HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies.HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson’s correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels.In this study, the effect of TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels.After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcγRIIa H/R131 with IgG2 and IgG3 levels; iii) Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual’s humoral response against malaria. Plasmodium falciparum malaria is a major cause of worldwide mortality and morbidity. Host genetic factors have been shown to influence malaria infection intensity and clinical malaria. Several candidate genes have been associated with resistance against severe malaria, whereas linkage or association analyses mapped several loci controlling mild malaria and/or parasitemia [. The HBB locus has been shown to be a major locus based on a genome association study for severe malaria, whereas haemoglobin S (HbS) and haemoglobin C (HbC) have been associated with protection against mild and severe malaria in a large number of studies. It should be stressed that a limited number of genes have been associated with mild or severe malaria in several independent studies; these include FCGR2A that encodes the human IgG receptor FcγRIIa.asitemia . Noticeaasitemia ,3, whereasitemia -10. The P. falciparum IgG antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Passive transfer of IgG has provided protection against the P. falciparum blood stages in humans. In vitro, human IgGs that recognize either infected erythrocytes or merozoites act in cooperation with monocytes to eliminate the parasite [HBB, IL4, TNF, and FCGR2A, which have also been associated with both malaria resistance and IgG levels [HBB, IL4, IL12, TNF, LTA, and NCR3 with mild malaria or parasitaemia has been previously reported in a population living in Burkina Faso [HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the IgG subclass patterns of antibodies against P. falciparum antigens in the same population by using a family-based approach.Anti-parasite . Cytophiparasite ,13. Inteparasite . In thisparasite . In addiparasite -18. Furtparasite . Some caG levels -25. Thisina Faso ,9,10,26.The study population consisted of 220 individuals belonging to 34 families living in urban district of Bobo Dioulasso in Burkina Faso, in which infected mosquitoes were detected only during August, September and October; the numbers of infective bites per person and per year was 30. Blood samples were taken from individuals in July at the end of the dry season (P1) and in December at the end of rainy season (P2). The mean age of sibs was 12.1 + 6.2 years (range one to 34 years). The study population and the area of parasite exposure have been described elsewhere ,27. PhenP. falciparum parasitaemia, fever (axillary temperature more than 37.5°C) and clinical symptoms ; in that case no threshold of parasitaemia was used. In the absence of classical symptoms of malaria, and once others pathologies could not be eliminated, only children (age < 15 years) with more than 5,000 parasites per ml and older subjects with more than 2,000 parasites per ml were considered as having had a malaria attack. Each episode of illness was treated according to the recommendation of the CNRFP  of Burkina Faso. Parasitaemia was checked at the end of the treatment. Subjects who presented at least one mild malaria attack during the survey were considered in the analysis affected, while the others were considered unaffected.Febrile episodes were extensively recorded by active case detection during 24 months. For patients with fever, a thick blood film was prepared by the standard procedures. Diagnosis of mild malaria attack was based on P. falciparum asexual forms were retained to determine parasitaemia. Parasitaemia was defined as the number of parasitized erythrocytes observed per μl in thin blood films.Determination of parasitaemia was described in a previous study . BrieflyMaximum parasitaemia was based on a logarithmic transformation of the highest parasitaemia that was measured in each individual during the survey . Mean ofSince age influenced mild malaria attack, maximum parasitaemia, and the mean of adjusted parasitaemia, the age effect was further taken into account. Linear regression was carried out to calculate the residual for maximum parasitaemia and the mean of adjusted parasitaemia, whereas logistic regression was used to calculate the residual for mild malaria. The residuals were used as phenotypes for linkage and association analyses.P. falciparum blood-stage extracts were previously performed; the data sets previously reported was used [P. falciparum W2 strain (Southeast Asia) was maintained and synchronized. Schizonts were isolated from infected red blood cells based on a treatment with saponin, were sonicated on ice in phosphate-buffered saline containing protease inhibitors. Sonicates were centrifuged, and the supernatants were filtered through a 0.22 μm-pore-size membrane, and were aliquoted and stored at −70°C until use. Enzyme-linked immunosorbent assay (ELISA) plates (Nunc) were coated with 1 μg of P. falciparum extract/ml in sodium carbonate buffer . Plates were saturated with 3% bovine serum albumin in phosphate-buffered saline. Serum dilutions were incubated for 16 h at 4°C . The following monoclonal antibodies were used: anti-IgG1 , IgG2 and IgG3 , and IgG4 . Total IgG was detected using a goat F(ab′)2 anti-human IgG (Jackson Laboratories). The anti-IgG1 and -IgG were conjugated to alkaline phosphatase, the anti-IgG2 and -IgG3 were biotinylated, and the anti-IgG4 was unlabelled. F(ab′)2 anti-mouse IgG conjugated to alkaline phosphatase was used for IgG4 detection. Signal amplification was performed for IgG2 and IgG3 detection by using streptavidin and biotinylated alkaline phosphatase (Pierce); the sensitivity of the assay was 30-fold higher than that of the assay using the same monoclonal antibodies conjugated to alkaline phosphatase. After 2 h of incubation at room temperature, enzymatic activities were revealed by p-nitrophenyl phosphate (Sigma) (1 mg/ml) in Tris buffer (pH 9.6). The optical densities were read at 405 nm using a DIAS automatic plate reader (Dynex Technology). A pool of 200 sera equally diluted was used to draw a standard curve; the same serum pool was used for IgG, IgG1, IgG2, IgG3 and IgG4 measurements. Fifty samples that were randomly selected were titrated; the titration curves were parallel to the standard curve for each IgG subclass. All tests were done in duplicate, and antibody levels were calculated by using the standard curve and were expressed as arbitrary units (AU). To allow for zero values in further analyses, a logarithmic transformation was applied based on log (1 + AU) (LAU) to the AU. The mean LAU and standard deviation were calculated at P1 and at P2. To correct the individual LAU for the visit effect, the LAU was standardized at P1 and at P2, and the mean of adjusted LAU (MALAU) was calculated for each subject, as previously described. The influence of age and sex on MALAU was further evaluated using polynomial regression; the age was considered as a continuous variable. There was an effect of age on IgG subclass and IgG levels (P < 0.01), whereas there was no influence of sex. Age was retained for adjustment for each IgG subclass. The residual was the phenotype used in the statistical analyses. IgG, IgG1, IgG2, IgG3 and IgG4 phenotypes were calculated for 193 individuals.The measurements of IgG subclasses directed against was used . BrieflyBlood samples were taken by venipuncture. The haemoglobin genotypes were identified by electrophoresis of red blood cell lysates on acetate membrane at an alkaline pH. Acetate sheets were stained with Ponceau red. This yielded discrimination of haemoglobin A (HbA), S (HbS) and C (HbC).IL4, FCGR2A, NCR3, TNF, LTA, and IL12B polymorphisms, the data sets previously reported were used [NCR3, TNF, LTA, and IL12B polymorphisms have been obtained by sequencing with a CEQ 8000 automated fluorescent sequencer , whereas FCGR2A H/R131 genotypes was determined by using PCR and allele-specific restriction enzyme digestion methods as previously described [DNA was extracted from mononuclear cells separated by the Ficoll–Hypaque density gradient as described . Samplesere used ,10,29,30The correlation among IgG subclasses was evaluated using Pearson's correlation coefficient. In addition, combined association and linkage analyses were carried out using the family-based association test (FBAT) approaches . The FBAP < 0.0001). In particular, IgG1 and IgG3 levels were highly correlated (r = 0.631). The best correlations were between IgG levels and IgG1 levels on the one hand and IgG levels and IgG3 levels on the other hand (r >0.7).Table LTA + 80, TNF-1031, TNF-238, TNF1304, and NCR3-412 were associated with parasitaemia or mild malaria in this sub-population  and previously reported family-based associations with parasitaemia and mild malaria are shown in Table ulation  . Moreoveulation  .P. falciparum crude extract was further assessed. The additive, the dominant, and the recessive models were used for each SNP. The FBAT analysis gave a number of significant results at the nominal level of 0.05, although there was no association of HbS, IL12Bpro, and IL12B 3’ UTR, IL4-590, NCR3-412, NCR3-3790, TNF-238, TNF-308, and TNF-1031 polymorphisms with the IgG responses  and IgG against P. falciparum extract in a population living in an endemic area in Burkina Faso. Most of the candidates have been associated with parasitaemia, mild or severe malaria [The aim of this study was to determine the effect of several candidate gene polymorphisms on the IgG subclass responses against  malaria .P. falciparum IgG subclass levels was observed, the best correlation being between IgG1 and IgG3 levels. The results are similar to a study in Thailand, where anti-malarial IgG3 levels were correlated to IgG1 and IgG2 levels [IL4-590 polymorphism does not explain, nevertheless, variation in the level of IgG subclasses, suggesting that other IL4 polymorphisms should be investigated. In addition, other cytokines, such as IL2, IL6, or IL10 are also known to enhance the IgG production in vitro by B cells activated by polyclonal activators or plasmodial antigens [in vitro, and to enhance IgG2 production [There was positive correlation between all measured antibody levels. Anti-malarial IgG levels were correlated with all anti-malarial IgG subclasses. The best correlations were the correlations of anti-malarial IgG levels with anti-malarial IgG1 and IgG3 levels. A correlation between all anti-2 levels . The res2 levels ,35. Cyto2 levels . IL4-590antigens ,38. Besiantigens . Interesoduction . Althougoduction . These oIL4 and IL12B polymorphisms, while others reported an association of anti-malarial IgG levels with IL4-590 [NCR3 polymorphisms, although those may alter the production of IFN-gamma by NK cells. The association of LTA + 80 with all anti-malarial IgG subclass levels was significant at the nominal level of 5%, but was no more significant after applying a multiple test correction; this polymorphism may, nevertheless, require further investigation to confirm or invalidate the trend.The family-based associations of candidate polymorphisms on the IgG subclass levels were further investigated. There were some significant results after applying a multiple test correction. There was no association of IgG subclass levels with  IL4-590 . There wHBB polymorphisms alter the