Plasmodium falciparum infection, peripheral parasite counts do not always correlate well with the sequestered parasite burden. As erythrocytes parasitized with mature trophozoites and schizonts have a high tendency to adhere to the microvascular endothelium, they are often absent in peripheral blood samples. The appearance of schizonts in peripheral blood smears is thought to be a marker of high sequestered parasite burden and severe disease. In the present study, the value of schizontaemia as an early marker for severe disease in non-immune individuals with imported malaria was evaluated.In P. falciparum malaria between 1 January 1999 and 1 January 2012 were included. Thick and thin blood films were examined for the presence of schizontaemia. The occurrence of WHO defined severe malaria was the primary endpoint. The diagnostic performance of schizontaemia was compared with previously evaluated biomarkers C-reactive protein and lactate.All patients in the Rotterdam Malaria Cohort diagnosed with Schizonts were present on admission in 49 of 401 (12.2%) patients. Patients with schizontaemia were more likely to present with severe malaria, a more complicated course and had longer duration of admission in hospital. Schizontaemia had a specificity of 0.95, a sensitivity of 0.53, a negative predictive value of 0.92 and a positive predictive value of 0.67 for severe malaria. The presence of schizonts was an independent predictor for severe malaria.Absence of schizonts was found to be a specific marker for exclusion of severe malaria. Presence of schizonts on admission was associated with a high positive predictive value for severe malaria. This may be of help to identify patients who are at risk of a more severe course than would be expected when considering peripheral parasitaemia alone. Plasmodium falciparum infection [Sequestration of infected erythrocytes in the microvasculature is one of the main pathological features of severe nfection -4. Partinfection . The prenfection , but onlnfection -9. In adP. falciparum malaria, presence of schizonts is currently not a well-established WHO criterion for severe disease [P. falciparum malaria.Although it is stated in the current WHO malaria guidelines that the presence of schizontaemia may be associated with a poor outcome in  disease ,10. To fP. falciparum malaria between 1 January 1999 and 1 January 2012 were included in this observational study. Of all patients identified, demographic, clinical and laboratory data were collected using a standardized form and stored in an electronic database. Patients with a mixed infection of P. falciparum with another Plasmodium species were included in this study, but pure non-falciparum Plasmodium infections were excluded. Multiple malaria episodes in a single patient were only regarded as separate cases if caused by true re-infection; recrudescent infections of P. falciparum were excluded.The Harbour Hospital is a 161-bed general hospital located in Rotterdam, The Netherlands. It comprises the Institute for Tropical Diseases, which serves as a national referral centre. All patients in the Rotterdam Malaria Cohort diagnosed with Available laboratory examinations included red and white blood cell counts, haematocrit, platelet counts, C-reactive protein, serum electrolytes, total bilirubin, serum creatinine and urea, sodium, potassium, liver enzymes, blood glucose and venous plasma lactate.Plasmodium vivax and Plasmodium ovale.The standard procedure to diagnose malaria comprised a quantitative buffy coat (QBC) analysis, a rapid diagnostic test (RDT) for malaria antigens Binax NOW® Malaria Test , and thick and thin blood smears using freshly collected blood specimens from finger pricks. The malaria rapid test and the QBC analysis were performed according to the manufacturer’s instructions. QBC capillaries were examined independently by two technicians by microscopic analysis of two complete rows of the region between the bottom of the capillary and the polynuclear leukocyte layer using an Olympus BX-60 fluorescence microscope equipped UV-filter and 50x objective and 12.5x oculars . These two lanes represented about 100 microscopic fields (at 625x magnification) and took an average examination time of 5 min. Subsequently, the polynuclear and mononuclear cell layer was screened for schizonts, gametocytes, malaria pigment and elderly trophozoites of Plasmodium parasites as well as Schüffner’s dots and Maurer’s clefts in infected erythrocytes. Thick and thin smears were examined with regular light microscopes at a total magnification of 1,250x. The Plasmodium species was identified on morphological characteristics in the thin and thick blood smears. Since a real-time quantitative PCR became available several years ago, a substantial proportion of the samples were additionally confirmed by PCR (without discrepant findings compared to classic light microscopy).Thick blood smears were stained with Field’s stain  and thin smears with Diff Quick stain . Both staining procedures had been optimized for optimal staining of Plasmodium falciparum schizonts were defined as clustered malaria pigment adjacent to two or more nuclei of P. falciparum in thick blood smears or QBC capillaries. Compared to thin and thick blood smears QBC analysis is the most sensitive test to detect Plasmodium parasites. Plasmodium falciparum schizonts can be identified in the erythrocyte layer in the QBC capillary, but identification within the leukocyte layer is difficult due to the intense fluorescent signal of the leukocyte nuclei. For this reason, this layer is also screened for quenched fluorescence by malaria pigment. If malaria pigment was detected in the leukocyte layer of the QBC capillary, the thick and thin blood smears were thoroughly examined for the presence of schizonts by analysis of at least 200 microscopic fields at 625x magnification.Parasitaemia was determined by parasite counting as described before . PlasmodPatients were classified as having severe malaria if they met one or more of the WHO criteria for severe malaria, as published , either P. falciparum schizonts identified in QBC and/or blood smear examinations from a blood sample taken on admission.Schizontaemia was defined as presence of one or more The degree of immunity to malaria was estimated as previously described . In brieU test and the Fisher’s Exact test. Trends between categorical variables were done with the Chi square test for Trend. Odds ratios were calculated using the approximation of Woolf. For clinical reference, the diagnostic performance of schizontaemia for severe disease was compared to that of the classic biomarkers serum C-reactive protein and plasma lactate [All data were not normally distributed (Kolmogorov-Smirnov test) and are therefore presented as medians and range. Univariate comparisons were performed using the Mann–Whitney  lactate -15. BinaPlasmodium species; 401 infections were caused P. falciparum infections including three patients who had a mixed infection . No patient with a mixed P. falciparum infection presented with schizontaemia on admission. These 401 patients with either a pure P. falciparum infection or a mixed Plasmodium infection with P. falciparum were included in this study.Between 1 January 1999 and 1 January 2012, a total of 562 cases of imported malaria were seen. Of these cases, 161 cases were caused by non-falciparum P. falciparum malaria outside of Africa was relatively low (n = 26), these patients tended to present with schizontaemia more frequently than patients who contracted malaria in Africa (Odds ratio 0.26 (95% CI 0.086-0.79), p = 0.0259). There were no differences between patients presenting with and without schizontaemia with regard to their immune status, ethnic background and use of malaria chemoprophylaxis, nor did they differ in duration of symptomatology. As detailed in Table Schizonts were present on admission in 49 of 401 12.2%) patients. The general characteristics of the patients with presence and absence of schizonts on admission are shown in Table .2% patieThe diagnostic accuracy of schizontaemia as a predictor for severe malaria was characterized by a high specificity, negative predictive value and an acceptable positive predictive value. When compared to the performance of plasma lactate, schizontaemia had a lower sensitivity but a higher specificity and positive predictive value for severe disease. When compared to the performance of C-reactive protein, schizontaemia had a higher specificity and positive predictive value for severe disease Table . Using bP. falciparum malaria and is a cause of major pathology in severe disease [P. falciparum parasites disappear from the peripheral blood after 24–26 hours to complete the asexual life cycle in the microvasculature, as the tendency to adhere to the endothelial wall increases during the maturation process [P. falciparum infection became common practice in the 1930s [Sequestration of parasitized erythrocytes in the microvasculature of vital organs is a cardinal feature of  disease -4. Maturhe 1930s .In the microvasculature, rupturing schizonts release up to 32 merozoites ,16, caus4 mature trophozoites and schizonts per μL of peripheral blood was found to have a high sensitivity (90%) and specificity (70%) for fatal outcome. The data of the present study confirm the prognostic value of schizontaemia for severe disease in predominantly non-immune travellers, comparable to that observed in semi-immune patients in region of malaria endemicity. However, it should be noted that besides differences in presumed immunity towards malaria, the current study also differed in its design with regard to outcome. In many studies in malaria-endemic regions, the primary outcome measure is usually survival or death and studies are restricted to these severe cases using the WHO severity criteria as an entry criterion. Since case fatalities are less common in the western non-endemic, resource-rich setting [In the present study schizontaemia was found to be a useful marker for severe malaria. Although schizontaemia was more frequently seen in patients with high-grade parasitaemia, the predictive power appeared to be independent of parasitaemia. In the study by Silamut and White  in adult setting  and malaUnfortunately, for the present study quantitative counts of the different parasite stages were not available. Although quantification would likely have resulted in a more precise estimation of its prognostic value, the specificity of schizontaemia was found to be a useful tool for exclusion of severe disease. The negative predictive value of schizontaemia is high, and comparable to that of C-reactive protein and lactate. Moreover, unlike C-reactive protein and lactate -15 schizP. falciparum malaria was found to be a specific marker for exclusion of severe disease; in contrast, presence of schizonts had a high positive predictive value for presence of severe disease and can thus help to identify patients that may be candidates in need of intensive monitoring and timely administration of preferably  artemisinin derivatives.Absence of schizonts on admission in travellers with imported The authors declare that they have no competing interests.MEvW contributed to the data acquisition and analysis and writing of the manuscript. MdeMM, KVJ and JvH participated in the data analysis and revising of the manuscript. RK is responsible for collection of patient materials, patient data and database management. PJvG designed the study, participated in the data acquisition and analysis and in writing and revising the manuscript. All authors have read and approved the final version.


Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the “rosetting” phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations.We used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress.PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other.Plasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like The results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation. P. falciparum malaria is a major cause of mortality and morbidity in young children in sub-Saharan Africa. In endemic areas, severe malaria is most prevalent in children under the age of five years, before they acquire immunity to severe disease as a result of repeated exposure. Severe malaria manifests in children in three partly overlapping syndromes; impaired consciousness (IC), respiratory distress (RD), and severe malarial anemia (SMA) [Severe life threatening ia (SMA) . Of thesia (SMA) .The pathophysiological process underlying each of these syndromes is still not understood. Disturbed microcirculation is thought to play a major role . In cerevar genes per parasite genome and undergoes antigenic variation. Switches in the expression of the repertoire of var genes results in a high degree of plasticity in the antigenic and adhesive properties of the infecting parasite population.PfEMP1, a parasite encoded protein expressed on the surface of the IE, interacts with host receptors on the microvascular endothelia as well as unparasitized erythrocytes (a phenotype referred to as rosetting) leading to sequestration of the IE in organs. PfEMP1 is therefore thought to play a central role in parasite virulence. PfEMP1 is encoded by about 60 var genes preferentially expressed in young and non-immune children to be associated with severe malaria especially in children with IC [Epidemiological studies have shown a subset of  with IC -10. This with IC -18 and 2 with IC ,5,18.var expression patterns and disease severity, through mechanisms involving sequestration and endothelial activation we would expect to observe a relationship between the expression of the var subset associated with severe malaria and markers of endothelial activation.Beside sequestration of IE, severe malaria is characterised by systemic endothelial activation and widespread release of activation markers such as von Willebrand factor (vWF) , solublevar expression profiling of 217 clinical P. falciparum isolates, that expression of a specific subset of “group A-like” PfEMP1 types is associated with severe malaria [vars expressed by the infecting parasites on the surface of IE may therefore determine the level of endothelial activation as suggested by [Previously, we showed through  malaria . This wa malaria ,10. In p malaria ,4 and Af malaria , the sev malaria . The subested by .vars and IC [In this study, we explored whether a relationship exists between widespread endothelial activation (represented by plasma ang-2 levels), parasite VSA expression and severe malaria. Specifically, we investigated whether widespread endothelial activation could provide a causal link between the expression of the group A-like s and IC  on the os and IC  on the oEthical approval for this study was obtained from Kenya Medical Research Institute (KEMRI) Ethical Review Committee (SSC 1131), and written, informed consent was obtained from parents/guardians of the study participants.P. falciparum infection and admitted to the hospital ward with impaired consciousness (Blantyre coma score (bcs) <5 in patients aged ≥8 months or ≤3 in patients aged under 8 months) [The method of sample collections was described in detail in ,10. Seve months) , respira months) , severe vars was PCR amplified, cloned into TOPO T-cloning vector, transformed into E. coli, and 100 colonies from each sample sequenced. The sequences were classified as described in [vars belong to cys2 and block sharing group 1 [The method for amplification of DBLα-tag from cDNA has been described in  and sequribed in ,32. Brieribed in . The seqribed in . Group A group 1 . Each suvar expression (ranging from high to low cys2 expression). The median MFI value of this IE surface recognition by IgG against the eight isolates was calculated for each participant [Rosette frequency data was obtained as described previously . IE surfticipant .Ang-2 level in the acute plasma was determined by a commercial ELISA Kit from R & D  using the manufacture’s protocol.P. falciparum parasitemia were used to determine both the dynamic range and the concentration of PfHRP2 using recombinant PfHRP2 protein obtained from MyBioSource (Cat no MBS232321). These plasma samples were then used to construct a standard curve. The ELISA assay was performed in duplicates in dilutions ranging from 1: 50 to 1:2000 and concentration (ng/ml) calculated from the linear range of the standard curve. Samples with OD readings greater than 20% standard deviation in the duplicate well was repeated or excluded from the analysis.Plasma PfHRP2 level was determined from the acute plasma using ELISA. Three plasma samples from patients with high Stata version 11 was used for all statistical analyses and P ≤0.05 was considered significant. Correlations between variables were evaluated using Spearman’s rank correlation coefficient or the 2-sample Wilcoxon rank-sum (Mann–Whitney) test. Linear regression was used when the outcome is a continuous variable and logistic regression when binary. All the regression analyses were adjusted for age. All independent variables that were significant in a univariate analysis were included in models with, either IC, or RD variable. Before use in a regression analysis, parasite density both peripheral and total (PfHRP2) and ang-2 were log-transformed. Rosette frequency and group A-like expression were arcsine-transformed as described in ,10. For We tested the normality of residuals in linear regression models by examining the interquartile range of the standardized residuals. None of the models had severe outliers, giving no sufficient evidence to reject normality at a 5% significance level.2 improvement test to assess the effect of adding subsequent explanatory variables to the fit of a simpler regression model.We used the Hosmer-lemeshow goodness of fit test to examine the goodness of fit for all logistic regression models with all the models reported showing adequate fit (P > 0.05). We also used the likelihood ratio (LR) χvar expression profiles of the infecting parasites from these children were determined [The samples used were from a published study  that invtermined  togethertermined  present termined .We used ang-2 as a marker of widespread endothelial activation because unlike sICAM-1  and vWF U test Z = 6.5, P < 0.0001, N = 213).Consistent with previous studies ,25, analSince severe malaria in African children presents in three overlapping syndromes , these svar, in relation to ang-2 levels. A significant positive correlation was found between ang-2 and both group A-like var expression  expression: rosetting and group-A like var subgroup expression and IC [We explored further whether the previously observed relationship between the group A-like n and IC  may invon and IC .var expression and ang-2 as independent variables. First, each of them was used as the only explanatory variable in an age-adjusted model and then in combination to adjust for one another. As shown in Figure var subgroup expression remained significantly associated with IC. Similarly, ang-2 remained significantly predictive of IC when adjusted for group A-like var expression . These results suggest that the group A-like var expression and ang-2 are independently associated with IC.We used age-adjusted logistic regression models predicting IC with group A-like var expression, the relationship between IE antibodies and IC appears to be independent of ang-2. Overall, a similar observation was made when CM (bcs ≤ 2) was considered as the outcome . PfHRP2 density . To test2 = 2.2, P = 0.14). Taken together these and the results shown in Figure Previously, we found that RD showed evidence for an association with peripheral parasitaemia and rosette frequency within this dataset . Above, var genes and malaria with IC on the one hand and rosetting frequency with RD