one P. falciparum infection that was likely of low parasite density given that the participant was afebrile. Others have found low parasite density to be a determinant of decreased sensitivity in survey settings P. falciparum parasites, particularly in low density infections. These subjects could have also had a recent infection that cleared, but persistent antigenemia. While not assessed in this study, microscopy and individual PCR are more sensitive than pooled PCR RDTs may also miss infections of low parasite density and many do not detect non-falciparum species One limitation with measuring parasitemia in a cross-sectional survey is that regardless of the sensitivity of the test, only one moment in time is captured. In low endemic settings such as Swaziland, prevalence may be so low that it will be difficult to track progress toward elimination. As a measure of past infection, serology has been proposed as a useful way to estimate exposure in low endemic settings Serological data may additionally help to identify other epidemiological risk factors for malaria. Travel to Mozambique and residence in the southeastern region were found to be associated with seropositivity. The first finding is consistent with Swaziland's passive surveillance and case investigation data, which have found travel to Mozambique to be a risk factor for infection One potential limitation in our study is that cutoff for seropositivity was based on an assumption of a bi-modal distribution of seropositives and seronegatives within the population sampled. Samples from representative seronegatives  were not available and in practice may be difficult to obtain. Our data suggest a clear temporal trend of a decline in malaria transmission 20 years prior. However, the potential non-specific response seen among younger age groups may suggest that this method is not specific enough to show cessation of transmission, as has been shown in other settings Findings of this study have important implications on Swaziland's strategic planning for malaria elimination. First, the findings of extremely low parasite prevalence and recent exposure suggest that the high IRS coverage  were arranged into four strata. Of 598 EAs, 172 were randomly selected based on probabilities proportional to population size. Geo-coordinates of all households within selected EAs were recorded An MIS with a stratified two-stage cluster sample, cross-sectional design was used to generate representative estimates of intervention coverage and malaria disease burden. Given Swaziland's goal of elimination, several modifications were made to the traditional MIS design P. falciparum HRP2 Detection Rapid Card Test, Premier Medical Corporation Ltd) and Whatman 903 filter paper. Participants testing positive by RDT were transported to the nearest health facility for care.A single finger prick was performed to collect blood for RDT  mixed with 24 negative spots. To determine species, PCR-positive samples underwent an AluI restriction digestion and were compared to digestion patterns of known controls Samples from RDT-negative participants were tested using a three-stage PCR-based pooling strategy as previously described A sub-sample of all participants with a dried blood spot was selected for serologic testing. Those less than one year of age were excluded due to potential persistence of maternal antibodies. All participants one to nine years of age were included because results in this age group were expected to be most reflective of recent changes in transmission. Based on reported declines in incidence, age-stratified analyses were expected to be powered by a lower seroprevalence in younger age groups. Therefore, participants ten to 49 years of age were randomly sampled by district based on the maximum number of participants in the younger age categories. Participants 50 years of age and older were excluded.Plasmodium falciparum FVO strain blood stage antigens merozoite surface protein-1 (MSP-142), and apical membrane antigen-1 (AMA-1), provided by Walter Reed Army Institute of Research ELISA assays were performed similarly to previously described methods The sample size was generated to provide an estimate of insecticide-treated bed net (ITN) use in children less than five years of age. Based on previous surveys, response rate and design effect were estimated at 90% and 1.3, respectively, allowing for a sampling error of 12% Survey data were entered into personal digital assistants (PDAs) and downloaded into Microsoft Access (2007). Analyses were performed using SAS (version 9.2) and STATA (version 11.0). Point estimates and confidence intervals were calculated incorporating survey procedures and weights to adjust for multi-stage clustering and changing selection probability.42.For serologic analyses, raw optical densities were standardized by dividing values by a positive control on all plates. Samples with a coefficient of variation >0.3 between duplicates were repeated. To determine seropositivity cutoffs for each antigen, standardized optical densities were fitted to a mixture model that assumed a bi-modal normal distribution, and seropositivity was defined as three standard deviations above the mean of the lower distribution Evidence for temporal changes in exposure was explored by assessing the relationship between age and seroprevalence. To formally assess a temporal change in exposure, a catalytic conversion model assessing seroconversion rate was fitted to the data, allowing for a change in the seroconversion rate at a single time-point. The time point at which a change in seroconversion occurred was assessed by maximum likelihood with confidence intervals based on the chi-squared distribution on one degree of freedom P-values less than .05 were considered statistically significant. To identify potential foci of transmission, spatial cluster analysis was performed with SatScan (version 9.0), using the Poisson model and allowing for elliptical clusters. Due to sampling among participants ten to 49 years of age, age was adjusted for as a categorical variable . Maximum geographical cluster size was set at 50% of the population, allowing for nested clusters. Statistically significant clusters were reported, including nested clusters with a relative risk greater than that of the parent cluster. Maps were produced using ArcGIS software (version 10.0).To analyze relationships between seropositivity and baseline characteristics, chi-squared, t-test, logistic regression, or two-sample test of proportions was performed as appropriate. For the household and women's questionnaires, oral informed consent was provided by a household head and women 15 to 49 years of age, respectively. Written consent was not performed because data along with oral consent was entered electronically into PDAs, and there were no sensitive questions and thus minimal risks for participants. Written informed consent for blood testing was obtained from participants or a parent or guardian for children less than 14 years of age. Ethical approval for the study, including the use of oral consent for the questionnaires, was obtained from the review committees at the Swaziland Ministry of Health, University of California, San Francisco, and United States Centers for Disease Control and Prevention.