anti-malarial IgG production. This result is consistent with previous reports also providing evidence for an involvement of HbC in the immune response against infected erythrocytes through enhanced IgG production [HbS and HbC have been associated with protection against mild and severe malaria, and it has been proposed that these polymorphisms could increase the elimination of the parasite through acquired immune mechanisms . There woduction ,44. ThisP. falciparum malaria in a population living in Burkina Faso [et al. found that the FcγRIIa R131 allele carriers in the Fulani had higher anti-malarial IgG3 levels than the individuals homozygous for the FcγRIIa H131 allele. One might speculate that more IgG3 will be present in the circulation of individuals with the FcγRIIa R131 allele, because the FcγRIIa H131 allele has a higher binding than the FcγRIIa R131 allele [The FcγRIIa H131 allele has been associated with protection from severe malaria in several populations [ina Faso , and in ina Faso ,46. The ina Faso ,47. On tina Faso ,23. Toge1 allele . Neverth1 allele . It is l1 allele .TNF polymorphisms have been related to severe malaria. SNPs at position −1031,-857,-308,-238 and −863 in the promoter region of TNF gene exhibit differential associations to malaria and TNF production in different populations suggesting that individual TNF responses may be genetically determined [TNF1304 within intron 3 was associated with variation in mild malaria and parasitaemia [TNF-308A and TNF-238A allele have been associated with high anti-P. falciparum antibodies [TNF-308 and TNF-238 polymorphisms. TNF-863A and TNF-857 T were, however, associated with high anti-malarial IgG3 and IgG levels, respectively. Moreover, there was an association of low IgG3, IgG4, and IgG levels with TNF1304, which has been associated with mild malaria and parasitaemia in the same population [TNF1304 on malaria phenotypes, although the effect of the polymorphism on the molecular function or the gene expression has not been reported. In contrast, the molecular effect of polymorphisms within the TNF promoter has been studied. Other authors reported that the transcription factor OCT-1 binds specifically to the TNF-863A and TNF-857 T alleles, that the transcriptional promoter activity of a haplotype harbouring TNF-863A and TNF-857 T was higher than the one of other haplotypes in Japan [TNF polymorphism with IgG response could be that TNF gene is important in the development of humoral response as an autocrine B-cell growth factor [In previous studies, termined ,51. TNF1tibodies ,25. In tin Japan ,51. An eh factor .TNF-857T, and TNF1304A were associated with high levels of anti-malarial IgG. The FcγRIIa H131 allele was associated with high levels of anti-malarial IgG2 and low levels of anti-malarial IgG3. TNF-863A was associated with high levels of anti-malarial IgG3, whereas TNF1304 was associated with variation in the levels of anti-malarial IgG3 and IgG4. It should be stressed that all these polymorphisms have been associated with parasitaemia, mild malaria or severe malaria; this suggests that their protective effect may be partly due to their effect on the IgG subclass production.Our results indicate that HbC, FCGR2A: Fc-gamma Receptor IIA; FDR: False discovery rate; HbC: Haemoglobin C; HbS: Haemoglobin S; IL: Interleukin; IgG: immunoglobulin G; LTA: Lymphotoxin α; NCR3: Natural cytotoxicity receptor 3; P. falciparum: Plasmodium falciparum; TNF: Tumor necrosis factor; UTR: Untranslated region.The authors declare that they have no conflicting interests.SA calculated the IgG subclass phenotypes, evaluated the correlation between IgG subclass phenotypes, and carried out most of the FBAT analyses. AA and SG participated in the FBAT analyses, and the multiple test correction. FF participated in the design of the study, and revised the results and the manuscript. PR performed the design of the study, supervised the IgG phenotype determination and the statistical analyses, and wrote the manuscript. All authors read and approved the final manuscript.Family-based association of polymorphisms with parasitological and clinical phenotypes.Click here for file


In a linked Perspective, Roly Gosling and Michelle Hsiang discuss the importance of non-falciparum malaria species to regional and global health.Please see later in the article for the Editors' Summary PLOS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1001575Douglas NM, Lampah DA, Kenangalem E, Simpson JA, Poespoprodjo JR, et al. (2013) Major Burden of Severe Anemia from Non-Falciparum Malaria Species in Southern Papua: A Hospital-Based Surveillance Study. PLoS Med 10(13): e1001575. Plasmodium species in southern Papua, Indonesia.Ric Price and colleagues use hospital-based surveillance data to estimate the risk of severe anemia and mortality associated with endemic PLOS Medicine, Ric Price and colleagues compare the burden of anemia in different plasmodia species in a robust hospital-based surveillance study in Eastern Indonesia Plasmodium falciparum. While the study is observational, and limited in its ability to control for co-morbidities , the numbers are impressive, with more than 200,000 outpatient and inpatient episodes with hematological assessment. Their findings reveal a significant burden of severe anemia (defined as hemoglobin less than 5 g/dl) due to P. vivax, P. malariae, or mixed species infections (adjusted population attributable fraction 12.2%), in addition to P. falciparum monoinfection (15.1%). Notably, severe anemia in infants attributable to P. vivax was 30.4% compared to 20.5% for P. falciparum. Patients with severe anemia were substantially more likely to be admitted to the hospital  and to die .In this week's P. falciparum receives the most attention because it causes the most deaths, largely in Africa, despite P. vivax being the most geographically widespread species of human malaria. More than 2 billion people are at risk of contracting P. vivax, stretching from Latin America, northern Africa, Arabia, Central Europe, to Asia and the Pacific P. falciparum, because a wider variety of Anopheles species with varied habits spread the infection; and because the dormant liver stage, the hypnozoite, can cause relapses, from days to years later and is notoriously hard to treat. There is a misconception that P. vivax is a benign disease despite it being well established that P. vivax causes severe chronic illness and newer studies linking it to severe illness and death P. falciparum yet continue to battle to eliminate P. vivaxP. falciparum have been a strong argument for global investment in control and elimination of P. falciparum, including drug-resistant P. falciparumP. vivax control and elimination can start to be built, supporting an investment case for the elimination of P. vivax. Indonesia, where this study took place, is working with 14 other countries in the Asia Pacific, the Asia Pacific Malaria Elimination Network (APMEN) P. vivax, which is the dominant species of Plasmodia in the region, and is in need of such economic arguments.Numerous countries have successfully eliminated P. falciparum endemic areas and found that treatment of anemia during an acute attack of malaria improves hemoglobin recovery and does not increase the risk of treatment failure or death For severe anemia due to non-falciparum infections, some clinical management issues arise. Patients are likely to require blood transfusion, which carries risk for a variety of transfusion-related reactions and transfusion-associated infections such as HIV and hepatitis P. vivax specifically, antimalarial treatment and anemia are inextricably connected. Radical cure requires the use of a hypnozoitocidal agent, of which the only one widely available is primaquine. This 8-aminoquinoline causes hemolysis that can be severe in patients with underlying glucose-6-phosphate dehydrogenase (G6PD) deficiency, found commonly in malaria endemic areas P. vivax.For P. falciparum endemic areas was cautioned following a study of routine iron and folate supplementation to children in Zanzibar, Tanzania that showed an association with severe illness and death related to iron supplementation and P. falciparum infection On a population level, chronic anemia is well known to cause poor growth and cognitive deficits in children. Chronic anemia can also predispose to severe anemia in the setting of a malaria infection. Community preventative treatment of children with iron supplementation to prevent anemia in P. vivax are less well established in the region, where outdoor biting and resting Anopheles are common. More research is needed on methods to reduce transmission of all plasmodia species in the Asia Pacific, such as the use of active case detection to find asymptomatic infections, drug based strategies including IPT regimens, mass drug administration to reduce the reservoir of human infections, development of sensitive rapid diagnostic tests for P. vivax or other non-falciparum species, and alternative vector control methods There is also a need to evaluate the impact of standard and new malaria control interventions on morbidity and mortality associated with non-falciparum malaria. Community benefits of vector control using insecticide-treated bed nets (ITNs) for P. falciparum, to previously under-recognized, but important, clinical and public health issues related to the other human infecting plasmodia. Let us hope that investment follows suit.The study by Price and colleagues requires our thinking to expand beyond


To present a new approach for estimating the “true prevalence” of malaria and apply it to datasets from Peru, Vietnam, and Cambodia.Bayesian models were developed for estimating both the malaria prevalence using different diagnostic tests , without the need of a gold standard, and the tests' characteristics. Several sources of information, i.e. data, expert opinions and other sources of knowledge can be integrated into the model. This approach resulting in an optimal and harmonized estimate of malaria infection prevalence, with no conflict between the different sources of information, was tested on data from Peru, Vietnam and Cambodia.Malaria sero-prevalence was relatively low in all sites, with ELISA showing the highest estimates. The sensitivity of microscopy and ELISA were statistically lower in Vietnam than in the other sites. Similarly, the specificities of microscopy, ELISA and PCR were significantly lower in Vietnam than in the other sites. In Vietnam and Peru, microscopy was closer to the “true” estimate than the other 2 tests while as expected ELISA, with its lower specificity, usually overestimated the prevalence.Bayesian methods are useful for analyzing prevalence results when no gold standard diagnostic test is available. Though some results are expected, e.g. PCR more sensitive than microscopy, a standardized and context-independent quantification of the diagnostic tests' characteristics (sensitivity and specificity) and the underlying malaria prevalence may be useful for comparing different sites. Indeed, the use of a single diagnostic technique could strongly bias the prevalence estimation. This limitation can be circumvented by using a Bayesian framework taking into account the imperfect characteristics of the currently available diagnostic tests. As discussed in the paper, this approach may further support global malaria burden estimation initiatives. Within this context, being able to estimate with confidence the malaria prevalence in a given country/district is essential for targeting