on the other [In this study we tested whether a marker of widespread endothelial activation, plasma ang-2 levels throw light on our previously reported associations between expression of group A-like he other . Previouhe other . In addihe other . As retihe other ,26,42, the other ,28 givesvar subgroup showed a significant but weak association with ang-2 this association dropped out when severe and non-severe cases are considered separately  and 13 (DC13) can bind to brain endothelial cells (via EPCR) in a manner that is not dependent on the induction of adhesion molecules such as ICAM-1 ,17,43 th malaria . Therefovar expression  is likely to be an important determinant of endothelial activation. Parasite density may contribute to endothelial activation by increasing the amount of released microparticles, free haemoglobin, and other molecules. Recently, levels of plasma PfHRP2 (thought to represent parasite load ,52, thouvar gene expression measurements and assessment of rosetting phenotype come from circulating parasites rather than sequestered ones. Since sequestration and microvascular obstruction occur in multiple small areas of the microvasculature and not in a uniform manner [Our m manner ,55,56, wm manner .var expression assays that more directly measure the levels of domain cassettes known to bind to this receptor.Further studies are needed to determine whether systemic endothelial activation observed in malaria is causally related to cytoadherence or invoked by circulating factors such as microparticles and other IE derived toxins. In Future studies it will be important to explore the role of cytoadhesion to endothelial protein C receptor and use PfEMP1: Plasmodium falciparum erythrocyte membrane protein one; IC: Impaired consciousness; RD: Respiratory distress; SMA: Severe malarial anaemia; IE: Infected erythrocyte; CM: Cerebral malaria; EC: Endothelial cell; HBMEC: Human brain microvascular endothelial cell.The authors declare that they have no competing interest.var sequencing, rosetting and IE surface antibody measurements. AA, EK, and MM performed the plasma Ang-2 ELISA: AA, GF and PCB performed the statistical analysis. AA and PCB wrote the manuscript with input from all authors. All authors read and approved the final manuscript.PCB, KM: designed the study and provided overall study supervision. GMW, JNM, MO, and PCB: processed samples, performed The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/170/prepubFigure S2. The relationship between ang‒2 and base‒excess.Patients characteristics. Click here for file


Plasmodium falciparum antigens can serve as a marker of recent transmission and serosurveillance may be feasible on a large scale.Critical to sustaining progress in malaria control is comprehensive surveillance to identify outbreaks and prevent resurgence. Serologic responses to P. falciparum whole parasite lysate using serum eluted from dried blood spots. The expected mean annual increase in optical density (OD) value for individuals with a documented prior history of recent malaria was determined using mixed models. SatScan was used to determine the spatial clustering of households with individuals with serological evidence of recent malaria, and these households were plotted on a malaria risk map.Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys in two study areas in Southern Province, Zambia, one in 2007 and the other in 2008 and 2009. Blood was collected and stored as dried spots from participating household members. A malaria rapid diagnostic test (RDT) was used to diagnose malaria. An enzyme immunoassay (EIA) was used to detect IgG antibodies to asexual stage RDT positivity differed markedly between the study areas and years: 28% of participants for whom serologic data were available were RDT positive in the 2007 study area, compared to 8.1% and 1.4% in the 2008 and 2009 study area, respectively. Baseline antibody levels were measured in 234 participants between April and July 2007, 435 participants between February and December 2008, and 855 participants between January and December 2009. As expected, the proportion of seropositive individuals increased with age in each year. In a subset of participants followed longitudinally, RDT positivity at the prior visit was positively correlated with an increase in EIA OD values after adjusting for age in 2007  and in 2008 . RDT positivity at the concurrent visit also was associated with an increase in EIA OD value in 2007  but not in 2008 . Households comprised of individuals with serologic evidence of recent malaria overlapped areas of high malaria risk for serologic data from 2009, when parasite prevalence was lowest.P. falciparum antigens using blood collected as dried blood spots can be used to detect temporal and spatial patterns of malaria transmission in a region of declining malaria burden, and have the potential to identify focal areas of recent transmission.Serological surveys to whole asexual  Increased funding for malaria control and elimination has led to implementation of comprehensive control programmes and concomitant reductions in the burden of malaria in many parts of sub-Saharan Africa ,2. ZambiPlasmodium falciparum can serve as a proxy measure of malaria transmission [19 and AMA-1 identified infection within the previous four months among children younger than six years of age in The Gambia [P. falciparum lysate were measured by enzyme immunoassay in two community-based cohorts in southern Zambia to assess the utility of serological surveys to identify temporal and spatial patterns of recent malaria transmission in a region with declining malaria burden in southern Zambia.Despite this impressive achievement, the incidence of malaria increased in five of nine provinces of Zambia in 2010 ,7. The gsmission -13 and me Gambia . Serologe Gambia  or oral e Gambia ,17. IgG Anopheles arabiensis is the primary vector responsible for malaria transmission [2 region to the east of Macha Hospital  for malaria and preparation of dried blood spots (DBS). The DBS were collected on filter paper , dried overnight and stored individually with desiccant in a sealed plastic bag at −20°C. DBS collected in 2007 were stored with desiccant at room temperature and DBS collected from February to September in 2008 were initially stored at room temperature but subsequently stored at −20°C. All samples collected after September 2008 were stored at −20°C.P. falciparum histidine-rich protein 2. This RDT was reported to detect 82% of test samples with P. falciparum at a concentration of 200 parasites/μL and 98% of test samples with a concentration of 2000 parasites/μL, with false positives in 0.6% of negative samples [A RDT  was used to detect  samples . IndividThe study was approved by the University of Zambia Research Ethics Committee and the Institutional Review Board at the Johns Hopkins Bloomberg School of Public Health.P. falciparum asexual stage lysate diluted in PBS and incubated overnight at 4°C. Plates were washed with PBST three times, blocked with 5% skim milk in PBS for 1 hour at 37°C, and washed again with PBST four times. Eluted samples were plated in triplicate and incubated for 1 hour at 37°C. Negative control serum  and positive control serum  were diluted 1:200 in 5% skim milk PBST and included in triplicate on each plate. The plates were subsequently washed with PBST four times and incubated for one hour at 37°C with peroxidase-labeled goat anti-human IgG . Following four washes, the plates were incubated for 15 minutes with 100 μL of ABTS solution (KPL). Absorbance was measured at 405 nm and IgG levels were expressed as the optical density.Antibodies to whole asexual parasite (NF54) lysate were measured by enzyme immunoassay using serum extracted from DBS. A circle of 7 mm (corresponding to approximately 10 μL blood volume) in diameter was punched out of the DBS and half of the circle  was used to extract serum. The half circle was soaked in 500 μL (1:200 dilution of serum) 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1 hour at room temperature. Immulon 2HB flat-bottomed 96-well plates  were coated with 1 μg/mL of whole A threshold optical density (OD) value of 0.57 was established to distinguish