Plasmodium vivax infection in the country, although CQ resistant P. vivax (CRPv) has started to challenge the efficacy of the drug. The present study was conducted to assess the current status of CRPv at Serbo, Jimma zone, south-west Ethiopia.Ethiopia has the highest proportion of vivax malaria, approximately 40% of all malaria infections, in contrast to African countries. Chloroquine (CQ) is the drug of choice for the treatment of in vivo therapeutic efficacy test was conducted from October 2007 to January 2008. Recurrence of parasitaemia and the clinical condition of patients were assessed on each visit during the follow-up. The levels of haemoglobin (Hb) in the study participants were determined. The patients' blood drug levels were measured using HPLC. Data was analysed using SPSS for windows version 10.0. HPLC data was computed using Chem Station for LC 3D systems software.A 28-day Of the total 84 patients included in the study, 78 completed their 28-day follow-up, six of whom being excluded for different reasons. In three children , parasitaemia reappeared within the 28-days follow-up in spite of adequate absorption of the drug and absence of malaria symptom. In addition, on the day of recurrence of parasitaemia the levels of chloroquine-desethylchloroquine (CQ-DCQ) were above the minimum effective concentration (≥100 ηg/ml) in all the three cases, showing that treatment failure could not be attributed to low level of drug in the patients blood.P. vivax. Moreover, as former report from the same country, Debrezeit, also showed the occurrence of CRPv, survey on CRPv malaria should be made in P. vivax endemic areas in order to estimate the level of burden across the country.Reappearance of the parasite within the 28 days of follow-up is due to parasite resistance to CQ. The 3.6% (95% CI = -0.038 - 0.0758) prevalence of CRPv malaria in the study area signals the need for launching monitory activities for CQ resistant  In Ethiopia, malaria is seasonal and unstable, causing frequent epidemics . It usuaPlasmodium falciparum and P. vivax are the two species commonly known to cause malaria in Ethiopia accounting for 60% and 40% proportion, respectively  observed was nearly similar to the study conducted at Debrezeit, Ethiopia (5%) . HoweverP. vivax malaria in the study area. However, the emergence of CRPv malaria in the study area signals the need for regular monitoring activities of CQ resistant P. vivax in malaria endemic area of the country. The trend observed in Indonesia is good example to estimate the severity of the burden. A decade ago, the level of CRPv in Indonesia was similar to the current situation of Ethiopia. After 14 years, however, the level of CRPv increased to about 84% [CQ is still drug of choice for treatment of bout 84%  in some P. vivax endemic areas for current estimate of the level of burden.This study confirmed the emergence of CRPv strains in Serbo town, Jimma zone, southwest Ethiopia. Moreover, as former report from the same country, Debrezeit, also showed the occurrence of CRPv, country wide survey on CRPv malaria should be made in The authors declare that they have no competing interests.TK was fully involved in all phases of the study, including data collection and monitoring both in the field and in laboratory during HPLC analysis, data analysis, interpretation, and write-up of the manuscript; BP designed the study project, supervised the study and critical revised the manuscript. KB was involved in statistical analysis of data and critical revision of the manuscript for publication; TB validated the HPLC method, analyzed and interpreted the HPLC data of the blood samples. All authors read and approved the final manuscript.


P. falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical human genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity that would be invaluable for vaccine development. We investigated the effect of single nucleotide polymorphisms (SNPs) on malaria pathology in a case- control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Twenty nine polymorphisms in cytokine and toll-like receptor (TLR) genes as well as the sickle cell trait (HbS) were assayed on the Sequenom iPLEX platform. Our results confirm the known protective effect of HbS against severe malaria and also reveal a protective effect of SNPs in interleukin-10 (IL10) cerebral malaria and hyperpyrexia. Furthermore, IL17RE rs708567 GA and hHbS rs334 AT individuals were associated with protection from uncomplicated malaria and anaemia respectively in this study. Meanwhile, individuals with the hHbS rs334 TT, IL10 rs3024500 AA, and IL17RD rs6780995 GA genotypes were more susceptible to severe malarial anaemia, cerebral malaria, and hyperpyrexia respectively. Taken together, our results suggest that polymorphisms in some immune response genes may have important implications for the susceptibility to severe malaria in Cameroonians. Moreover using uncomplicated malaria may allow us to identify novel pathways in the early development of the disease. P. falciparum infections progress to severe or fatal episodes ; Severe Malarial Anaemia (SMA) ; hyperpyrexia (axillary temperature ≥ 40°C); hyperparasitaemia ; convulsions before/during admission; respiratory distress  and hypoglycaemia (blood glucose <2.2mmol/L); Anaemia (haemoglobin < 11 g/dL or haematocrit < 33%). Participants with co-existing severe or chronic medical conditions  unrelated to a severe malarial infection were excluded. Uncomplicated Malaria (UM) was defined as a clinical illness characterized by an axillary temperature ≥ 37.5°C associated with a Plasmodium positive blood film, haemoglobin ≥ 8g/dL and full consciousness, in the absence of clinical signs and symptoms of severe malaria and/or evidence of vital organ dysfunction.Patient samples were collected as part of epidemiological studies of severe malaria in children from the Centre, Littoral and South-West regions of Cameroon, conducted between 2003/5 and 2007/8 ,45,46. TControls (n=891) consisted of apparently healthy children  and adults  also belonging primarily to the Bantu and Semi-Bantu ethnic groups . ChildrePlasmodium species was identified on the thin blood smear. A smear was only considered negative if no malaria parasites were seen in 50 high-power fields. With each positive smear, the level of parasitaemia was estimated by counting the parasites against at least 200 leucocytes and using the corresponding leucocyte count to calculate the number of parasites/µl blood .Interestingly, this association with hHbS was only found in the Semi-Bantu and not Bantu   . AT/AA, OR=41.33, 95%CI 6.48-263.63, p=8.56 x 10-7] and SMA   and the TLR9 rs187084 SNP  were associated with susceptibility to malaria . In addition, a number of genotype associations, albeit weak, were also observed with UM and hyperparasitaemia  while IRF1 rs2706384 heterozygotes  and CT/CC genotypes of TLR1 rs4833095  were more refractory to hyperparasitaemia.Sickle celled individuals were more likely to develop malaria infection [rs334 TT vs  x 10-8] . The CT/ malaria  followinsitaemia . IndividIL10 rs1800890 SNP , both in healthy adult and in children with uncomplicated and severe malaria in Burkina Faso [IRF1 polymorphisms on severe malaria may be too small to be detected with this sample size, or may affect only a sub-phenotype of severe malaria.Since the transcription factor IRF1 is a critical effector molecule in IFN-gamma signalling, variants may affect human susceptibility to malaria. This is supported by reports of an association between an IRF1 polymorphism and human immunodeficiency virus (HIV) - infection in Kenya , with prina Faso . Howeverina Faso  as was tina Faso . FurtherIL17, with Toll-like receptor modulation of murine CM thought to depend on the genetic background of the host [Signalling pathways in murine malaria are thought to include the host . The dowthe host . Howeverthe host  and Indithe host . NeverthIL17 may be associated with uncomplicated malaria in the Cameroonian population while SNPs in, IL10 IL17RD, IRF1, TLR1 and TLR9 may be linked with severe malaria. These suggest that polymorphisms in human genes may have important implication for outcome to paediatric malaria in Cameroon, possibly by altering the transcription of cytokine genes. Moreover using uncomplicated malaria may allow us to identify novel pathways in the early development of disease. However, it is possible that an apparent association with malaria can arise from linkage disequilibrium between the typed SNP and a primarily associated polymorphism. Constructing a detailed map of polymorphisms around these candidate genes and describing a profile of linkage disequilibrium among them will be helpful to identify the causal variant(s). In addition, an appropriately designed family-based study may provide useful linkage information, have more power to detect association and allow haplotypes to be constructed with more confidence.Our genetic-association studies confirm and extend the strong protective effect of the sickle cell trait against malaria as well as show that sticklers are more susceptible to severe malarial anaemia. Our data demonstrate the first evidence that polymorphisms inFile S1Material S1, Membership of The Malaria Genomic Epidemiology Network . Table S1, Minor Allele Frequencies and Test of Hardy-Weinberg Equilibrium in selected candidate SNPs across ethnic groups. Table S2, Results of genotype associations between selected SNP and syndromes of malaria in the two major ethnic groups.(DOCX)Click here for additional data file.