control/elimination efforts, monitoring the progress towards established goals, e.g. the Millennium Development Goals, and documenting achievements Though malaria remains a major public health problem worldwide, particularly for the poorest countries The other assumption made by Hui and Walter is that diagnostic tests are conditionally independent given the true (but latent) prevalence of infection. From a practical point of view, such assumptions can be incorrect and several approaches to circumvent them have been suggested The performance of different diagnostic techniques depends on the malaria species, the parasite density, previous treatment, gametocytaemia and the quality of the diagnostic method Given that the diagnostic tests' characteristics, i.e. sensitivity and specificity, can be variable and context-specific and that no gold standard is available, combining all available information can be an interesting approach. Indeed, results obtained by different diagnostic tests are related more or less closely to the ”true” malaria prevalence so that it may be useful to estimate it by considering the results obtained by all tests available. As already shown P. falciparum infection, i.e., a case is defined as a currently infected individual, and that the sensitivity and specificity of the two diagnostic tests are unknown. The class of models in which the infection status is unknown is sometimes referred to as latent class models, i.e. the infection status is latent as it exists but is not evident or detected by a diagnostic test. In the analysis of the diagnostic test characteristics, the following notations can be used  and “|”meaning conditional on (or “given”):The concept of analyzing multiple diagnostic tests can be explained by as an example assuming that only two diagnostic tests are available. The model also assumes that there is a single true but unknown prevalence of infection status D: D = 1: infected, D = 0: not infectedtest result: Yj = 1: positive, Yj = 0: negativesensitivity of test j (Sej): P(Yj = 1 | D = 1)specificity of test j (Spj): P(Yj = 0 | D = 0)prevalence of infection in the study population (π): P (D = 1)Assuming independence of Y1 and Y2 and given the infection status, then:Expanding this to all possible outcomes of two tests' results in a set of 4 equations ( = model):This provides 3 independent equations  and 5 parameters to estimate. In mathematical terms this is consequently not estimable but it would be when fixing in a deterministic way some parameters or using in a probabilistic way prior information for some of them, e.g., inclusion of the experts' opinions. This approach allows the incorporation of knowledge, such as historical information from experiments similar or related to the one under study, an educated guess about outcomes or even subjective beliefs of the investigator (i.e. expert opinion). These prior probabilities are then updated in a rational way after data collection. Bayesian statistics have recently experienced an explosive growth, with many applications in different areas. Powerful computers and software packages as R and WinBUGS are now common and Bayesian statistics theory is now commonly applied in the development of powerful algorithms and models that process data in new ways Extending the example to 3 tests will result in 7 independent equations and 7 parameters to estimate, meaning that the equations are “estimable”. With 4 or more tests, there are more equations than parameters and the number of estimable parameters exceeds that to estimate. The model (set of equations) is then over specified. For many years, it has been assumed that two (or more) diagnostic tests are conditionally independent given the true (but latent) prevalence of infection Since none of the diagnostic tests included in this study can be considered as the gold standard, a Bayesian approach can be used, i.e. combining data with prior information to estimate the malaria prevalence and the test characteristics. In this paper, a multinomial Bayesian model adapted from Berkvens et al. Within the multi-diagnostic Bayesian framework, there are mostly more parameters to estimate than equations, especially when conditional dependence is taken into account. This requires the inputs from experts for some of the parameters, i.e. the sensitivity and specificity. They are asked to provide both their estimations and an expression of uncertainty . In this study, prior information on the test characteristics was obtained from four experts at the Institute of Tropical Medicine, Antwerp, Belgium, and was expressed as conditional probabilities.The match between the experts' opinions, any other prior information and the observations can be evaluated through the Bayesian p-value (Bayes-p), the Deviance Information Criterion (DIC) The correspondence between the pD and DIC values calculated in the posterior mean of the multinomial probabilities and in the posterior mean of the parameters of the model (parent nodes) was checked. The trend of the Bayes-p P. falciparum. The credibility intervals for differences between the characteristics of the diagnostic tests in different conditions with both limits having the same sign  can be interpreted as the equivalent of a significant difference in a frequentist approach.The analysis was done in WinBUGS 4 and R 2.11.0. Three chains, 20,000 iterations, following a burn-in of 5,000 were used to assess the convergence of the results. The sensitivity and specificity of the diagnostic tests and the malaria prevalence were estimated by the model. The prevalence was defined as the proportion of individuals infected by P. falciparum infections, an antibody detection ELISA test was used using a specific antigen for Plasmodium. falciparum  . For the detection of the specific Plasmodium species DNA, a semi-nested multiplex PCR was used as described by P. falciparum amplicon of 395 bp were considered positive while samples showing no amplification or a PCR product of a different size (indicating infection with another species) were considered negative. Samples showing mixed infections of P. falciparum with other species were considered positive. A parasitological diagnosis was performed using standard microscopic reading of thick and thin blood films. Details of slide preparation and reading procedures were published elsewhere To detect specific antibodies against The 3 diagnostic tests were performed on blood samples obtained from individuals living in 3 different countries, Vietnam, Cambodia and Peru, the latter contributing with 2 sites while samples from Cambodia were obtained in the same site but at 2 different time points. Therefore, 5 different datasets were used for this analysis. All the named institutional review boards or ethics committees specifically approved the study. Each study protocol had been reviewed by the ethical committee of the ITM and University of Antwerp as well as by respective national ethical committees for each country .Anopheles. dirus sensu stricto being the main vector. The survey was part of a community-based trial aimed at determining the effectiveness of long-lasting insecticidal hammocks (LLIH) in preventing forest malaria Samples were collected during a cross sectional survey carried out in November-December 2004 (end of the rainy season) in 33 rural communities located in 2 forested districts of Ninh Thuan Province, Central Vietnam. Malaria transmission in the study area was relatively low but perennial with 2 peaks (May-June & October-November), with the sylvatic species The cross sectional survey was carried out in April-May 2006 (end of rainy season), in the peri-urban area of Jaen city, the capital of Cajamarca Dept, Northern Peru. Households were chosen randomly and all family members screened for malaria parasites in order to reach a total sample size of 504 individuals (age 6 months–50 years). This study was submitted for ethical approval to the Ethics Review Board of the Universidad Peruana Cayetano Heredia, Lima, Peru (Code SIDISI: 051675).This study was carried out in several communities  near Iquitos City, the capital of Loreto Department, situated in the Peruvian Amazon, within the activities of the Multi-Country Malaria Project “Malaria control on the cross border areas of the Andean Region: A community based approach” - PAMAFRO together with the National Malaria Control Program. Blood samples were collected in all febrile patients presenting at their health posts or health centers between November and December 2006 (beginning of the rainy season). This study was submitted for ethical approval to the Ethical Review Committee of the Universidad Peruana Cayetano Heredia (EC-UPCH), Lima.A malariometric surveys were conducted in August-September (rainy season – survey 1 (S1)), and in November-December 2005 (end of rainy season - survey2 (S2)) in 12 villages located in the forest areas of north-east (Rattanakiri -6 villages) and west Cambodia . A representative sample of the inhabitants of each village has been examined . The protocol was approved by the Commission of Medical Ethics of the Prince Leopold Institute of Tropical Medicine (Ref: 05 10 4 491).In all surveys, selected individuals were explained in the local language the study objectives, methodology, risks and benefits, and were asked to give their informed consent. In both Peruvian studies, written informed consent was given by all study participants.In Vietnam and Cambodia, verbal informed consent was given by all study participants. The institutional review board of the Institute of Tropical Medicine approved this verbal consent.Positive patients were treated according to the national guidelines. Blood samples for microscopic examination (thick and thin blood film) and for later genotyping and serology on filter paper (Whatman filter paper grade 3) were collected. PCR analysis was not carried out on the Cambodia samples.The apparent prevalence was relatively low in all sites, with ELISA giving the highest estimates , as confSeveral significant differences were observed between the test characteristics in the 5 different conditions. Notably, the sensitivities of microscopy and ELISA were statistically lower in Vietnam than in Peru-Iquitos, Peru-Jaen and Cambodia (S1 and S2). Similarly, except for the ELISA in Cambodia S2, the specificity estimates for microscopy, ELISA and PCR were significantly lower in Vietnam compared to the corresponding ones in the other sites. The estimated true prevalence was significantly higher in Vietnam than in the other 4 sites among which Peru-Iquitos had the highest prevalence .A comparison between the “true” and apparent prevalence provides the degree of bias when this is estimated with only one diagnostic test. In Vietnam and Peru, microscopy was closer to the “true” estimate than the other 2 tests  and 5 whAn analysis of three tests for the detection of a malaria infection and for estimating its prevalence was conducted using a Bayesian framework. Bayesian techniques become exceedingly useful for improved interpretation of diagnostic test performance in both the medical and veterinary fields The characteristics of the diagnostic tests employed were estimated without a gold standard. This contrasts with the common practice of estimating the sensitivity and specificity of a test by comparing its results with those obtained by microscopy The expert opinions originally provided for the 3 tests were not in agreement with the data so that constrains (i.e. the level of “uncertainty”) on the sensitivity of microscopy