seronegative from seropositive individuals based on the mean OD value plus three standard deviations from filter paper spotted with serum from ten individuals never exposed to malaria. Individuals with OD values above this threshold were defined as seropositive. Results were included only if the standard deviation of the triplicate values was within 20% of the mean OD value. The mean standard deviation for the triplicate values was 0.020 in 2008 and 0.026 in 2009.P. falciparum antigens, expressed as OD values and adjusted for age, were analyzed using data from participants enrolled in the longitudinal cohort. Malaria infection was defined in two ways: 1) a positive RDT result on the day of the study visit was defined as concurrent malaria; and 2) a positive RDT result at any previous study visit was defined as prior malaria. The change in antibody level over the one year study period was expressed as the mean difference in OD values between study visits. To account for within-subject correlation, a linear regression model was fitted adjusting for within subject correlation, with random intercept and robust standard errors.Associations between RDT positivity and antibody levels to P. falciparum. A control was defined as an individual whose change in OD value from the first to the last study visit at the end of the study year did not exceed the coefficient from the linear model. Spatial clustering of cases was analyzed with SatScan™ software (version 9.0) using the Kulldof spatial statistic [Using the coefficient for the change in the OD value in individuals with a prior documented history of recent malaria, participants enrolled in the longitudinal cohort in 2008 and 2009 were categorized as either a case or control. A case was defined as an individual whose change in OD value from the first to the last study visit at the end of the study year exceeded the coefficient from the linear model, and thus represented someone with a greater than expected increase in OD value and serological evidence of recent exposure to tatistic  and a Betatistic ,24 and rP. falciparum antigens were measured in 234 participants between April and July 2007 , 435 participants between February and December 2008 , and 855 participants between January and December 2009 . Most participants enrolled in the longitudinal cohort in 2008 continued to be followed in 2009, although three longitudinal households were replaced and six new households were added in 2009. The median age of the study participants was 12.2 years  in 2007, 14.2 years  in 2008, and 14.0 years  in 2009). Slightly less than half of the participants were male (47%)  was compared to participants with two or three RDT positive results . As there was no statistically significant difference in mean EIA OD between individuals with a single or multiple RDT positive results, RDT results during any prior study visits were dichotomized as negative (no prior positive RDT result) or positive (at least one prior RDT positive result) for the analysis.The effect of documented malaria during the study period on antibody levels to P. falciparum antigens was not examined.In 2008, 99 individuals enrolled in the longitudinal cohort had at least two study visits. Of these, nine participants had a single RDT positive result and one participant had two RDT positive results. In 2009, 168 individuals enrolled in the longitudinal cohort had at least two study visits. Only two were RDT positive and none had multiple RDT positive results. Due to the small number of RDT positive results in 2009, the association between RDT positivity and antibody levels to P. falciparum antigens and a concurrent or prior documented episode of malaria was assessed, adjusting for age. In 2007, participants with a positive RDT result at the concurrent visit had a 0.177 higher OD value  than RDT negative participants, adjusting for age , was used to assess whether serology could be used to identify spatial clusters of malaria transmission in the study area for 2008 and 2009. In 2008, 55 participants had samples collected at the beginning (February) and end (December) of the study period. The median age at the first study visit was 11 years  and the median interval between the first and the last study visit was 296 days (range: 284 to 305 days). Of these 55 participants, 43 (78%) with a median age of 14 years  had OD values that increased more than 0.116. In 2009, 74 individuals had samples collected at the beginning (January) and end (December) of the study period. The median age at the first study visit was 13 years  and the median interval between the first and last study visit was 305 days (range: 281 to 324 days). Of these 74 participants, 10 (14%) with a median age of 21 years  had an OD values that exceeded 0.116.Participants with OD values that exceeded 0.116 over the one year study period were classified as cases and clusters of households with cases were identified. The primary household clusters identified in 2008 (within a 9.2 km radius) were not statistically significant (p = 0.15), but household clusters identified in 2009 (within a 5.4 km radius) achieved marginal statistical significance (p = 0.06) Table . Study hP. falciparum antigens measured by enzyme immunoassay increased with increasing age and were correlated with prior malaria infection as documented by RDT in this region of declining malaria transmission in southern Zambia. In the year with the lowest parasite prevalence, spatial clustering of individuals with serologic evidence of recent infection overlapped with a region at risk for malaria transmission as previously determined by ecological factors [Antibody levels to whole, asexual stage  factors . These fP. falciparum and clinical immunity to malaria after repeated exposure, with earlier acquisition of seropositivity and clinical immunity in regions with higher transmission intensity [19[Residents of malaria endemic areas acquire antibodies to ntensity . Immunitntensity . In soutntensity ,11. Althnsity [19.P. falciparum was recently described [P. falciparum may not be suitable to detect short term changes in population immunity in regions with rapid declines in malaria transmission. This study attempted to identify recent exposure to P. falciparum by measuring an increase in OD values above the expected value for having RDT positive result during one year study period. The findings suggest that recent exposure to P. falciparum can be detected through increases in OD values. Prior studies measured antibody responses to single parasite antigens, such as AMA1 or MSP119, and samples were collected in cross-sectional studies [The use of serology as a marker of malaria transmission intensity and recent exposure to escribed . A poten studies . In cont19, AMA1 and MSP2 were measured; the broader antibody response to whole parasite lysate in our study may account for the larger cluster sizes.Spatial clusters of individuals with serological evidence of recent infection was identified and these clusters were validated by comparing them to a previously reported malaria risk map of the study area . Prior sP. falciparum antigens were positively correlated with prior RDT positivity. Serological surveys to whole asexual P. falciparum antigens using blood collected as dried blood spots can be used to detect recent malaria infection and identify focal areas of transmission, particularly in a region of declining malaria burden.Antibodies to whole, asexual stage The authors declare that they have no competing interests.TK participated in the coordination of the study, performed the data analysis and drafted the manuscript. SC performed the enzyme immunoassays and reviewed the manuscript. TS participated in the spatial analyses and reviewed the manuscript. HH coordinated the data and specimen collection and reviewed the manuscript. SM supervised the laboratory assays and reviewed the manuscript. PET supervised the field activities and reviewed the manuscript. GG participated in the spatial analyses and reviewed the manuscript. WJM conceived of the study, participated in its design and coordination, and participated in the preparation of the manuscript. All authors read and approved the final manuscript.