In 2010, the World Health Organization revised guidelines to recommend diagnosis of all suspected malaria cases prior to treatment. There has been no systematic assessment of malaria test uptake for pediatric fevers at the population level as countries start implementing guidelines. We examined test use for pediatric fevers in relation to malaria endemicity and treatment-seeking behavior in multiple sub-Saharan African countries in initial years of implementation.PfPR2–10<5%), moderate (PfPR2–10 5–40%), high (PfPR2–10>40%). Among febrile under-fives surveyed, 16.9% (95% CI: 11.8%–21.9%) were tested. Compared to hospitals, febrile children attending non-hospital sources  and community health workers  were less often tested. Febrile children in high-risk areas had reduced odds of testing compared to low-risk settings . Febrile children in least poor households were more often tested than in poorest , as were children with better-educated mothers compared to least educated .We compiled data from national population-based surveys reporting fever prevalence, care-seeking and diagnostic use for children under five years in 13 sub-Saharan African countries in 2009–2011/12 . Mixed-effects logistic regression models quantified the influence of source of care and malaria endemicity on test use after adjusting for socioeconomic covariates. Results were stratified by malaria endemicity categories: low (Diagnostic testing of pediatric fevers was low and inequitable at the outset of new guidelines. Greater testing is needed at lower or less formal sources where pediatric fevers are commonly managed, particularly to reach the poorest. Lower test uptake in high-risk settings merits further investigation given potential implications for diagnostic scale-up in these areas. Findings could inform continued implementation of new guidelines to improve access to and equity in point-of-care diagnostics use for pediatric fevers. For many years presumptive anti-malarial treatment for febrile children was promoted in malaria-endemic African countries due to lack of diagnostic tools, resulting in widespread malaria over-diagnosis The shift from presumptive treatment of febrile children to test-based case management has great potential to improve malaria surveillance, rational drug use and appropriate management of febrile illnesses Despite this investment, evidence to date regarding malaria diagnostic test practices in sub-Saharan Africa is largely derived from adherence studies in limited health facility settings Malaria transmission intensity has long been known to influence the management of acute febrile illnesses in children, resulting in common malaria over-diagnosis in malaria-endemic settings Similarly, it is likely that treatment-seeking behavior greatly influences whether a febrile child gets tested for malaria. Microscopy has historically been concentrated at hospitals and higher-level health facilities In 2009, Roll Back Malaria (RBM) recommended asking a question on malaria diagnostic test use in national population-based surveys National population-based cross-sectional surveys from DHS, MICS, MIS and ACT Watch conducted in sub-Saharan Africa since 2008 were systematically reviewed for inclusion in this study . 84 surv13 DHS and MIS met inclusion criteria, which spanned the period 2009–2011/12 . Survey Malaria diagnostic test use is measured by asking caregivers of children under five with reported fever in the past two weeks if “At any time during the illness did (name) have blood taken from his/her finger or heel for testing?” This question does not differentiate between diagnostic tests, and is assumed to refer to either microscopy or mRDT.There were two main covariates: source of care and malaria endemicity. Source of care is measured by asking caregivers of febrile children if they sought advice or treatment for the illness, and if so, from where care was sought. Multiple responses are allowed, and response categories are standardized across countries with some modifications to account for different health system structures. This covariate was categorized as: (1) hospital (2) non-hospital formal medical (3) community health worker (CHW) (4) pharmacy (5) other (6) no care sought. Hospital, CHW, and pharmacy include any such listed response. Non-hospital includes any formal health system source that is not a hospital or CHW, including health centers or posts, outreach or mobile clinics, and private doctors. Some countries include additional sources for this category, such as maternities or municipal clinics. Other includes shops, traditional practitioners, relatives, and non-specified sources. ‘Hospital’ and ‘non-hospital’ categories were further dichotomized into public or private sources to analyze test uptake across different managing authorities.The questionnaire does not explicitly record where testing occurred, but plausibly happened where care was sought. If the child visited multiple sources , it was assumed testing occurred at the highest level attended and the covariate was coded using a hierarchical stepwise approach. We conducted a sensitivity analysis by comparing adjusted odds ratios with a covariate constructed by excluding febrile children visiting both hospital and non-hospital sources. In this approach, 732 febrile children visited multiple sources in 13 countries; 367 were excluded that visited both hospital and non-hospital sources. No significant difference was found between approaches (data not shown).Plasmodium falciparum parasite rate in 2–10 year olds (PfPR2–10) for every 5×5-km pixel for the year 2010. Malaria endemicity estimates were linked to survey datasets through geocoded PSUs. All individual observations were assigned their PSU-level malaria risk value, which was then categorized into one of five malaria endemicity classes: malaria free; unstable transmission; and low (PfPR2–10<5%), medium (PfPR2–10 5%–40%), and high (PfPR2–10>40%) stable endemic transmission.Malaria Atlas Project estimates of malaria endemicity were included in the model, which are described elsewhere Socioeconomic covariates associated with child survival intervention uptake were incorporated in the model. These included child’s age and sex, maternal age and education, household wealth and density, and residence Among 29,245 febrile children under five surveyed in 13 countries, 300 had missing values for the outcome, 312 for source of care, 752 for malaria endemicity, and one for maternal education. 