and ELISA in Vietnam had to be relaxed. This adaptation allowed agreement between prior information and actual data and resulted in a significantly lower sensitivity for the microscopy in Vietnam compared to the other regions, confirming the high variability of this test's sensitivity that depends both on the parasite density and on the experience and skills of the slide reader. Indeed in Vietnam, the parasite density is usually low and PCR data indicate a high proportion of sub-patent infections, as well as mixed infections. Therefore, sensitivity and specificity of a given test can vary according to the setting and such variability can explain the wide confidence intervals reported in other studies In Peru-Iquitos, comparable results were obtained with both microscopy and PCR. This finding can be attributed to the sampling of symptomatic patients in whom parasite density is usually higher than in individuals, often healthy, selected randomly for a population survey. The latter infections are asymptomatic, hence with a lower parasite density, or even sub-patent, i.e. undetectable by microscopy.Combining several diagnostic tests does not imply that the results of each separate test do not have any value. Although positive serological tests may reflect persisting antibodies in non-infected individuals, and hence the lower specificity, they have an advantage of providing an indication of individuals having had a past infection, a valuable information in areas where malaria transmission is very low.The approach based on the Bayesian framework may be useful to re-examine data obtained with several malaria diagnostic tests The recent shift from control to elimination with eventually eradication as a goal will require a rigorous assessment of the disease  free state. Combining information from different diagnostic tests may respond to this need and provide an assessment on the uncertainty related to a disease  situation. Moreover, the approach based on the Bayesian framework could be used for future studies and the obtained “latent” prevalence could then fit nicely within the global malaria initiatives such as the malaria atlas project (MAP)


The highlands of Ethiopia, situated between 1,500 and 2,500 m above sea level, experienced severe malaria epidemics. Despite the intensive control attempts, underway since 2005 and followed by an initial decline, the disease remained a major public health concern. The aim of this study was to identify malaria risk factors in highland-fringe south-central Ethiopia.Plasmodium infection status (positive or negative) and six variables. Both fixed- and random-effects differences in malaria infection were estimated using median odds ratio and interval odds ratio 80%. The odds ratios and 95% confidence intervals were used to estimate the strength of association.This study was conducted in six rural kebeles of Butajira area located 130 km south of Addis Ababa, which are part of demographic surveillance site in Meskan and Mareko Districts, Ethiopia. Using a multistage sampling technique 750 households was sampled to obtain the 3,398 people, the estimated sample size for this study. Six repeated cross-sectional surveys were conducted from October 2008 to June 2010. Multilevel, mixed-effects logistic regression models fitted to Overall, 19,207 individuals were sampled in six surveys . Six of the five variables had about two-fold to eight-fold increase in prevalence of malaria. Furthermore, among these variables, October-November survey seasons of both during 2008 and 2009 were strongly associated with increased prevalence of malaria infection. Children aged below five years (adjusted OR= 3.62) and children aged five to nine years (adj. OR= 3.39), low altitude (adj. OR= 5.22), mid-level altitude (adj. OR= 3.80), houses with holes (adj. OR= 1.59), survey seasons such as October-November 2008 (adj. OR= 7.84), January-February 2009 (adj. OR= 2.33), June-July 2009 (adj. OR=3.83), October-November 2009 (adj. OR= 7.71), and January-February 2010 (adj. OR= 3.05) were associated with increased malaria infection.The estimates of cluster variances revealed differences in malaria infection. The village-level intercept variance for the individual-level predictor  and final  were lower than that of empty .Malaria control efforts in highland fringes must prioritize children below ten years in designing transmission reduction of malaria elimination strategy. About half of the total population living between altitudes of 1,500 and 2,500 m above sea level (masl) is at risk of malaria and the areas experience epidemics in Ethiopia . MoreoveSince 2005, Ethiopia has scaled up malaria control programs using key malaria interventions such as effective case management , and vector control options  in endemic areas . Subsequently, the program obtained fruitful results in reducing malaria burden between 2006 and 2008. In addition, the 2011–2015 National Strategic Plan highlights the intent to eliminate malaria in specific geographical areas with historically low malaria transmission; and achieve near zero malaria deaths in the remaining malarious areas of the country . PrevaleAccordingly, identifying factors influencing malaria infection both at individual-and village-levels (clusters of household) appears useful in guiding targeted malaria interventions at highland areas with malaria low prevalence and at high epidemic risk. In addition, the Ministry of Health of Ethiopia recommended evidence-informed decision to incorporate high altitude areas  in malaria control program . Thus, ukebeles  in Butajira area using the demographic surveillance system site at Butajira Rural Health Programme (BRHP) [This study was conducted in six rural e (BRHP) , locatedkebeles. A recent study found a very low household ITN ownership  with the higher proportion at low (54.2%) than high (3.5%) altitudes. Most of those households  had at least a family member who slept under a net the night prior to the survey [Malaria is one of the important causes of sicknesses in Butajira area. Between 2004 September and 2010 August, 32.3%  were microscopically confirmed malaria cases from Butajira and Enseno Health Centres, and Butajira Hospital. On average, more than 10 thousand malaria suspected cases visited these public health facilities between 2002 and 2010. Indoor residual spraying (IRS) operation was performed mainly for epidemic control in the low altitude areas of the present study area. During 2009/2010, a spraying of houses was done to control malaria outbreak in Hobe and Bati Lejano e survey .The sample size required for this study was estimated as follows. Estimation of the sample size for malaria prevalence was based on 4.1% prevalence from three Ethiopian regions. The study used a sample size estimated  to measure malaria prevalence in this study area ; this sakebeles , two from each of the three different altitudinal strata, were selected. These strata were low, mid-level, and high altitude areas. These localities with varying altitude were found to be suitable for the present study. The target of our survey was 4,816 households residing in those six kebeles. Sixteen villages (or clusters of households) with 750 households were randomly selected from the six kebeles for the surveys using probability proportion to size (PPS) sampling method. Since it was expected that lower malaria prevalence in the highlands, proportionally selected more households from the highlands. Six cross-sectional surveys were done on the same households for two consecutive years. Blood specimens were collected from October 2008 to June 2010 on a quarterly basis. The surveys were conducted in October-November 2008 (a month after the main rainy season), in January-February 2009 (dry season), in June-July 2009 (main rainy season), in October-November 2009, in January-February 2010, and in June 2010.This study used community-based repeated cross-sectional survey. Six rural Plasmodium parasites.This study obtained data from household head interviews of sampled households and blood film collection and examinations of family members. Trained data collectors conducted the interviews using a pretested and structured questionnaire to obtain baseline socio-demographic and household characteristics. Standard procedures were followed for blood specimen collection, processing, microscopic examination and reporting of malaria parasites . Thin anAll positive slides and 10% of slides with negative results were sent to another microscopist blinded to the microscopy results to ensure quality of light microscopy. To ensure maximum response rate of participants, households with absentees were revisited once more. The altitude readings of the sample households were recorded using hand-held Global Positioning System (GPS) (Garmin eTrex ®). The principal investigator (AW) and two data collectors (postgraduate students from Addis Ababa University) conducted the GPS recording.Teff (Eragrostis tef), pepper, Enset (Ensete ventricosum), and Khat (Catha edulis). House construction materials were types of roofing and presence of window.This study used a dataset of relative household wealth index data computed in another study . The proSuitability of the data for factor analysis was assessed before performing PCA. Thus, both the Kaiser-Meyer-Oklin (KMO) value and Bartlett’s Test of Sphericity results were supporting the factorability of the correlation matrix. PCA result was repeated with alterations until the resulting model was suitable for the survey data. Finally, 11 indicators were selected to run the final PCA. PCA revealed the presence of two components with eigenvalues above 1, explaining 36.0% and 9.8% variance in the dataset, respectively. The first principal component  represented 36.0% of the variance in the sample and was used to generate the wealth index of the study households.The 11 variables with greatest weights were loaded on the first principal component: possession of motorcycle (0.937), sewing machine (0.869), truck (0.869), television (0.809), grain-mill (0.646), lantern-kerosene (0.610), phone (0.575), electricity line (0.568), bicycle (0.423), types of sleeping places (0.356) and cart (0.355). The wealth index varied from −0.256 to 13.27. Then, all households were ordered into three wealth groups: the “lowest” ranked group , followed by the “middle” ranked group , and finally the top third in the “higher” ranked group . Data collectors assessed physical condition of houses and recorded the data. Training manual was distributed to help in categorizing the houses into three including dilapidated, houses with their walls allowing mosquito entry and good condition.Plasmodium parasites in blood films upon microscopic examination was the primary outcome of this study. The results expressed as parasite prevalence, i.e. the percentage of Plasmodium positive subjects. The factors used to explain outcomes are defined at two levels: individual  and village .Finding Variables were categorized and coded as follows. Age grouped into four classes and coded as <5, 5–9, 10–14, and ≥15 years. Altitudinal location of households was classified into three including low , mid-level , and high  altitudes. There were three wealth groups including lowest, middle, and higher; and three house status categories such as dilapidated, walls with holes, and good condition. The survey seasons are October-November 2008, January-February 2009, June-July 2009, October-November 2009, January-February 2010, and June 2010. In all cases the last categories were considered as reference