P. falciparum infections among HIV-infected pregnant women in Bangui, the capital of the Central African Republic.Co-infection with malaria parasite and HIV is an emerging public health problem in tropical areas, particularly in pregnant women, and management of the concurrent effects of these two infections is challenging. Co-trimoxazole is a sulfamide preparation used to prevent opportunistic infections in HIV-infected patients, and many studies have reported that it has significant activity against malaria. As the efficacy of intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine (SP) against malaria is decreasing, co-trimoxazole might be an alternative for preventing malaria among HIV-infected populations. The aim of this study is to compare the effectiveness of SP-IPT, which is recommended for the prevention of malaria during pregnancy in the Central African Republic, with that of a daily dose of co-trimoxazole against 3, will be eligible. All the women will provide written consent before being enrolled in the study and will then be randomly allocated to either SP-IPT (25 mg of sulfadoxine and 1.25 mg of pyrimethamine) or daily co-trimoxazole doses (960 mg per dose). The primary end-point is the placental malaria parasitaemia rate at delivery. Other main outcome measures include the number of malaria episodes during pregnancy, safety, and treatment compliance. Furthermore, the frequency of molecular resistance markers dhfr and dhps will be measured.The MACOMBA study  is a multicentre open-label randomized clinical trial conducted at four maternity hospitals in Bangui. All HIV-infected pregnant women presenting for an antenatal clinic visit between the weeks 16 and 28 of amenorrhoea, with a CD4 count of more than 350 cells/mmIn this trial, we seek to confirm whether co-trimoxazole is operationally suitable to replace SP-IPT in order to prevent malaria among pregnant women infected with HIV in the Central African Republic.NCT01746199.ClinicalTrials.gov Identifier:  Malaria and HIV infection are important global public health problems. In 2008, malaria affected more than 2 billion people, representing 40% of the world’s population. That same year, some 247 million episodes of malaria were recorded, in addition to almost 1 million deaths, of which nearly 20% were children. Each year, more than 50 million women living in malaria-endemic regions become pregnant, 20% for the first time, and more than half develop complications. In 2008, an estimated 33.4 million people were living with HIV infection, and 2 million people, including 280,000 children, died from AIDS-related diseases. Sub-Saharan Africa is the most severely affected region: in 2007, it was estimated to contain 67% of all people living with HIV in the world, 75% of all women infected with HIV, and 72% of all deaths from AIDS [Effective integrated care of patients with malaria and HIV infection in regions where the two coexist is therefore an international health challenge.Concurrent HIV infection and malaria during pregnancy is an emerging problem in mother and child health, with an estimated 1 million pregnancies per year among women with both infection and malaria. In 11 of the 43 countries of Africa in which malaria is endemic , the prevalence of HIV infection among pregnant women seen at antenatal visits varied from 10% to more than 25% .As the clinical and biological symptoms of malaria are enhanced by co-infection with HIV, pregnant women with HIV infection more often have episodes of malaria that evolve into complicated forms .et al. [et al. [The World Health Organization (WHO) recommends that pregnant women in endemic regions undergo four antenatal examinations and receive two doses of sulfadoxine-pyrimethamine (SP) at each visit (directly observed treatment (DOT)), beginning at week 16 of pregnancy . Steketeet al.  however, [et al.  reportedPlasmodium falciparum malaria is well recognized. Thus, Mermin et al.[Ascertaining HIV serological status has become a prerequisite for better malaria prevention. The efficacy of co-trimoxazole in preventing in et al. reportedin et al., showed in et al.-16, and in et al..P. falciparum malaria is increasing.Therefore, preventive treatment with SP for all HIV-positive people (including pregnant women) who are receiving treatment containing co-trimoxazole is superfluous and is even contraindicated in light of its common side-effects (particularly severe skin reactions). Few studies, however, have described the efficacy of co-trimoxazole in preventing malaria among pregnant women, particularly in areas where the frequency of therapeutic failures with SP in cases of P. falciparum to SP in many sub-Saharan African countries [dhfr) and dihydropteroate synthase (dhps). The number of mutations correlates with the extent of P. falciparum resistance to SP in vitro. In studies carried out in Bangui, the prevalence of therapeutic failure was estimated to be 23.8% after 14 days of follow-up among children with uncomplicated malaria [P. falciparum to pyrimethamine in vitro was reported to be 38.3% [in vitro of P. falciparum strains [dhfr at Ile-51, Arg-59 and Asn-108 and the double mutation at Gly-437 and Glu-540, alone or in combination, are associated with therapeutic failure of SP [The emergence of an increase in the frequency of resistance of ountries ,19, inclountries ,21, chal malaria , while tbe 38.3% , and there of SP .P. falciparum has increased due to the HIV epidemic [P. falciparum to both co-trimoxazole and SP because of their similar modes of action, although this hypothesis has not been proven [dhfr and dhps genes to SP and co-trimoxazole in vitro showed partial cross-resistance to the two drugs [dhfr and Gly-43 of dhps correlated with P. falciparum resistance to co-trimoxazole [Pregnancy and HIV infection increase the risk for emergence of mutated strains that are resistant to SP, partly because a wide variety of types and clones are found in parasitaemia among pregnant women (genetic diversity) , but alsepidemic . There in proven ,28. A stwo drugs . Mutatiomoxazole .The current prevalence of malaria among women at first presentation to an antenatal clinic in Bangui is estimated to be 23% . Since 2In 2011, we assessed data from maternity centres of Bangui and found that more than 75% of pregnant women accepted HIV testing. This finding is in accordance with the recommended threshold of acceptance . Our pre3 from the second trimester and throughout pregnancy [To prevent opportunistic infections, co-trimoxazole is given to all women at WHO clinical stages 2, 3 and 4 and those at stage 1 with a CD4+ count <350/mmregnancy .In the Central African Republic, two treatment schemes are recommended for the management of HIV infections during pregnancy :3, regardless of their symptoms. Treatment begins at any time during pregnancy. First-intention treatment should comprise zidovudine (AZT) plus lamivudine (3TC), with nevirapine (NVP) or efavirenz (EFV). Breastfed infants should receive NVP or AZT throughout breastfeeding.•Antiretroviral treatment is offered to all pregnant women with severe or clinically advanced disease, regardless of their CD4+ count, and for those with a count of ≤350 cells/mm•Women who are ineligible for antiretroviral treatment are given a preventive regime to reduce HIV MTCT: (i) AZT from week 16 of amenorrhoea, (ii) AZT, 3TC and NVP during labour and delivery and (iii) AZT and 3TC for 7 days after delivery. Breastfed infants receive a dose of NVP at birth and a daily dose from birth to 1 week before weaning. Infants who are not breastfed are given a single dose of NVP at birth, followed by one dose per day of NVP or AZT up to the age of 6 weeks.P. falciparum isolated from HIV-positive pregnant women express more dhfr and dhps resistance markers.Our main hypothesis is that co-trimoxazole is more efficacious than SP-IPT against placental parasitaemia, perhaps due to a higher plasma concentration of co-trimoxazole attained with daily doses. If we validate this hypothesis, co-trimoxazole could be recommended as prophylaxis for HIV-positive pregnant women, regardless of their CD4+ cell count. In this study, we will also test the hypothesis that the strains of 3.The main objective of this randomized trial is to confirm that co-trimoxazole is more efficacious than SP against placental parasitaemia (primary end-point) among HIV-positive pregnant women with a CD4+ count >350 cells/mmWe will also compare the two treatments with regard to adherence, the occurrence of adverse events, the frequency of maternal anaemia (hemoglobinaemia <10 g/dl), malaria episodes during pregnancy, abortions, stillbirths, prematurity (birth <37 weeks of amenorrhoea) and low birth weight (<2500 g), placental malaria, umbilical malaria transmission, and mother-to-child HIV transmission.The MACOMBA study  is a multicentre randomized controlled open-label trial to compare SP-IPT with co-trimoxazole. The study will be open-label, because it will be impossible to blind the pregnant women and health-care workers involved to which group the women are assigned. Furthermore, we seek to compare the two strategies under real-life conditions.The trial will be performed by the Institut Pasteur of Bangui, supported by Institut Pasteur of Paris. The four main maternity hospitals in Bangui are involved in this study.3 and they have no opportunistic diseases . These women will not be re-tested for HIV, but their CD4+ count will be checked at this stage of inclusion in the study.HIV-seropositive pregnant women will be recruited in the four maternity clinics. All pregnant women attending maternity clinics will be offered an HIV test as part of the standard procedure of HIV counselling . Inclusi3 and no sign of WHO stage 2, 3 or 4, agreement to attend all antenatal consultations for the study, willingness to adhere to all the requirements of the study (including voluntary counselling and testing for HIV) and signed informed consent.The inclusion criteria are age ≥18 years, HIV positivity; gestation of 16 to 28 weeks; CD4+ count >350 cells/mmThe exclusion criteria are psychological instability that might interfere with compliance, hypersensitivity to sulfamides or dermatological disease  that would increase the risk for severe reaction to the drugs being tested; severe anaemia (Hb <7 g/dl), known hepatic cardiac or renal disease, or