36 had missing values for two or more variables. Listwise deletion was used to exclude observations with any missing value from the analysis.Mixed-effects logistic regression models were used to quantify the influence of covariates on malaria test use in pooled and individual country datasets. PSUs were nested within country identifiers and normal distribution of the random effects was assumed. Covariates were included as categorical fixed effects nested within PSUs. Crude odds ratios of main covariates  were initially estimated for their effect on the outcome. Main covariates were then included simultaneously in one model and, subsequently, odds ratios were adjusted for the effect of all covariates, as listed above. We tested for an interaction between maternal education and malaria endemicity in the final model, and separately for an equivalent interaction between maternal education and residence. Results were stratified by malaria endemicity categories and separately by residence to examine effect differences across contexts. The level of statistical significance was set to 0.05. National point estimates were tabulated using sample weights pre-specified in datasets, and proportions for the pooled dataset were estimated using meta-analytical methods. Standard error estimation accounted for data clustering in survey designs. Stata 12  was used for all analyses.We also crudely estimated total pediatric fevers attending and tested at different sources of care in 2010 across studied countries to further contextualize findings in our discussion. This was done by applying proportions tested from our analysis to published estimates of total pediatric fevers updated to 2010 105,791 children under five years old were surveyed in 13 countries . 27,916 Febrile children in high-risk areas were less often tested than those in low-risk areas . CompareSource of care was consistently and significantly associated with malaria test uptake after controlling for other covariates . Comparep = 0.315), and lower uptake at public  compared to private  non-hospital sources (p = 0.022).Crude analyses indicate a non-significant difference in test uptake among febrile children attending public  versus private  hospitals across studied countries  when incorporated into the final model. To further explore this result, the final model was stratified by low (PfPR2–10<5%), medium (PfPR2–10 5%–40%), and high (PfPR2–10>40%) stable malaria transmission categories. In high-risk settings, the odds of testing febrile children with mothers who attended primary or at least secondary education was 1.71 (95% CI: 1.44–2.02) and 2.23 (95% CI: 1.76–2.82) times higher, respectively, than if the mother had no education after controlling for other covariates  are less often tested than older ones (six to 11 months). This finding has not been previously reported to our knowledge. Mean age of malaria onset is about six months Our study further demonstrates a socioeconomic dimension to malaria testing. Independent of other factors, febrile children in poorest households are less often tested, as are those with poorly educated mothers. Rural settings are associated with less testing, as are large households in the adjusted analysis. This is likely due to lower test availability where marginalized families often seek care. Integrated community case management is a promising approach to improve equitable access to testing and care These results should be viewed in light of some data limitations. First, findings indicate differences in test uptake across population groups but do not explain reasons for observed practices. Second, surveys only measure whether blood was taken for testing, and as such, do not differentiate between microscopy and mRDT. Higher coverage at certain locations, such as hospitals, may be due to long-standing microscopy availability rather than targeted mRDT roll out. Moreover, testing practices could differ for microscopy and mRDT, particularly since mRDT requires less training and time to use effectively Finally, a recent validation study found caregiver recall of testing was not highly sensitive (61.9%) but had reasonable specificity (90.0%) when compared to direct facility observation of malaria diagnostic test receipt Based on 105,791 children under age five years surveyed in 13 countries in 2009–2011/12, our findings demonstrate low and inequitable testing of pediatric fevers as countries start to implement new guidelines. Malaria diagnostic testing has become increasingly important in the context of malaria control and elimination to improve surveillance, rational drug use and appropriate fever management Table S1National results for the effect of source of care, malaria endemicity and socioeconomic covariates on test uptake. Table legend: CI refers to confidence interval. AOR refers to adjusted odds ratio. Mixed-effects logistic regression models in individual country datasets, adjusted for data clustering and all listed covariates.(DOCX)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


Several studies conducted in Northeast Tanzania have documented declines in malaria transmission even before interventions were scaled up. One explanation for these reductions may be the changes in socio-environmental conditions associated with economic development, and in particular improvements in housing construction.This analysis seeks to identify (1) risk factors for malaria incidence among young children and (2) household and environmental factors associated with mosquito vector numbers collected in the child’s sleeping area. Both analyses focus on housing construction quality as a key determinant.Anopheles gambiae and funestus mosquitoes collected per household across three occasions.For 435 children enrolled in a larger trial of intermittent preventive treatment for malaria in infants in the Korogwe District in Tanga, Northeastern Tanzania, detailed information on their dwelling characteristics were collected in the last year of the trial. Principal components analysis was used to construct an index of housing structure quality and converted to quintile units for regression analysis. Univariate and multivariate random effects negative binomial regressions were used to predict risk factors for child malaria incidence and the mean total number of indoor female A. gambiae and funestus numbers by nearly two-fold. Compared to those residing in the lowest quality houses, children residing in the highest quality houses had one-third lower malaria incidence rates, even when wealth and rural residence were controlled for. Living in the highest quality houses reduced vector numbers while having cattle near the house significantly increased them.Building materials have substantially improved in Korogwe over time. Multivariate regressions showed that residing in rural areas (versus urban) increased malaria incidence rates by over three-fold and mean indoor female Results corroborate findings from other studies that show associations between malaria incidence and housing quality; associations were concentrated amongst the highest quality houses. Recently, researchers have documented substantial declines in malaria incidence in eastern sub-Saharan Africa in areas that historically have had high malaria burdens. In coastal Kenya, pediatric malaria admissions have fallen steadily since 1999 and especially dramatically in Kilifi and Kwale While some suggest that these improvements are attributable to increases in access to quality malaria diagnosis and treatment and preventive measures (e.g. insecticide-treated nets (ITNs), indoor residual spraying) in the region. However, intervention coverage rates have only recently increased: greater household ownership of ITNs, ITN use among children under 5, and the proportion of children receiving artemisinin combination therapy (ACT) treatment in Tanzania started in 2004, and only surpassed 50% coverage in 2010 see . ITN vouAlternatively, the early decline in malaria may be related to the rapid changes in socio-environmental conditions associated with economic development in the area. As standards of living improve, individuals are able to spend more on lifestyle improvements, including housing structure improvements and health