groups.Plasmodium infection, the outcome variable. This model computes the regression using a two-stage system of equations, which involves the lowest level and higher level equations. In this case, individual as the lowest level that explains the individual variation within each village; and the village-level that explains variation across villages were entertained as suggested [Data entry and cleaning was done using Epi Info version 6 (Centers for Disease Control and Prevention (CDC), Atlanta, Georgia (USA). Descriptive statics was performed using IBM® SPSS® Statistics version 20.0. Descriptive statistics was performed to describe characteristics of predictor variables. Multicollinearity was checked using linear regression as recommended and no multicollinearity was evident. Multivariate analysis was done using STATA version 11.0 . Mixed-effects logistic regression was fitted using selected independent variables to estimate individual uggested .Multilevel analysis is a statistical tool applied to data with nested sources of variability, which involve units at lower level nested within units at a higher level . MoreovePlasmodium infection. MOR and IOR-80 are recommended for estimating variability of binary outcomes to overcome contextual and interpretational problems with ICC in a binary outcome [latent variable method that converts the individual level variance from the probability scale to the logistic scale, on which the area level variance is expressed. Thus, in this study, the method assumes that the propensity for malaria infection detected is a continuous latent variable underlying the binary outcome . This meant, every person has a certain propensity for getting malaria infection but only persons whose propensity crosses a certain threshold detected as Plasmodium positive. The unobserved individual variable follows a logistic distribution with individual level variance equals to π2/3  [Intra-class correlation coefficients (ICC), median odds ratios (MOR) and 80% interval OR (IOR-80) were computed to estimate village-level variance in  outcome . In mult outcome . This st., 3.29) . However., 3.29) .MOR was computed to translate the area level variance in the widely used odds ratio scale. The MOR is defined as the median value of the odds ratio between the area at higher risk and the area at lowest risk when randomly picking out two areas the MOR can be conceptualised as the increased risk that (in median) would have if moving to another area with a higher risk . The MORp<0.05.In order to integrate the area level fixed effect and the random residual variations using the 80% interval odds ratio (IOR-80%) is suggested . In inteTo assess whether a model predict the outcome variable beyond what would be expected by chance, the familiar Chi-Square likelihood-ratio test of a difference between models is used. The degrees of freedom for the Chi-Square are the differences in the number of parameters for the models being compared .In this study, individual-level predictor model  compared against the full model . By subtracting, the difference (969.7091 and 886.97871) is 55.9 and this value showed statistically significant with (15–4) = 9 df, so the full model leads to prediction that is significantly better than chance.Ethical approval of the study was obtained from the Faculty of Medicine at Addis Ababa University, and the Ethiopian Ministry of Science and Technology. Individual informed consent was obtained from adults, and from the parents or guardians of children aged less than 18 years. In addition, minors gave verbal assent. Blood specimens were collected as recommended using an alcohol swab and disposable blood lancets by trained staff . All peoOverall, 19,207 individuals were sampled in six surveys . Most of the participants were 15 years old and above with a mean (±SD) age of 20.5 (±17.2), and the range was between one month and 99 years. Above half (51.3%) of the participants were females. A total of 3,416 participants were included in the baseline survey conducted during October-November 2008. In the consecutive five follow-up visits, there were 3,205 (January-February 2009), 3,227 (June-July 2009), 3,210 (October-November 2009), 3,127 (January-February 2010), and 3,022 (June 2010) participants sampled  and the rest due to Plasmodium falciparum  and mixed infections  [A recent study estimated 0.93%  of the participants had malaria infection. Most of the infections were due to lciparum .4%, n=22Plasmodium infection. Similarly, survey seasons including October-November 2008 (unadj. OR=7.95), January-February 2009 (unadj. OR=2.35), June-July 2009 (unadj. OR=3.76), October-November 2009 (unadj. OR=7.68), and January-February 2010 (unadj. OR=2.93) showed higher malaria prevalence , children aged five to nine years (unadj. OR= 3.40), low altitude (unadj. OR= 5.12), mid-level altitude (unadj. OR= 3.63) and houses with holes (unadj. OR= 1.57) had increased risk of having Multilevel logistic regression of the fixed effects showed that age, altitudinal location, house status, and seasons were related to higher risk of malaria infection. These variables had about two-fold to eight-fold increase in prevalence of malaria. Furthermore, among these variables, October-November survey seasons of both during 2008 and 2009 were strongly associated with increased prevalence of malaria infection (Table The estimates of cluster variances (or random effects) revealed differences in malaria infection. The village-level intercept variance for the individual-level predictor  and final  were lower than that of empty . The ICC value for the final model was 95.7%. Moreover, the MOR values for the empty (2.34±0.21), individual-level predictors (2.23±0.0.34) and final (1.19±0.0.05) models were large  coverage and poor ITN conditions in the study area . Other sehaviour .The finding of high malaria risk in low altitude can be explained by the presence of suitable high ambient temperature and topography that favours mosquito abundance ,36. A stThe increased risk of malaria in houses with their walls having holes can be due to increased access of mosquitoes to bite humans. Housing conditions allowing mosquito entrance were indicated as malaria risk factors ,38. AnotIn conclusion, this finding showed increased malaria infection is associated with survey seasons, age of participants, altitudinal location and housing conditions in highland-fringe areas with low transmission settings. The current malaria control efforts could benefit through application of targeted interventions to villages of high malaria cases by prioritizing children aged below ten years in highland-fringe areas of low endemicity. Subsequently, seasonal transmission reduction could be operational in low transmission like Butajira area. Future studies should consider designing more frequent observations and incorporate household spray status and ITN use concurrently.The authors declare that they have no competing interests.AW contributed to conception and design, acquisition of data, analysis and interpretation of data, and drafting the manuscript. WD, AA and BL substantially contributed to conception and design of the study and reviewing the manuscript, revisiting it critically for important intellectual content. BL, AA, WD and AW reviewed the paper and all authors approved the final version.


The burden of malaria has decreased in parts of Africa following the scaling up of control interventions. However, similar data are limited from high transmission settings.A cohort of 100 children, aged six weeks to 10 months of age, were enrolled in an area of high malaria transmission intensity and followed through 48 months of age. Children were given a long-lasting insecticide-treated bed net (LLIN) at enrolment and received all care, including monthly blood smears and treatment with artemisinin-based combination therapy (ACT) for uncomplicated malaria, at a dedicated clinic. The incidence of malaria was estimated by passive surveillance and associations between malaria incidence and age, calendar time and season were measured using generalized estimating equations.Reported compliance with LLINs was 98% based on monthly routine evaluations. A total of 1,633 episodes of malaria were observed, with a median incidence of 5.3 per person-year (PPY). There were only six cases of complicated malaria, all single convulsions. Malaria incidence peaked at 6.5 PPY at 23 months of age before declining to 3.5 PPY at 48 months. After adjusting for age and season, the risk of malaria increased by 52% from 2008 to 2011 . Asymptomatic parasitaemia was uncommon  and rarely observed prior to 24 months of age.In Tororo, despite provision of LLINs and prompt treatment with ACT, the incidence of malaria is very high and appears to be rising. Additional malaria control interventions in high transmission settings are likely needed.Current Controlled Trials Identifier NCT00527800 Malaria causes more than 500 million clinical cases and is responsible for about one million deaths annually, mostly among African infants and young children . Uganda As utilization of malaria control interventions, including long-lasting insecticide treated bed nets (LLINs), indoor residual spraying of insecticides, and prompt treatment with artemisinin-based combination therapy (ACT) has increased, many reports have shown a substantial drop in malaria transmission, malaria-associated hospitalizations, and malaria-associated deaths ,4-8. HowConsiderable knowledge regarding the natural history of malaria, acquisition of anti-malarial immunity, and impact of interventions has been gained from longitudinal cohort studies. Beginning with Koch’s seminal studies in Papua New Guinea in the late 1800s, these studies have consistently revealed that, in endemic populations, the incidence and severity of malaria decreases considerably after the first years of life, with a corresponding rise in the prevalence of asymptomatic carriage of parasites -15. AlthThe Tororo Child Cohort (TCC) study was conducted in Tororo, a rural district in south-eastern Uganda with an entomological inoculation rate (EIR) estimated at 562 infective bites per person year (PPY) in 2002 . DetailsSubjects were followed for all medical problems at a dedicated study clinic open seven days a week, and parents/guardians were encouraged to bring their children to the clinic whenever they were ill. After hours, care was available through the adjacent hospital paediatric ward. Children presenting with new medical problems underwent a standardized medical evaluation using algorithms to guide therapy for illnesses. Medications with anti-malarial activity were avoided for the treatment of non-malarial illnesses. Monthly assessments were done in the study clinic to ensure compliance with study protocols, measure adherence to LLINs through recall of LLIN use in the prior evening, and perform routine blood smears. Study participants were withdrawn for: 1) movement out of the study area, 2) inability to be located for >60 consecutive days, 3) withdrawal of informed consent, 4) inability to adhere to the study schedule and procedures, or, 5) inability to tolerate the drugs used for malaria treatment.Subjects who presented with a documented fever (tympanic temperature ≥38.0°C) or history of fever in the previous 24 hours had blood obtained by finger prick for a thick smear. If the thick smear was positive for malaria parasites, the patient was diagnosed with malaria regardless of parasite density. At the time of their first episode of uncomplicated malaria, study