any other severe disease.Potential study participants will be identified by midwives at antenatal clinics. Women eligible for participation in the study will be invited for additional counselling by a research midwife to ensure that they are fully informed orally and by written material about the nature of the study. After providing informed consent to participate in the study, the women will be randomly allocated to either the SP-IPT or co-trimoxazole group.Randomization of women will be centralized and stratified according to maternity clinic and gravidity (primigravidae versus multigravidae). For each maternity clinic, two random lists for SP-IPT and co-trimoxazole in a ratio 1:1 will be generated for primigravidae and multigravidae, with the R software (version 2.14.1). Once a pregnant woman is confirmed to be eligible for the study, the field investigator will telephone the coordination staff at the Institut Pasteur of Bangui to indicate the gravid rank, and the site staff will assign women to a treatment arm according to the randomization list, respecting the chronological order of inclusion.The drugs to be used in this study will be supplied by the International Dispensary Association in the Netherlands. Doses of SP-IPT and co-trimoxazole will be administered from week 16 to the end of pregnancy. Three doses of SP will be administered under direct observation, equivalent to 25 mg of sulfadoxine and 1.25 mg of pyrimethamine per kilogram of body weight. The first dose will be given between weeks 16 and 28 of amenorrhoea, the second 1 month later and the third 1 month after the second.A daily dose of one co-trimoxazole tablet (containing 160 mg trimethoprim and 800 mg sulfamethoxazole) will be given until delivery . CompliaAll women will be given iron supplementation (200 mg) and folic acid (0.4 mg) and will receive an insecticide-impregnated bed net .During follow-up, any suspected malaria episode will be followed up immediately with parasitological evaluation (blood smear). Treatment with quinine at a dose of 24 mg/kg body weight will be given at three doses per day 8 hourly apart for 7 days. The tablet form of quinine will be given in the absence of severe clinical signs. If the malaria episode is associated with severe clinical symptoms, quinine (perfusion in 10% serum glucose) will be administered parenterally at a dose of 24 mg/kg body weight per day until clinical evaluation shows that oral quinine can be taken. Patients who experience adverse effects of quinine will be treated with artemether or artemisinin combined treatment according to national guidelines.A woman who fulfils all the inclusion criteria but is found to have symptomatic malaria parasites at recruitment will be treated for the malaria episode and will not be included in the study. She will be reconsidered for inclusion later, when she is no longer parasitaemic. All women with asymptomatic malaria parasites at baseline will be included and randomized to either the SP-IPT or co-trimoxazole group. If symptomatic malaria or persistence of asymptomatic malaria parasitaemia is seen 8 days later, the woman will be given curative treatment, as stated above Figure .All women included in this study will undergo regular clinical surveillance (once a month) and will be encouraged to notify the doctor involved in the study of any adverse events. The adverse effects attributed to SP-IPT and co-trimoxazole will be addressed according to cutaneous toxicity, classified according to the grades of the French National Agency for AIDS Research. Symptomatic medications will be given, and the study may be interrupted at the discretion of the clinicians.All adverse events associated with HIV infection or antiretroviral treatment will be managed by the national service for prevention of MTCT.3 will be sent to the centre for antiretroviral treatment at the Hôpital Communautaire or Hôpital de l’Amitié with a referral to the service for infectious diseases.All women whose HIV infection evolves to clinical stage 2 or who have a CD4 count interval of ≤350 per mmAt baseline, the following data will be collected: socio-demographic characteristics, clinical data, obstetric history, gestational age estimated from the date of the last menstrual period and/or measured by uterine fundal height, and biological end-points , malaria diagnosis, and blood cell count). During follow-up, standard clinical examinations will be performed monthly. Adverse events will be recorded and any suspected malaria episode will be investigated by laboratory testing. Blood will be taken from women presenting with a malaria episode, and CD4 count and HIV VL will be determined.dhfr and dhps) will be performed for all episodes of Plasmodium infection.Polymerase chain reaction (PCR) for malaria diagnosis and identification of resistant strain markers . At this point, MTCT of HIV and possible passage of the malaria parasites though umbilical blood circulation will be assessed.Plasmodium DNA extraction. Early screening of HIV and malaria parasites transmission in infants will be determined from heel-prick and umbilical blood respectively. Placental blood will be collected immediately after delivery. For this procedure, a block of tissue (1 × 1 × 1 cm) will be excised from the basal side of the placenta, resulting in a large pool of intervillous blood at the excision site. The placental tissues will be pressed to prepare thin and thick blood smears  minicollect tubes at inclusion, at scheduled and unscheduled antenatal clinic visits if a malaria infection is diagnosed, and after delivery. These blood samples will be tested for blood cell counts and HIV VL estimation. Finger pricks for thick and thin blood smear preparation and antigen Pf/Pan-SD Bioline™ rapid diagnostic testing will be performed for any suspected episodes of malaria during follow-up. Maternal blood will be also collected on filter paper for Malaria rapid diagnostic tests and thick smear analyses will be performed at the study sites, while other laboratory analyses will be performed at the Institut Pasteur of Bangui.CD4+ cells will be counted by cytometry , and blood cell counts will be conducted by the ABX PENTRA 60 .HIV VL will be estimated at the Institut Pasteur of Bangui with the ABI PRISM 7000 .Generic HIV DNA Cell kits  will be used for PCR to assess maternal-child transmission of HIV infection.P. falciparum. Nested species-specific PCR will be performed to screen maternal and placental blood samples for malaria parasites as described previously [The antigen Pf/Pan-SD Bioline™ rapid diagnostic test will be conducted according to the manufacturer’s guidelines. Thick blood smears will be air-dried, stained with 4% Giemsa and analysed under a light microscope (× 100 oil immersions) to detect asexual forms of eviously . The greeviously ,37.Plasmodium resistance markers to pyrimethamine and sulfadoxine (dhfr and dhps) will be performed on blood samples with a positive malaria result. The genotypes pfdhfr 51, 59 and 108 and pfdhps 437 and 540 will be determined by nested PCR [Molecular studies to identify sted PCR .Plasmodium.The primary end-point of the trial is placental parasitaemia at delivery. Data on this variable will be coded as positive or negative results. Other outcome measures are adherence to co-trimoxazole treatment, adverse events related to co-trimoxazole and SP-IPT, the incidence of malaria episodes during pregnancy and complications due to malaria: anaemia (hemoglobinaemia <10g/dl), abortion (delivery <28 weeks of amenorrhoea), stillbirth, prematurity (birth <37 weeks of amenorrhoea) and low birth weight (<2500 g body mass), mother-to-child HIV transmission and umbilical blood parasitaemia. At delivery, maternal, placental and newborn umbilical blood will be sampled to detect the asexual form of On the basis of previous studies ,17,39,40The primary end-point will be analysed as intention-to-treat; deaths and women lost to follow-up will be considered failures. Analysis of the ‘per protocol’ population will be conducted among women who complete the study until delivery and undergo the recommended initial treatment.dhfr mutations, maternal anaemia and maternal morbidity or mortality. Student’s t test or the Mann-Whitney test will be used to compare continuous variables in the two arms. A logistic regression model will be used, with adjustment for maternity clinic, gravidity, age, CD4 count and anaemia to compare the two groups of women for the number of placental malaria infections, malaria episodes during pregnancy, and identified resistance.The data will be recorded in an ACCESS™ database  and will be analysed with STATA™ software . The analyses will be carried out after stratification according to the maternity clinic and gravidity (primigravidae versus multigravidae). The chi-squared test or Fisher’s exact test will be used to compare categorical variables between the two groups, including the number of positive placental malaria infections (primary end-point), cumulated incidence of malaria episodes during pregnancy, proportion of parasite strains with The National Ethics Committee of the Central African Republic and the Clinical Research Committee of the Institut Pasteur in Paris approved the study protocol.unpublished data). If the frequency of mutations in malaria parasites is found to be higher in the SP arm of this study, all seropositive women will be offered treatment with co-trimoxazole. The results of this study will be communicated to health policy-makers and experts who make recommendations about malaria prevention among HIV-positive women.In this trial, we seek to confirm that co-trimoxazole is operationally suitable to replace SP-IPT in order to prevent malaria among pregnant women infected with HIV in the Central African Republic. Studies of alternatives to SP in preventing malaria during pregnancy have been designed in Benin ,42. The This study is currently being implemented.The authors declare that they have no competing interests.AM, MV and MK were involved in the conception and design of the study. AS, ES, SG, GGBB, SM, AB, JMM, OS and LL have made substantial contributions to the design and are involved in the conduct of the study. AM, MV and MK drafted the manuscript. All authors mentioned in the manuscript are members of MACOMBA study group. They read and approved the final manuscript.