inputs (preventive and curative measures). A number of studies have documented individual and household risk factors for malaria that highlight the importance of housing structure. For example, malaria infection among children in the Ethiopian highlands was associated with having an earthen roof, animals sleeping in the house, open eaves, an attached kitchen, and having one sleeping room Anopheles gambiae sensu latu (A. gambiae) and Anopheles funestus (A. funestus) mosquitoes  in the bedroom of study participants in an larger intervention study This study has two main objectives: (1) identify risk factors associated with malaria incidence among young children and (2) identify key household and environmental factors associated with the number of female This study was approved by the ethics review boards of the National Institute for Medical Research of Tanzania and the London School of Hygiene and Tropical Medicine. Witnessed, written informed consent was obtained from the caretaker of enrolled study children.This study uses data from a larger, longitudinal trial study of intermittent preventive therapy in infants (IPTi) in the Tanga Region of northeastern Tanzania. The IPTi study area and trial methods have previously been described elsewhere For this study, a subset of 435 children was randomly chosen from the 1240 children still enrolled in the IPTi trial in the last year of observation in the Korogwe District. The random sampling was stratified across three groups based on the cumulative number of malaria episodes over the course of the trial: 0 malaria episodes, 1–2 episodes, and ≥3 episodes. In the last year of the IPTi trial (2007–2008), a survey was conducted for this subset of children to collect individual and household characteristics, household infrastructure, geo-location, surrounding environmental features, and characteristics of the sleeping area of the index child enrolled in the IPTi study. In addition, a historical account of housing improvements was collected by asking caregivers for the type of roofs and walls that their housing unit was made of at the time of different life events: when they were first married, when they had their first child, when they had their last child, and at the time of survey.To collect information about exposure to mosquitoes, an indoor rapid vector assessment exercise was conducted in 2008 for all households of the sample subset of 435 children. For a detailed explanation of mosquito catch data, see Bousema et al. Using data collected on house materials at the time of major life events, the historical trend in housing improvements for the chosen subset of households was constructed. The cumulative proportion of study households changing from thatched roofs to iron roofs and from dirt floors to concrete floors was calculated across urban and rural areas separately.A. gambiae and funestus mosquitoes  are plotted against house quality quintiles . The median of mean vector numbers was also plotted against the presence of cattle near the house–one risk factor emerging from multivariate regression outputs.To assess the association between house quality and malaria incidence, the number of positive blood slides per child per year (standardized for varying time periods of observation) was plotted against an index of the quality of the house resided in at the time the household survey was conducted in the last year of the IPTi trial. Principal components analysis (using only the first component) was conducted using the set of variables collected for house and room construction materials to construct a house quality index. The component items are as follows: roof material, wall material, wall texture, presence of eaves, floor finishing, degree of screening on windows, and presence of a ceiling. This index was then converted to quintile categorical indicators for use in data analysis. Auxiliary analysis showed that higher order principal components yielded little additional explanatory power in regression analyses and thus were excluded, and that univariate and multivariate associations hold when using different numbers of categories for the house index. To assess the association between house quality and mosquito numbers, the median number of female Two types of regression analyses were conducted. First, we estimated univariate and multivariate negative binomial regressions to assess individual and household risk factors for malaria incidence. The number of slide positives per child per year was predicted by the child’s age, mother’s education, household wealth, infrastructure conditions , and rural/urban location. Dummy variables for currently living in a house corresponding to housing indices were included . Historic measures of housing improvements were not used as too few households changed building materials during the years when the IPTi trial was conducted. Malaria prevention measures that would influence the child’s exposure, including use of repellent and insecticide-treated nets were controlled for, as was the arm of the IPTi study that the child was randomized to. Only statistically significant explanatory variables at the 5% level in univariate analysis were included in the multivariate model. To account for spatial autocorrelation across geographic areas, village-level random effects were included. Within villages, households were classified as being located in “urban” hubs or outlying “rural” areas as identified by study surveyors when conducting household visits.A. gambiae and funestus mosquitoes collected from each household from three occasions was predicted using a negative binomial regression. Sensitivity analyses showed that mean total female A. gambiae and funestus mosquito numbers per household yielded more precise estimates of standard errors than regressions predicting each occasion’s collected number separately. A histogram of the per household mean total number of female A. gambiae and funestus mosquitoes did not reveal an excessive number of zeros. Risk factors for mosquito numbers include indicators for house quality, the presence of cattle near the house, distance to the nearest water body, infrastructure conditions, and rural/urban location. Only statistically significant explanatory variables at the 5% level in univariate analysis were included in the multivariate model. Village-level random effects were again included in all regressions to account for spatial autocorrelation.Second, the mean total number of indoor female Culex . Only a small portion was female A. gambiae  and A. funestus . The 435 children in this subset experienced about 1.5 episodes of malaria on average, or 0.8 episodes per child per year when adjusted for observation time. However, malaria incidence was highly skewed as 61% of children did not experience a single episode of malaria. When averaged over the three occasions in each household, the median number of all mosquitoes caught was about 19 . Most mosquitoes were female There has been a noticeable improvement in the quality of houses in the study area. The incidence of malaria (without regression controls) decreased as the housing quality index increased . In partA gambiae. In contrast, in rural areas, vector numbers were a mix of A. gambiae and funestus, and generally decreased as house quality increased, except for houses in quintile 4. More vectors were collected