participants ≥4 months and ≥5 kg were randomly assigned to receive open-label artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP). Each first daily dose of study drugs was directly observed at the study clinic. Study participants received the assigned treatment for all subsequent episodes of uncomplicated malaria. Episodes of uncomplicated malaria in children less than four months of age or weighing <5 kg as well as episodes of complicated malaria were treated with quinine. Clinical treatment failures occurring within 14 days of initiation of therapy for uncomplicated malaria were treated with quinine. Those occurring within 14 days of initiation of therapy with quinine were treated with quinine plus clindamycin. All children with malaria were followed up on days 1, 2, 3, 7, 14, 21, and 28 following diagnosis. Thick blood smears were collected on all malaria follow-up days apart from day 1 .Thick blood smears were stained with 2% Giemsa for 30 min. Parasite densities were calculated from thick smears by counting the number of asexual parasites per 200 leukocytes , assuming a leukocyte count of 8,000/μl. A blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites. For quality control, all slides were read by a second reader, and discrepancies were settled by a third reader. Laboratory technicians were blinded to the study participants’ treatment assignments.Data were double entered into an Access database. Data analysis was done using Stata version 11 . The observation period began one day after enrolment and ended when the child turned four years of age or on the day the child was prematurely withdrawn from the study.Incident episodes of malaria were defined as all treatments not preceded by another treatment in the prior 14 days. The incidence of malaria was calculated as the number of episodes per person years at risk. Asymptomatic parasitaemia was defined as a positive routine blood smear in the absence of fever that was not followed by the diagnosis of malaria in the subsequent seven days. As asymptomatic parasitaemia was assessed using active surveillance, the monthly prevalence of asymptomatic parasitaemia was ascertained for each month of observation. Geometric mean parasite densities were calculated for symptomatic malaria and asymptomatic parasitaemia.Generalized additive regression with smoothing splines were used to visualize the effects of age, calendar time, and season on the incidence of malaria and prevalence of asymptomatic parasitaemia . InferenInformed consent was obtained from the parent or guardian of all study participants. The study protocol was approved by the Uganda National Council of Science and Technology and the institutional review boards of the University of California, San Francisco, Makerere University and the Centers for Disease Control and Prevention.A total of 100 children aged between six weeks and <10 months of age (median 5.5 months) were enrolled between August 2007 and January 2008. Children were followed for a median of 3.5 years. Of 100 children enrolled, 96 were followed through one year of age, 88 through two years of age, 83 through three years of age, and 79 through four years of age. Children were prematurely withdrawn for the following reasons: 13 moved out of the study area, three were unable to comply with study procedures, two withdrew consent, one had treatment failure following quinine + clindamycin, one was unable to be re-administered study drugs due to drug toxicity, and one died. At the time of monthly routine assessment, 98% of study participants were reported to have slept under an LLIN the prior evening  incident cases were complicated . There were 12 treatment failures within 14 days of treatment: five early treatment failures associated with complicated malaria following treatment with AL due to single convulsions (two) or severe anaemia ; two episodes of recurrent malaria 14 days after treatment with AL; and five treatments with quinine + clindamycin following treatment with quinine. The only death occurred at home in a 35-month-old boy one day after he was diagnosed with his 27P < 0.001) and 26% higher from Apr-Jul  compared to the incidence from Aug-Oct .The median incidence of malaria was 5.4 episodes PPY Table , with 25P < 0.001). Significant heterogeneity in the prevalence of asymptomatic parasitaemia was observed, with 10% of children having nearly 55% of the total episodes and 45% of children having no episodes of asymptomatic parasitaemia during follow-up (P < 0.001 overdispersed compared with Poisson). Asymptomatic parasitaemia was more common in children with the most malaria; all children having more than 10 episodes of asymptomatic parasitaemia were in the highest quartile of incident malaria, and only one child with less than two episodes of malaria per year had an episode of asymptomatic parasitaemia.Of 6,951 blood smears obtained outside of the first 14 days of malaria follow-up, 475 demonstrated parasitaemia in children without fever. Of these, 186 met the specified definition of asymptomatic parasitaemia, as 289 were followed by malaria within seven days. Overall, the monthly prevalence of asymptomatic parasitaemia was 5.0% . There was no significant trend for parasite densities for either malaria or asymptomatic parasitaemia over age  of malaria cases were uncomplicated. The incidence declined as children reached three years of age, suggesting the development of natural immunity to malaria , althougMany reports across Africa have shown significant declines in malaria-related deaths and hospitalizations over the past decade, and it is routinely suggested that malaria-control interventions - including usage of LLINs and ACT - are responsible for these declines -8. HowevAnopheles gambiae complex (93.5%), as found in 2002 [L1014S mutation has increased from 29% in 2002 to 75% in 2008 in mosquitoes collected from Tororo [The reason for a lack of decline, and indeed a significant increase in the incidence of malaria in Tororo is unknown. The increase may be due to changes in drug resistance, vector species, insecticide resistance, and/or climate/rainfall, leading to increases in exposure to malaria vectors. Considering drugs, AL and DP have both shown outstanding efficacy in numerous recent studies, and there is no convincing evidence of resistance to these agents in Africa. Although we did not collect detailed information on mosquito species or biting habits, preliminary data from the East African International Centers of Excellence in Malaria Research (ICEMR) estimates the EIR in Tororo to be 379 infective bites PPY (unpublished data from CDC light traps collected between Oct 2011 and Sep 2012), near the EIR of 562 infective bites PPY estimated by human landing catches in 2002 . The maj in 2002 . Notably in 2002  to 42% i in 2002  and 62% m Tororo ,27, suggm Tororo . In thesm Tororo , and chem Tororo , will beImportantly, even though more than 1,600 cases of malaria were diagnosed in the 100 children enrolled in this cohort, remarkably few cases of malaria were associated with complications. Only six episodes met criteria for complicated malaria, and all these cases were due to a single convulsion (danger sign) and did not meet criteria for severe malaria. There were no cases of cerebral malaria or respiratory distress. This result is in sharp contrast to historical reports suggesting that 2% of all clinical attacks of malaria are severe . ConsistAs others have reported, children in this cohort appeared to acquire anti-disease immunity as they aged. The incidence of malaria declined in children after 30–36 months of age, and asymptomatic parasitaemia was more common in older children . HoweverThere were several limitations to this study. The cohort was enrolled by convenience sampling, limiting the generalizability of the findings. Although the analysis attempted to adjust for calendar time, children enrolled early in the study may have differed from those enrolled later. Detailed information was also unavailable regarding individual- and household-level exposure. A further limitation was the assessment for LLIN use by self-report, and not by directly observed LLIN usage, which may have overestimated LLIN coverage . FinallyIn conclusion, despite reports of decreasing malaria morbidity and mortality across many parts of Africa, the incidence of malaria continues to be very high in Tororo, Uganda, even in a setting with LLINs and ACT, and this incidence appears to be rising. Thus, additional malaria control interventions among young children living in high transmission settings are needed. Fortunately, with prompt diagnosis and effective treatment, malaria-related complications can be limited even in high transmission settings.The authors declare that they have no competing interests.GD, JT, PJR, and MRK conceived and designed the study. MKM, AK, and EA participated in data collection. PJ, BG, and GD participated in the data analysis. All authors participated in the writing of the manuscript, read and approved the final manuscript.


Plasmodium knowlesi can cause severe malaria in adults; however, descriptions of clinical disease in children are lacking. We reviewed case records of children (age <15 years) with a malaria diagnosis at Kudat District Hospital, serving a largely deforested area of Sabah, Malaysia, during January–November 2009. Sixteen children with PCR-confirmed P. knowlesi monoinfection were compared with 14 children with P. falciparum monoinfection diagnosed by microscopy or PCR. Four children with knowlesi malaria had a hemoglobin level at admission of <10.0 g/dL (minimum lowest level 6.4 g/dL). Minimum level platelet counts were lower in knowlesi than in falciparum malaria . Most (81%) children with P. knowlesi malaria received chloroquine and primaquine; median parasite clearance time was 2 days (range 1–5 days). P. knowlesi is the most common cause of childhood malaria in Kudat. Although infection is generally uncomplicated, anemia is common and thrombocytopenia universal. Transmission dynamics in this region require additional investigation. Plasmodium knowlesi is increasingly recognized as a frequent cause of potentially fatal human malaria in adults in Malaysian Borneo  of 152 adults with malaria were infected with P. knowlesi  on the northeast tip of Sabah, Malaysia, a coastal rural area which has been largely deforested. KDH services 5 subdistricts , with a total population of 85,000 persons. The Rungus are the most common ethnic group on the mainland, and minority groups include ethnic Chinese, Bajaus, Dusuns, and Balabaks.P. knowlesi infections and the difficulties of diagnosing this species by microscopy, it became policy at KDH to send slides that had been determined by microscopy to show presence of P. malariae to the Sabah State Reference Laboratory for PCR confirmation. In addition, KDH sends ≈15% of all blood films positive for other Plasmodium spp. for PCR confirmation.Ministry of Health policy in Sabah requires that all patients with a blood film result that indicates malaria be admitted to the hospital and discharged only after smear results are negative for 2 consecutive days. Since January 2009, in response to increasing reports of Plasmodium spp. during January 1–November 30, 2009. The patient’s age, sex, ethnicity, and address were recorded for all positive samples. Microscopy results were matched with PCR results.We retrospectively searched laboratory microscopy records for all blood smear results positive for Medical records were retrieved for all children <15 