The World Health Organization recommends that malaria treatment should begin with parasitological diagnosis. This will help to control misuse of anti-malarial drugs in areas with low transmission. The present study was conducted to assess the prevalence of parasitologically confirmed malaria among children under five years of age presenting with fever or history of fever in rural western Tanzania. A finger prick blood sample was obtained from each child, and thin and thick blood smears were prepared, stained with 10% Giemsa and examined under the light microscope. A structured questionnaire was used to collect each patient's demographic information, reasons for coming to the health center; and a physical examination was carried out on all patients. Fever was defined as axillary temperature ≥ 37.5°C.P. falciparum infection. Of the children with P. falciparum infection, 52.7%  had fever and the remaining had no fever. The geometrical mean of the parasites was 708.62  parasites/μl and 25%  of the children with positive P. falciparum had ≥ 1001 parasites/μl. On Univariate  and multivariate  analysis, only children above one year of age were associated with malaria infections.A total of 300 children with fever or a history of fever (1 or 2 weeks) were recruited, in which 54.3%  were boys. A total of 76  of the children had fever. Based on a parasitological diagnosis of malaria, only 12%  of the children had Only a small proportion of the children under the age of five with fever had malaria, and with a proportion of children having non-malaria fever. Improvement of malaria diagnostic and other causes of febrile illness may provide effective measure in management of febrile illness in malaria endemic areas. In Tanzania, malaria is a leading cause of health service attendance and the disease contributes to approximately 40% of all morbidities reported in children under five presenting in outpatients . One of At present, episodes of malaria related fever among children under five are reported to be in decline in Africa , and basThere is a strong need for clinicians to base their treatment decisions on laboratory confirmed cases of malaria. Parasitological diagnosis will enable clinicians to report more accurately whether patients presenting with fever or history of fever actually are parasitaemic . EvidencThe cross-sectional survey was conducted amongst children attending the outpatient clinic at the Nyasa Health Center located in Nzega District, Tabora region, in western Tanzania. The study was conducted between August and October 2010. Nzega district is located in a holoendemic area and intense malaria transmission is observed during the long rain season (February-May).The inclusion criteria for the study subjects were: (1) children who sought treatment at the health center and had fever/history of fever in the past 1-2 weeks, (2) Parents/guardians consent for their children to participate in the study, (3) Children aged between 2 months and 60 months, and (4) no history of treatment with anti-malarial drugs in the past two weeks.Parents/guardians of eligible children gave written informed consent to allow their children to participate in the study. Children under five were consequently recruited into the study until the sample size of 300 was reached. A qualified clinician used a pre-tested Swahili translated questionnaire to collect patient's demographic information and the reasons why he/she was brought to the health center. The clinician conducted a physical examination on all patients and checked for pallor, spleen enlargement, jaundice and other abnormalities. The axillary body temperature was measured using a digital clinical thermometer. Fever was defined as body temperature ≥ 37.5°C.Plasmodium species was declared as positive. Slides were declared as negative if no asexual parasites were found after examining 100× objectives. P. falciparum parasite density was determined according to the number of parasites per 200 WBC (white blood cells), assuming a total WBC count of 8,000/μL.A finger prick blood sample was obtained and one thin and two thick blood smears were prepared and stained with 10% Giemsa. The diagnosis and management of malaria was based on initial readings of one of the thick blood smears at the health centre. Management of malaria was done in accordance with the Tanzania Malaria Treatment Guidelines. The final reading of the Giemsa stained slides was done at Weill-Bugando University College of Health Sciences which was based on a rigorous quality control system. A second microscopist re-examined all the stained blood smears and any disagreement between the first and second microscopist was resolved by a third microscopist. In addition, the third microscopist examined 10% of positive and negative slides which were selected randomly. The microscopist identified malaria parasite stages and speciated the malaria parasites. The Giemsa stained smears were examined at 100× objectives under oil immersion and a slide with the presence of trophozoites or any other asexual form of P-value < 0.2 be included in the multivariate model. Stepwise backward logistic regression was used to determine whether these variables were independent predictors of malaria in children. Odds ratios with 95% Confidence intervals were used to measure the strength of association at statistical significance level of P < 0.05.Data were analysed using Stata Version 11 . An estimate of relative frequency and means for demographic data and outcome of interest was derived. In order to determine independent predictors of malaria in febrile children, numerous variables were assessed, including demographic characteristics, symptoms and clinical signs. Univariate analysis was employed to identify variables with a The study received ethical clearance from the faculty of medicine, Weill-Bugando University College of Health Sciences, Mwanza, Tanzania.A total of 300 children under five with fever or history of fever participated in the study. The median age of the enrolled children was 13 months (range-minimum-maximum: 8 - 23.5 months). Of the 300 children under five, 54.3%  were boys and 45.7%  were girls. Parents/guardians reported fever or history of fever as part of the illness in all the children, although during physical examination only 25.3%  had an axillary temperature ≥ 37.5°C.2 (1) = 2.7248 P = 0.099). Table Parasitological diagnosis of malaria parasites revealed that out of the 300 children under five, only 12%  had positive slide readings. Of these, 52.7%  had fever and 42.7%  had no fever on physical examination. The distribution of positive malaria slide readings between girls and boys was not statistically significant  parasites/μl. Among the P. falciparum-positive children, 52.8%  had 1-500 parasites/μl, 22.2%  had between 501 -- 1000 parasites/μl and 25%  had ≥ 1001 parasites/μl. There was no statistical significance on the distribution of positivity and parasite density by age  and sex . Table All the P = 0.044) and multivariate  analysis, only children above one year of age were associated with malaria infections.Table P. falciparum was the most predominant species in the study area as well as many parts of Tanzania and Africa [P. falciparum malaria observed in our study was higher than 6% reported in Pakistan [P. falciparum-positive blood film [P. falciparum malaria among the study population could be attributed in part to the difference in malaria transmission patterns, season of conducting the study and the use of malaria prevention tools [In this study almost 12% of all the study population had malaria positive slides. In all the children under five with malaria, d Africa ,27. The Pakistan  and loweood film . The varon tools ,31.P. falciparum infections had no fever on physical examination. Similar findings have been reported from malaria endemic areas [In our study, a proportion of children under the age of five with ic areas ,28. The ic areas . Becauseic areas .Several factors have been described to predict malaria in children under five living in malaria endemic countries ,34. AmonIn our study, a proportion of children under the age of five years presented with fever but had malaria negative blood slides. This indicated that there could be other causes of fever among the febrile children under five in the present study area. At a time when the proportion of malaria-related fevers are reported to be significantly declining in many parts of Africa , thus inP. falciparum malaria observed in the present study participants, despite the area being located in holoendemic area [Our study is subject to limitations. The cross sectional nature and the small sample size of the children under the age of five years, especially those with fever and malaria parasites, may have contributed to the lack of temporal association between malaria and other variables. In addition, a relatively small sample of the children under five who participated in the study may not be a complete representation of all the children under five who seek health services at the study health centre. Furthermore, the present study was conducted during the dry season, during which malaria transmission is known to be low. This may have contributed to the low prevalence of mic area ,31,34. FP. falciparum malaria and a proportion of the febrile children had malaria negative blood slides. Age (above one year) was the only association with malaria infections. To avoid the current practice of treating every fever with anti-malarial drugs, it is important to improve malaria diagnosis and the diagnosis of non-malaria fever. We recommend that health policy makers consider increasing microscopic diagnoses or malaria Rapid Diagnostic Tests (RDTs) to rural health facilities.In conclusion, only a small proportion of febrile children under five were parasitologically positive for The authors declare that they have no competing interests.HDM, EJK and EEM designed the study. WM carried out the clinical examination of the children and interviewed the parents. BRK and HDM conducted the data analysis and drafted the manuscript. All authors read and approved the last version of the manuscript for submission.