in houses with cattle nearby.The median total number of vectors caught per household (mean of three occasions for each household) by housing index quintile is plotted in Regression results predicting child malaria incidence are shown in A. gambiae and funestus mosquitoes collected per house across three occasions. In univariate analyses, houses in the highest quality quintile, without cattle nearby, with piped water and electricity, and that are located in urban areas all had significantly lower mean vector numbers. In multivariate analysis, rural location  and having cattle near the house  significantly predicted higher mean A. gambiae and funestus numbers. A house in quality quintile 5 was significantly associated with lower vector numbers .A. gambiae and funestus catches were found to be significantly lower in urban hubs. This finding could represent “urbanization” at the village level where the density of housing and the pollution of breeding sites preferred by A. gambiae and funestus mosquitoes reduces malaria transmission. In The Gambia, significant reductions in vectors were also found among houses located within the town center compared to outlying areas This analysis of risk factors for malaria among young children suggests that there may be some role of urbanization and housing materials in reducing risk, even when controlling for socioeconomics differences and preventive behaviors. The incidence of malaria among urban children was nearly one-fourth of those living in rural areas. This may be related to the number of mosquito vectors found indoors. The main urban centre of Korogwe is a small rural town with a population of between 40,000–45,000. In this study, we classified the centre of each village as an “urban” hub with surrounding rural areas. Even when including these “urban” hubs in an overall very rural setting, indoor female A. gambiae nor A. funestus is characterized by marked zoophilic behavior.The analysis also corroborates findings from other studies that show associations between malaria risk and poor housing materials Entomological surveys in nearby villages in Tanga, Tanzania show a large decline in mosquitoes overall between 1998 and 2009 caught in indoor light traps These results should be interpreted in light of additional caveats. Although analyses suggest that house quality was related to both malaria incidence and vector numbers, this relationship was only significant for the highest quality types. Even when examining multivariate regressions using different numbers of housing index categories, there were not significant differences between the majority of houses that were in lower quality tiers. This suggests that other risk factors not included in this analyses may better explain malaria incidence or vector numbers among these households and should be further explored in future analyses.Could house improvements be an intervention to reduce malaria transmission? The protective effects of housing structure improvements such as house screening, closing of eaves, and ceiling installation have been documented since the 19th century In low transmission settings where countries are moving towards elimination and malaria becomes highly clustered Surveying household conditions could aid in defining and identifying hotspots and “hotpops,” or geographic areas or sub-populations where malaria risk is concentrated and where interventions should be targeted


In heavily endemic malaria areas, it is almost inevitable that malarial infection will be associated with anaemia, although malaria may not be the prime cause of it. Anaemia is a major public health problem in Cameroon. We hypothesized that, factors other than falciparum malaria account for anaemia in the study area.Plasmodium falciparum positive children to determine the prevalence, risk factors and the perception of anaemia by the caregivers in a semi-rural community. The investigative methods included the use of a structured questionnaire, clinical evaluation and laboratory investigations.A longitudinal study was conducted among 351 At enrolment the overall prevalence of anaemia as assessed by Hb concentration (Hb < 11 g/dl) was 80.3% (282). Following treatment the prevalence of persistent anaemia was 6% and 46.2% of the children achieved haematological recovery by day 42. Exploratory multiple linear regression analysis showed the following; parasitaemia density (P < 0.01), enlarged spleen (P < 0.05), duration of fever > 2 days (P < 0.01), high white blood cell count (P < 0.001), sex (P < 0.05), iron status indicators (ferritin and transferrin) (P < 0.001), level of education of the caregiver (P < 0.05), management of onset of malaria by caregiver (P < 0.005) and wasting (P < 0.05) to be risk factors for anaemia in children with falciparum infection. Approximately 75.5% (265) of the caregivers had some knowledge about anaemia.The identified risk factors revealed the important contributors to the pathogenesis of anaemia in the Mount Cameroon region. Control efforts should therefore be directed towards proper health education emphasizing on proper health seeking behaviour and attitudes of the population. Plasmodium falciparum[With about 216 million cases of malaria and an estimated 3.3 billion people reported at risk, malaria is still a major public health challenge [alciparum.P. falciparum infection [Anaemia an indicator of both poor nutrition and poor health is a common and sometimes serious complication of nfection ,6. Anaemnfection .The highnfection . The comnfection .et al. [Data on the prevalence of anaemia are limited in this area. Cornet et al.  reportedet al. . Informaet al. .One strategy for reducing the morbidity and mortality that may be associated with anaemia is to identify the predictors that can be readily recognized to facilitate prompt therapeutic interventions especially in children residing in resource-poor settings. Family size, residence, younger age, duration of illness, a palpable spleen or liver, gender, history of fever, pale body, general body weakness, diarrhoea, soil-eating, stunting, malaria parasitaemia and recrudescent infections have been associated with mean haemoglobin levels -15. The et al. [P. falciparum parasitaemia positive and sickle test negative. Children who presented with fever, joint pains, headache, malaise, abdominal pain, nausea and vomiting were considered to be symptomatic . Following government guidelines, children with clinical malaria and malaria parasite positive slides were treated with a standard dose of 4 mg/kg body weight (bw) of Artesunate and 10 mg/kg bw of amodiaquine given once a day for 3 days.The study was carried out in Muea village a semi rural setting in the Mount Cameroon Region. The study area has been described in detail by Sumbele et al. . The chiThis longitudinal study was carried out from January to November 2006, to include the rainy season (April-September) which has been reported as the peak malaria transmission period in the locality . The invA structured questionnaire on the risk factors was administered to caregivers (parents or guardian) in their home settings to obtain information on child and household demographics; treatment seeking behaviour; knowledge, attitude and practices on anaemia; mosquito avoidance practices; anti-malarial drug use and information on type of food consumed in the previous