years of age who had received a diagnosis of malaria on the basis of microscopy results. Demographic, clinical, and laboratory details were extracted by using standardized data forms, which also included disease response to antimalarial treatment. Parasite clearance time was defined as the number of days until negative smear. Anemia and severe anemia were defined as hemoglobin levels <11 g/dL and <7.1 g/dL  At the Sabah State Reference Laboratory, parasite DNA was extracted, and nested PCR was performed for P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi by methods described , with accurate quantitation per microliter being recorded for most blood films that showed 2 or Fisher exact test. Normally distributed and non–normally distributed variables were compared by using Student t test and Wilcoxon rank-sum test, respectively. For comparison between P. falciparum and P. knowlesi cases, the analysis included children with PCR-confirmed P. knowlesi infection, and children with P. falciparum infection diagnosed by either microscopy or PCR. Children with mixed Plasmodium infections were excluded from analysis, as were children whose medical records could not be located.Data were analyzed by using Stata statistical software, version 10 . Proportions were compared by using the χP. malariae monoinfection or mixed infection. PCR was performed on samples from 157 (80%) of these patients and results were positive for P. knowlesi in 137 (87%); 125 (91%) of these were P. knowlesi monoinfections. For the remaining 20 patients who had been given a diagnosis of P. malariae monoinfection or mixed infection by microscopy, PCR found undifferentiated Plasmodium spp., negative results for Plasmodium spp., and 7 cases of P. falciparum infection.From January 1 through November 30, 2009, 220 patients at KDH were given a diagnosis of malaria on the basis of microscopy results . Of thesP. malariae with P. knowlesi). Microscopy results were also used if PCR result was negative  or if PCR result was positive for Plasmodium spp. (7 P. knowlesi infections). Using this method, we found 172 (78%) P. knowlesi monoinfections, 29 (13%) P. falciparum monoinfections, and 19 (9%) mixed infections  than were those with P. falciparum malaria . Median age was higher for those with P. knowlesi infection  than for those with P. falciparum infection .To estimate the final numbers of malaria species, we used positive PCR results when possible and microscopy results when PCR had not been performed  had P. falciparum monoinfection, and 7 (17%) had mixed infections  showed P. knowlesi, and 1 showed mixed P. knowlesi/P. vivax infection. Again, final numbers of malaria species were estimated by using PCR results (if available) or microscopy (if PCR had not been performed). Accordingly, 24 cases (59%) were P. knowlesi, 15 (37%) were P. falciparum, and 2 were mixed P. knowlesi/P. vivax infections. Children thus represented 14% (24/172) of all cases of P. knowlesi monoinfection.Of 220 patients with positive results by microscopy, 41 (19%) were <15 years of age. Microscopy showed that 24 (59%) children had fections . SamplesP. knowlesi monoinfection and children with P. falciparum monoinfection diagnosed by either microscopy or PCR. Medical records were unavailable for 2 children , leaving 16 children with PCR-confirmed P. knowlesi infection and 14 with P. falciparum infection for comparison  were 7–14 years of age. In contrast, children with P. falciparum malaria were significantly younger, with a mean age of 5.4 years .Half of the children with PCR-confirmed ere male . The meaAll children had fever or a history of fever. The duration of fever before hospital admission appeared shorter with knowlesi malaria than with falciparum malaria (median 4 days vs. 6.5 days), although this difference was not statistically significant. Although documentation of clinical features was limited, no child was in a state of shock, and no child was reported to be dyspneic, to have bleeding complications, or to have any other clinical feature or laboratory results that indicated severe malaria  parasites/µL. Only 2 children with P. falciparum malaria had parasite densities accurately determined at admission . Anemia was common with P. knowlesi and P. falciparum infections. At admission, 9 (56%) children with P. knowlesi infection were anemic (hemoglobin level <11.0 g/dL), and 4 (25%) had a hemoglobin level <10 g/dL. All children with P. knowlesi infection had a hemoglobin minimum level <11.0 g/dL, and 10 (63%) had a hemoglobin minimum level <10 g/dL. The lowest hemoglobin level was found in an 8-year-old boy: 8.3 g/dL at admission, 6.4 g/dL on day 3, and 7.2 g/dL on day 6, the day before discharge. His parasite count at admission  was the highest recorded in this study.In 11 (69%) children with P. knowlesi by microscopy . Among children with P. knowlesi infection, a lower hemoglobin minimum level was associated with higher parasite density at admission (p = 0.001). All children with P. falciparum infection had hemoglobin levels at admission <11.0 g/dL; 10 (71%) had hemoglobin levels <10 g/dL, and 5 (36%) had severe anemia (hemoglobin <7.1 g/dL). Severe anemia developed in a sixth child after admission. Four children received transfusions, with hemoglobin levels of 4.8–6.1 g/dL at admission. Children with P. knowlesi malaria took longer to reach their hemoglobin minimum level than did children with P. falciparum malaria .In addition, severe anemia  occurred in a second child with positive results for P. knowlesi malaria had thrombocytopenia. Fifteen children (94%) had a platelet count <150,000/µL at admission, and the remaining child exhibited thrombocytopenia within 1 day. The lowest platelet count recorded was 28,000/µL. Platelet count was not correlated with hemoglobin level. Thrombocytopenia was less common in children with P. falciparum; 7 (50%) had platelet counts <150,000/µL at admission or during hospitalization and the lowest platelet count was 47,000/µL. Children with P. knowlesi malaria had a lower lymphocyte count minimum than those with P. falciparum malaria .All children with P. knowlesi malaria were given oral chloroquine and primaquine for 3 days, and these children had a median parasite clearance time of 2 days .Most (81%) children with f 2 days . The lonP. falciparum malaria were given a variety of treatment regimens. Most (71%) received a regimen that contained quinine, 7 (50%) children received sulfadoxine/pyrimethamine, 6 (43%) received primaquine, and 5 (36%) were given chloroquine. Only 5 (36%) received artemisinin-based combination therapy. Parasite clearance times were significantly slower among children with P. falciparum malaria, with a median of 5 days until the first negative smear; in 4 children (29%), it took >10 days for parasites be cleared. Children with P. knowlesi malaria had significantly shorter hospital admissions  than did children with P. falciparum malaria .Children with P. knowlesi was the most common cause of malaria in adults and children in the Kudat region in Sabah, Malaysian Borneo. Although those with P. knowlesi infections had an older age distribution than did those with P. falciparum infections, the species still caused 63% of all malaria cases among children <15 years. Although nearly all previous reported cases of knowlesi malaria have been in adults, 14% of all knowlesi cases in Kudat occurred in children. In children, P. knowlesi most often caused uncomplicated malaria, which responded well to chloroquine. Nevertheless, knowlesi malaria was associated with substantial illness in children, with all PCR-confirmed P. knowlesi–infected children being anemic at admission or during hospital stay.P. knowlesi infection, comprising 7% of all knowlesi cases  children with P. knowlesi malaria had an adequate response to a 3-day regimen of treatment with chloroquine and primaquine, although the mean parasite clearance time of 2 days was longer than the 90% parasite clearance time of 10.3 hours that was recently reported in adults (P. falciparum malaria received many different treatment regimens and took significantly longer to clear their parasites. Less than half received the recommended artemisinin-based combination therapy, and only after alternative treatments failed. Children with P. falciparum malaria had significantly longer hospital stays, likely related at least in part to suboptimal treatment regimens. This finding highlights the importance of increasing the usage of artemisinin-based combination therapy for falciparum malaria in district hospitals in Sabah and elsewhere.Most children with P. knowlesi. Furthermore, although PCR was performed on samples from most children with P. knowlesi malaria, only 1 child with P. falciparum malaria had PCR performed. Our analysis, therefore, compared children with PCR-confirmed knowlesi malaria to children with falciparum malaria diagnosed by either microscopy or PCR. Some of those children with a diagnosis of falciparum malaria may have actually had knowlesi malaria, and the differences found between these 2 species may therefore be minimum estimates. The retrospective design of our study also limited our ability to collect standardized clinical information.Our study had several limitations. First, PCR was only performed for 73% of cases across all age groups, and this limited our ability to accurately determine the true proportion of disease caused by P. knowlesi has become the predominant malaria species in the Kudat region, estimated by results of microscopy, PCR, or both, as contributing to 87% of all malaria cases, a higher proportion than that reported elsewhere in Malaysian Borneo. The emerging dominance of P. knowlesi in Malaysian Borneo has been hypothesized to result from the following factors: changing patterns of human exposure to monkeys and vectors (P. vivax and P. falciparum as a result of a 40-fold reduction in the prevalence of these species in Sabah and Sarawak during 1960–2006 following intensive malaria control efforts (P. vivax in Kudat was particularly notable.This study demonstrates that P. knowlesi did not occur solely in those spending time in forested areas. Macaque monkeys, the natural hosts of P. knowlesi, are widely distributed in different habitats throughout the Kudat area and are frequently domesticated. The major vectors of P. knowlesi in forested areas of Sarawak, Anopheles latens mosquitoes, disappear with deforestation, but vectors capable of transmitting P. knowlesi, An. balabacencis mosquitoes (,P. falciparum malaria, pediatric knowlesi malaria was restricted to children of school age. Further studies will be required to characterize the transmission patterns, vectors, and risk factors for P. knowlesi in deforested areas of Malaysia.Previous reports of adult disease have been from communities adjacent to rainforests (P. knowlesi is the most common cause of malaria in adults and children in the Kudat region of Sabah, a rural coastal deforested region. Consistent with previous studies in adults (P. knowlesi in children most often caused uncomplicated malaria that responded adequately to chloroquine and primaquine. Anemia was common in children and knowlesi infection was associated with moderately severe anemia. Thrombocytopenia was universal and is characteristic of knowlesi malaria across all age groups. Larger prospective clinical studies are needed to describe more fully the epidemiology of P. knowlesi malaria in children, the full spectrum of clinical disease and the transmission patterns in nonforested areas such as Kudat.