seven days.Clinical evaluation was carried out by trained medical personnel from the Muea health centre in their homes and this consisted of general examination. Axillary body temperature was measured using a digital thermometer. A child was considered febrile when he/she had an axillary body temperature ≥ 37.5°C. Weight and height were measured using a Terraillon weighing scale  and a measuring tape, respectively. Ages of the children were obtained from their mothers and verified from their birth certificates. Height-for-age (HAZ), weight-for-age (WAZ) and weight-for-height (WHZ) standard deviation (SD) scores (z scores) were computed based on the National Center for Health Statistics (NCHS)-WHO growth reference curves using the nutrition module of the Epi Info 2000 programme .On D0 and D42 approximately 4–5 ml of blood sample was collected from the children by venipuncture into 5 ml sterile disposable syringes (Cathy Yougo) and dispensed into micro-containers or vacutainers containing ethylenediaminetetraacetate (EDTA) solution. Drops of whole blood were dispensed immediately on slides to prepare blood films. The caregivers and their children were instructed clearly on how to collect stool samples. Each child was then given a labelled, clean, wide-mouthed and screw-capped container to bring the stool sample. Labelled blood and stool samples were then transported on ice in a cool box to the University of Buea malaria research laboratory for further analyses.R Test Kit  following the manufacturer’s instructions. Anaemia was defined as Hb concentration < 11 g/dL and further categorized as mild (Hb between 10.1–10.9 g/dl), moderate (Hb between 7.0 -10.0 g/dl) and severe (Hb < 7 g/dl) anaemia [The thick and thin blood smears prepared on glass slides at the time of blood sampling were stained with Giemsa stain and examined following standard protocols . Parasit anaemia ,22. Whit anaemia  and Daci anaemia . Determi anaemia .Stool samples were examined for helminths by the Kato–Katz thick smear technique . Plasma Data was doubly entered and validated using SPSS version 11.5 and Epi-info soft ware. Analysis was done with SPSS version 17 . Data was summarized into means and standard deviations, and percentages were used in the evaluation of the descriptive statistics. Chi square (χ2) test was used to compare the prevalence of anaemia in the various groups of subject. The Epi Info package (Epi Nut module) was used to analyze WAZ, WHZ, and HAZ z-scores. Four exploratory multiple linear regression (MLR) models namely; biological (host and parasite factors ), socio economic, management and prevention and integrated models were created containing all the possible associations with haemoglobin as the dependent variable to assess the factors associated with the risk of developing anaemia in children with falciparum malaria. Exploratory analyses were used to reduce the number of potential confounders. The exploratory MLR model was run using the z scores of the variables. All results were considered to be statistically significant at 95% probability level (P < 0.05).The study received administrative approval by the Ministry of Public Health Cameroon. The Ethical Committee of the University of Buea issued the ethical clearance document. Additional authorisation was obtained from the local health committee and the village chief. Children participated in the study if a parent or guardian signed the informed consent form. The parents or guardian and their children were informed that their participation in the study was voluntary and could withdraw at any time without any explanation.P. falciparum infected children  with a mean age of 6.45 ± 7.9 years (6 months to 14 years) residing in the Mount Cameroon region were evaluated for the prevalence and risk factors of anaemia. More than half of the children were infected with soil-transmitted helminths (STH) and the prevalence of fever and malnutrition was low (Table Ascaris lumbricoides (49.0%) with a mean egg per gram of faeces of 549.5 ± 2008.8, followed by Trichuris trichiura  and hookworms .A total of 351 2 = 4.9, P = 0.02) higher prevalence of underweight (11.2%) when compared with those > 5 years (4.7%). Equally, malnutrition was significantly (χ2 = 5.01 P = 0.03) higher in males (28.0%) than in females (17.9%).Stunting, wasting and underweight were prevalent in 20.5%, 2.6% and 8.1% of the children while 6.9%, 0.3%, and 1.4% were severely stunted, wasted and underweight respectively. Children who were ≤ 5 years old had a significantly  in males (27.5%) and females (27.8%). Although not statistically significant (P > 0.05), the prevalence of abnormal transferrin, ferritin and iron values was higher in females  than males .Anaemia as assessed by Hb concentration (Hb < 11 g/dl) was prevalent in 80.3% 282) of the children. The prevalence of mild, moderate and severe anaemia in the study population was, 22.7% (64), 65.2% (184) and 12.1% (34) respectively. Children ≤ 5 years had a significantly (P < 0.01) higher prevalence of anaemia when compared with those greater > 5 years. Similarly the prevalence of anaemia in children with enlarged spleens was significantly (P < 0.01) higher than those with normal spleen . Although not significant the prevalence of anaemia was higher in malaria parasite negative children (34.3%) than their positive counterparts (26.7%) on D21 , D28 (1.7%) and D21 (33.3%) respectively  was prevalent in 6% (7/117) of the children. The prevalence of persistent anaemia in the different sexes were comparable (P > 0.05) even though male children were 0.9times (95% CI: 0.19 – 4.12) more likely to have persistent anaemia than females. Although not significant (P > 0.05), the prevalence of persistent anaemia was higher in children ≤ 5 years (7.0%) than those > 5 years (1.9%) old.On D42 post treatment, 46.2% (54/117) of the children  achieved haematological recovery. Male children were 0.7times (95% CI: 0.4 – 1.4) likely to achieve haematological recovery than their female counterpart. Haematological recovery was comparable (P > 0.05) in children ≤ 5 (39.5%) and those above 5 years (38.5%) of age.The exploratory MLR was run using the z scores of the variables and the tolerance statistics were all below 1. All the Variance Inflation Factors (VIF) were < 2. The lower values of the condition index (1.084 - 4.478) confirm that the collinearity diagnostic was successful.2 = 0.43, adjusted R2 = 0.37) which included iron indices, the variables with significant influence on the Hb concentration were spleen size , sex of the child , transferrin  and ferritin  concentrations.Two biological sub exploratory MLR models were run to examine host and parasite factors associated with Hb concentration. The first sub model examined host and parasite factors associated with haemoglobin concentration while the other examined in addition iron status indicators as risk factors for anaemia. In the first sub model, WBC count (P = 0.001), parasitaemia density (P = 0.002) and length of fever in days (P = 0.001) were significantly (P < 0.01) associated with Hb concentration. Furthermore a significant negative correlation of Hb concentration with parasitaemia density, length of fever in days and WBC count was observed Table . In the 2 = 0.06 and adjusted R2 = 0.01) only the level of education of the guardian/caregiver was identified as a risk factor (P = 0. 