Out of the 369 (28.2%) women with peripheral positive parasitemia, 211 (57.2%) had placental malaria and 14 (3.8%) had malaria parasites in their umbilical cord blood. The umbilical cord parasitemia levels were statistically associated with the presence of maternal peripheral parasitemia , placental parasitemia , high-density peripheral parasitemia , and high-density placental parasitemia . In Burkina Faso, the mother-to-child transmission rate of malaria appears to be low.Malaria congenital infection constitutes a major risk in malaria endemic areas. In this study, we report the prevalence of transplacental malaria in Burkina Faso. In labour and delivery units, thick and thin blood films were made from maternal, placental, and umbilical cord blood to determine malaria infection. A total of 1,309 mother/baby pairs were recruited. Eighteen cord blood samples (1.4%) contained malaria parasites  Plasmodium falciparum and development of malaria morbidity or mortality than others. These include children less than five years of age and pregnant women. Malaria contributes significantly to perinatal disease burden in terms of pregnancy loss, prematurity due to preterm labor, and intrauterine growth retardation [Plasmodium falciparum infection during pregnancy does not cause symptomatic malaria, but may lead to maternal anemia as well as placental and cord blood malaria infection, especially among primigravidae and secundigravida [In malaria high-transmission areas, some population groups are at considerably higher risk of infection with ardation . Malariaigravida –4. Plasmodium-infected erythrocytes in the intervillous space in the placenta, causing histologic changes including leukocyte-induced damage to the trophoblastic basement membrane. The placental infection does not reflect the existence of peripheral infection over a short period preceding the delivery or whether it is related to infection during pregnancy. Susceptibility to this may be correlated to high exposure to malaria and repeated episodes of parasitemia during the pregnancy [Placental malaria is defined as the accumulation of regnancy .Plasmodium falciparum from mother to fetus has long been well-described [Vertical transmission of malaria from mother to foetus through the placenta and umbilical cord is defined as umbilical cord blood parasitemia. The transplacental transmission of escribed , 7. The direct burden of neonatal malaria infection in terms of prevalence is not well-described in malaria endemic areas. In fact, the method used to identify congenital transmission is peripheral blood of newborns or umbilical cord blood. Malaria parasites have been detected only rarely in the peripheral blood of newborns, whether the blood specimen is collected at the time of birth or hours later , 9. In sPlasmodium falciparum has been found to be rare in malaria-endemic areas, ranging from about 1 to 5% [Studies published so far have documented contradictory levels of this burden. In countries without endemic malaria, congenital malaria has occurred in children born to women who have immigrated from malarious areas. However, transplacental transmission of  1 to 5% –15. In c 1 to 5% , 15–18.In Burkina Faso, the real prevalence of neonatal malaria is unknown but is estimated to be even higher. This assessment is based on presumptive malaria diagnosis. The present study was designed to determine the real burden of transplacental transmission, the risk factors associated with transplacental transmission, and the prevalence of cord blood and placental malaria parasitemia in malaria holoendemic area of Burkina Faso. Plasmodium falciparum. These results represent a pooled analysis of studies on malaria prevention in pregnant women , 19, 20 Anopheles gambiae, Anopheles arabiensis, and Anopheles funestus. The annual entomological inoculation rates range from 10 to 500 infective bites per individual. Plasmodium falciparum is responsible for more than 90% of malaria infections. The first study took place within six delivery units (DUs) of the Koupela health district, which is located around 120 km east of Ouagadougou. The second study was conducted in one DU of the Health District of Bousse. Both study sites have been extensively described elsewhere , 19, 20.The study participants were pregnant women who voluntarily consented to participate in trials of malaria prophylaxis during pregnancy. They were encouraged to deliver at the health facility where the study samples were collected for processing. Women delivering at the health facility, after giving informed consent, were asked a standard series of questions focused on sociodemographic characteristics, history of fever, antimalarial drug use, and the use of antimalarial chemoprophylaxis and bednets. Capillary blood was obtained by fingerstick for malaria blood film preparation. Placental blood films were prepared by identifying the maternal side of the placenta, wiping away excess blood, cutting into the surface, and placing pooled blood onto a slide. Umbilical cord blood samples were obtained by wiping away excess blood from a clamped cord, piercing it with a lancet, and placing a drop of expressed blood on a slide.A detailed clinical examination was done on each newborn infant within 24 hours of delivery. Neonates were weighted with an electronic digital scale (±10 grams) . The Dubowitz scoring system was used to estimate gestational age, using findings from physical and neurologic examinations. Scoring by APGAR index was performed at delivery but was not recorded in this study.μL. Parasite densities were calculated according to the following formula: number of parasites counted × 8000/number of leucocytes counted. Thin films were then used to determine species when thick films were positive. All slides were double-read by two independent microscopists. If the ratio of densities from the first two readings was greater than 1.5 or less than 0.67, or if fewer than 30 parasites were counted with a difference of more than 10 parasites between the two readings, the slide was evaluated a third time. The geometric mean of the parasite density of the two most concordant results of the three readings was taken as the final result. When the discordance was only in terms of positivity, the slide was also evaluated a third time and the definitive result was based on the majority verdict for positivity.All blood films  were stained with Giemsa and examined for parasites at the “Centre National de Recherche et de Formation sur le Paludisme” immunoparasitology laboratory in Ouagadougou. For thick films, parasites and leukocytes were counted in the same fields until 500 leukocytes were counted. Parasite densities were estimated using an assumed leukocyte count of 8,000 leukocytes/Blood film results were considered to be positive if any asexual-stage parasites were identified and negative if no parasites were seen in 100 high-power fields. Prematurity was defined as a gestational age <37 weeks as estimated by Dubowitz examination.We defined low birth weight as a birth weight of <2,500 g with a gestational age ≥37 weeks.t-test. Statistical results were considered significant, when the two-sided P value was <0.05.Data collected in the study questionnaire were verified, then double-entered, and validated with EpiInfo, version 6.04 fr . Data were analyzed using Epi-info and Stata 7.0. The analysis included data from births of all enrolled participants who delivered at the study centre clinic for whom data were available. Continuous, normally distributed data were described by mean and standard deviation and nonnormally distributed data by the median or geometrical mean and range. Proportions were compared using the Chi-square test and normally distributed continuous variables were compared using Student's The study was discussed with health authorities and local leaders to obtain their assent. The study was reviewed and approved by the Burkina Faso Ministry of Health ethical committee. Informed consent (by signature or thumbprint) was obtained after the consent document was read to the women in the local language. For illiterate mothers, the informed consent discussion process was witnessed by an impartial individual.In total, 1,374 women delivered at the health unit; 1,309 women delivered singleton infants and provided data in the form of peripheral blood smear, placental blood, and umbilical cord blood. The profile of enrolled women is summarized in Primigravidae comprised 35% of the total sample size, and primigravidae and secundigravida together made up 56% of the total sample size. The mean duration of gestation was 38.66 ± 3.89 weeks. The mode of delivery of babies was spontaneous for 97.5% of the mothers. Two women died during delivery as a consequence of eclampsia. Other complications during the delivery included peripartum haemorrhage, placental retention, and retroplacental haemorrhage. Fifty stillbirths (3.6%) and 18 (1.3%) miscarriages occurred at the DUs. The weight range of babies was 700 g to 4,700 g with a mean weight of 2,875 ± 450 g. Sixteen percent of the babies presented low birth weight at delivery .Plasmodium falciparum was detected in peripheral blood from 369 (28.2%) of the women at the time of delivery. Overall, the geometric mean of the maternal peripheral parasite density was 1,106.47 (95% CI 871.58–1404.67) parasites/μL. Plasmodium falciparum was detected in the placentas of 255 (19.5%) women; 28.5% of these belonged to primigravidae and 11.4% to multigravidae. The difference in infection between the primigravidae and the multigravidae was statistically significant (P < 0.0001). All of the women received malaria prophylaxis during pregnancy. Over one-third of the women were parasitaemic. Indeed, of the 1,309 pregnant women, Plasmodium falciparum was detected in 18 (1.4%) babies, and the geometric mean umbilical cord blood parasitemia was 315.69 (95% CI 87.02–1145.20) parasites/μL. Of these, 2.4% (11) belonged to primigravidae and 0.5% (3) to multigravidae. Of the 18 babies with Plasmodium-falciparum-positive umbilical cord blood parasitemia, 17 were positive for trophozoites and 1 was positive for schizonts only. Both schizonts and trophozoites were present in seven babies, and one newborn had a triple-positive association (trophozoite + schizont + gametocyte). Five newborns (27.8%) out of the 18 infected with Plasmodium falciparum had low birth weight. Umbilical cord blood parasitemia with Plasmodium falciparum, 14 (77.8%) were born from mothers with positive peripheral parasitemia. For two newborns with Plasmodium falciparum infection, no peripheral or placental infection was found in the mother. None of the babies with cord blood positive for malaria had fever within the first 24 h of life. Out of 369 (28.2%) women with peripheral positive parasitemia, 211 (57.2%) had positive placental malaria and 14 (3.8%) had malaria parasites in the umbilical cord blood. Eleven (5.2%) of the 211 (57.2%) women with positive placental smears for malaria had malaria parasites in the umbilical cord blood. Of the 18 babies infected with μL) decreased with increasing gravidity . Being born with only placental infection reduced the rate of umbilical cord parasitemia (11%). There was also an association between umbilical cord blood infection and placental infection (P < 0.001) . Being born with only maternal peripheral infection reduced the likelihood of having umbilical cord parasitemia (17%). There was an association between umbilical cord blood infection and maternal peripheral infection (< 0.001) . μL or placental parasite density ≥10,000 parasites/μL conferred a greater likelihood of umbilical cord parasitemia . Four cases of umbilical cord blood parasitemia occurred in pregnancies not complicated by maternal peripheral malaria infection. Maternal peripheral blood parasitemia was associated with umbilical cord blood parasitemia , and between cord blood/placental and maternal peripheral parasitemia. Five cases of umbilical cord blood parasitemia occurred in pregnancies not complicated by placental malaria infection. Only two newborns had mothers with neither peripheral malaria infection nor placental malaria infection. Earlier studies of transplacental malaria reported these associations [Plasmodium falciparum malaria parasites in umbilical cord blood denotes infection acquired antenatally or contamination with infected maternal blood at delivery is not clear [There were also associations between placental and cord blood parasitemia  was an important determinant of the likelihood of umbilical cord blood parasitemia . The density of placental parasitemia  was also an important determinant of the likelihood of umbilical cord parasitemia . High density parasitemia seems to facilitate umbilical cord infection. Conversely, previous reports, showing low rates of transplacental transmission of malaria, suggest that the placental barrier is very effective in the case of very low malaria parasite density [The density of maternal peripheral parasitemia . Finally, univariate analysis showed that being born from a first pregnancy was also linked to umbilical cord blood parasitemia (OR = 2.96, Our data indicate that the rate of mother-to-child transmission of malaria, defined as positive umbilical cord blood parasitemia, appears to be low. Clinicians are therefore advised to investigate other aetiologies of fever in neonates. The low level of parasitaemia in cord blood suggests that contamination probably occurs during the labour period. Maternal, placental, and high density parasitemia were all associated with umbilical cord parasitemia. Prevention of malaria during pregnancy with effective antimalarial drugs should reduce the risk of infection for newborns.