04) for anaemia. Children whose caregivers were illiterate had a higher prevalence of anaemia (90%) when compared with those whose caregivers had basic education (77. 8%).In the socio economic exploratory MLR model . Anaemia was more prevalent in those who used other traditional methods of management of malaria (90.2%) than in those who were taken to the hospital as soon as they took ill (73.0%) or those who went to a health facility only after other treatment(s) failed (81.1%).The caretaker management ability of malaria  was the only factor significantly (P = 0.005) associated with the Hb concentration in this model , wasting (P < 0.05), sex (P < 0.05), transferrin (P < 0.01) and ferritin (P < 0.001) concentrations, were significantly associated with haemoglobin concentration in children with falciparum infection Table .Approximately 75.5% (265) of the caregivers had some knowledge about anaemia and the majority (91.3%) of them were cognisant of pallor as the only symptom of anaemia. If and when a respondent discovered the child was anaemic, only 36.1% of them were likely to consult a physician while the rest did self medication. While 16.5% of the caregivers will self medicate with any available iron tablet, 83.5% of them used herbal remedies. About 63.9% of the respondents were aware of iron containing foods of which vegetables (57.3%) and plantains (25.9%) were the foods of choice. A diet recall in the previous seven days showed fish, plantain, huckleberry and red pepper to be frequently consumed foods.et al. [et al. [et al. [Anaemia is a major public health problem in Cameroon. In addition to measuring the haemoglobin concentration which is the most reliable indicator of anaemia at the population level , the cauet al.  in child [et al.  in pre - [et al.  and majo [et al. . Further [et al.  reportedet al. [Following treatment more than half of the children (63/117) never achieved haematological recovery. In line with Obonyo et al.  haematolet al. [P. falciparum parasitaemia to be the single most important factor associated with anaemia. Hyperparasitaemia has been considered as one of the possible manifestations of severe and complicated malaria, depending on the endemic area [While our findings showed a significant negative correlation (P < 0.01) between haemoglobin level and parasite density, others ,25 faileet al.  reportedmic area . AnaemiaIn addition to the density of parasitaemia, hypersplenism is also thought to contribute to the early anaemia of acute malaria through sequestering red blood cells in the spleen . Our stuet al. [et al. [P. falciparum parasitaemia may induce dyserythropoiesis thus contributing to the pathogenesis of malarial anaemia [Protracted infections with malaria parasite are associated with clinically significant RBC destruction . Childreet al.  and Gren [et al.  who repo anaemia . However anaemia .et al. [9/L [9/L, it may be possible that some of these children had concurrent bacterial infections which were not clinically apparent and were beyond the scope of this study. Nonetheless bacteraemia most commonly due to nontyphoid salmonella, has been strongly associated with severe anaemia [We found a significant inverse association between WBC count and haemoglobin concentration. Studies by Ladhani et al.  associatet al. , leucopoal. [9/L . In view anaemia ,36.Although no significant difference was observed in the prevalence of anaemia between the different sexes, anaemia was higher in males than in females. Males were 0.84 (95% CI: 0.59 – 1.20) times more likely to be anaemic than females. Similar observations were made in Kenya ,37, Tanzet al. [Ferritin and transferrin were significantly associated with haemoglobin concentration in the IVM model. Further observations highlighted a significant difference in the prevalence of anaemia between children with abnormal  and normal  ferritin and transferrin values respectively. Stoltzfus et al.  reportedet al. [et al. [et al. [Our study revealed the level of education of the caregiver to be a significant determinant of haemoglobin concentration in children with falciparum malaria in this semi rural setting of the mount Cameroon region. Furthermore a negative trend (r = − 0.16) in the relationship between haemoglobin concentration and education level was observed. Children whose caregivers were illiterate showed a high prevalence of anaemia (90%), when compared with those whose caregivers had basic education (77.8%). Correlation between education level and anaemia has also been reported by Kahigwa et al.  in Tanza [et al.  in Weste [et al.  in MalayThe significant association of the caretaker management ability of malaria and haemoglobin concentration may be linked to the effect of protracted febrile infection with the malaria parasite on the red blood cells. Pre-hospital antimalarial treatment of febrile children by caregivers/parents with mostly traditional herbs or drugs of questionable quality remains a significant common practice among individuals in the population as revealed by the questionnaire survey. Lack of proper education and poverty may be contributing factors to these attitudes and practices. In a previous related study , early tet al. [Wasting a manifestation of acute malnutrition  was signet al.  reportedet al. ,44. HoweEven though the majority of caregivers were aware of pallor as a symptom of anaemia, none could detect whether the child was anaemic before assessment of haemoglobin concentration. The efficiency and applicability of pallor examination in the detection of anaemia is important in clinical circumstances . Signs oIn addition to malaria, enlarged spleen, duration of fever > 2 days, high white blood cell count, sex, iron status indicators (ferritin and transferrin), level of education of the caregiver, management of onset of malaria by caregiver and wasting are important contributors to the pathogenesis of anaemia in the Mount Cameroon region. Improving the case management of malaria is likely to reduce the burden of anaemia hence useful health benefits. Control efforts should therefore be directed towards proper health education emphasizing on proper health seeking behaviour and attitudes of the population. In addition to malaria which is commonly considered to be a principal cause of anaemia additional diagnoses including malnutrition and helminths should be considered.aSignificant at P < 0.05 level, bSignificant at P < 0.01 level, csignificant at P < 0.001 level.D: Day; HAZ: Height-for-age; Hb: Haemoglobin; IVM: Integrated values mapping; MLR: Multiple linear regression model; STH: Soil-transmitted helminths; WBC: White blood cell; WAZ: Weight-for-age; WHZ: Weight-for-heightWe the authors declare that we have no competing interests.IUNS: PhD and Lecturer of Parasitology, Department of Zoology and Animal Physiology.MS: MSc and Assistant lecturer of Biochemistry, Department of Medicine.TNA: PhD and Professor of Medical Parasitology, Department of Microbiology and Parasitology, Dean Faculty of Science.IUNS was involved in all phases of the study, including study design, data collection, data analysis, interpretation, and write-up of the manuscript; TNA. designed and supervised the study and also revised the manuscript. MS was involved in the collection and laboratory examination of samples. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/123/prepub