Ascaris lumbricoides is common. Malaria parasites induce a pro-inflammatory immune response that contributes to the pathogenic sequelae, such as malarial anaemia, that occur in malaria infection. Ascaris is known to create an anti-inflammatory immune environment which could, in theory, counteract the anti-malarial inflammatory immune response, minimizing the severity of malarial anaemia. This study examined whether Ascaris co-infection can minimize the severity of malarial anaemia.Co-infection with malaria and intestinal parasites such as Ascaris co-infection on anaemia severity. Children were enrolled and tested for malaria, helminths and anaemia at baseline, four, and eight months. Six hundred and ninety subjects were analysed in this study. Generalized linear mixed models were used to assess the relationship between infection status and Ascaris and Plasmodium parasite intensity on severity of anaemia, defined as a haemoglobin less than 11 g/dL.Data from a randomized controlled trial on the effect of antihelminthic treatment in Nigerian preschool-aged (6–59 months) children conducted in 2006–2007 were analysed to examine the effect of malaria and Ascaris at baseline, 60% were co-infected four months later and 48% were co-infected eight months later, underlining the persistent prevalence of malaria-nematode co-infections in this population. Over the course of the study the percentage of anaemic subjects in the population ranged between 84% at baseline and 77% at the eight-month time point. The odds of being anaemic were four to five times higher in children infected with malaria compared to those without malaria. Ascaris infection alone did not increase the odds of being anaemic, indicating that malaria was the main cause of anaemia in this population. There was no significant difference in the severity of anaemia between children singly infected with malaria and co-infected with malaria and Ascaris.Malaria prevalence ranged from 35-78% over the course of this study. Of the malaria-infected children, 55% were co-infected with Ascaris co-infection neither exacerbated nor ameliorated the severity of malarial anaemia.In this cohort of Nigerian preschool children, malaria infection was the major contributor to anaemia status.  Plasmodium falciparum . Severely anaemic children also had a trend towards higher levels of Plasmodium parasites than children with mild anaemia, but the p-value was no longer significant after adjusting for multiple comparisons (p = 0.09). Similar results between anaemia groups were found at four months and eight months.Anaemia prevalence and degree of anaemia were significantly correlated with increasing malaria parasite intensity Table . The higps Table . In bothSchistosoma and Trichuris in addition to Ascaris) was examined in each anaemia group over time. As expected, parasite intensities changed over time (results not shown) but were not significant when comparing anaemia groups (Table Ascaris-only was considered (p = 0.20). Hookworm infection was not significantly associated with an increased odds of anaemia at baseline (p = 0.59) or at any of the three time points analysed (p = 0.66).Helminth parasite intensity  or malaria parasite densities (p=0.965) than children with malaria-only infection but did have significantly lower haemoglobin levels than uninfected or Ascaris-only infected children (p<0.001) (Table Ascaris-only infection had haemoglobin levels that did not significantly differ from children with no infection (p = 0.999). Similar results were seen at four months and eight months. The odds of having anaemia were greater in the co-infection group when compared to the uninfected and Ascaris-only group, but were the same when compared to the malaria only group   Table . Furtherup Table . This st0) Table .Ascaris and malaria was common in this cohort of children and this study investigated whether the severity of malarial anaemia was altered in co-infected children.Anaemia was highly prevalent in this cohort of preschool Nigerian children, with a rate of 84%. This is significantly higher than prior studies ,28, and Ascaris infection would be protective with respect to the severity of malarial anaemia. The rationale for this hypothesis was that the induction of an anti-Ascaris immunosuppressive response would dampen the anti-malarial inflammatory immune responses, thus reducing the contribution of the inflammatory immune response on malarial anaemia [Ascaris did not have any effect on the severity of malarial anaemia, in agreement with a previous study carried out in Cameroon [The initial hypothesis was that  anaemia . HoweverCameroon , and in Cameroon . In thisCirculating levels of tumour necrosis factor (TNF) , macrophet al.[The immunoregulatory cytokine IL-10 is a powerful antagonist to the contributory effects of TNF on malarial anaemia. An elevated ratio of IL-10 to TNF in the serum is associated with a decreased severity of anaemia in Kenyan children . Immunoret al. indicatiThe causes of anaemia in children living in resource poor settings are numerous. In this analysis, age and socio-economic status were significantly correlated with anaemia. Periods of rapid growth including early childhood , as in this cohort, and adolescence are notable for increased iron utilization which can lead to iron-deficiency and, in many cases, associated anaemia .Other factors that were not analysed may also contribute to anaemia, masking the effect of co-infection on the severity of malarial anaemia. Nutritional factors such as vitamin and iron deficiencies play a significant role in the development of anaemia in resource poor regions of the world, like Nigeria. Additionally, sickle cell disease ,51 and oAscaris co-infection had no effect on malarial anaemia status or severity. While de-worming is crucial, this study suggests that ensuring that preschool children have adequate access to diagnosis and treatment of malaria is paramount in protecting them from the devastating effects of severe anaemia. This study highlights the continued need for research and resource allocation to efforts targeted towards the elimination of malaria worldwide. Despite these findings, the importance of de-worming should not be overlooked as the devastating effects on growth and nutritional status that helminths produce cannot be understated.In this cohort, anaemia was highly prevalent and correlated with malaria infection status. CI: Confidence interval; Hb: Haemoglobin; HIV: Human immunodeficiency virus; IL: Interleukin; iRBC: Infected red blood cell; OR: Odds ratio; RBC: Red blood cell; RDT: Rapid diagnostic test; SMA: Severe malarial anaemia; TGF-β: Transforming growth factor-β; Th2: T-helper 2 cells; TNF: Tumour necrosis factor.The authors declare that they have no competing interests.FA participated in the interpretation of the data and drafted the manuscript. CM performed statistical analysis of the data and drafted the manuscript. PK, SM, and SA coordinated the study. CH designed and coordinated the study. JG conceived the design of the study, participated in the interpretation of the data and drafted the manuscript. TL conceived design of the study, participated in the interpretation of the data and drafted the manuscript. All authors read and approved the final manuscript.


The recently reported declining burden of malaria in some African countries has been attributed to scaling-up of different interventions although in some areas, these changes started before implementation of major interventions. This study assessed the long-term trends of malaria burden for 20 years (1992–2012) in Magoda and for 15 years in Mpapayu village of Muheza district, north-eastern Tanzania, in relation to different interventions as well as changing national malaria control policies.Plasmodium falciparum infections and other indices of malaria burden  were compared across the years and between the study villages. Major interventions deployed including a mobile clinic, bed nets and other research activities, and changes in national malaria control policies were also marked.Repeated cross-sectional surveys recruited individuals aged 0 – 19 years from the two villages whereby blood smears were collected for detection of malaria parasites by microscopy. Prevalence of P. falciparum infections initially decreased between 1992 and 1996 (from 83.5 to 62.0%), stabilized between 1996 and 1997, and further declined to 34.4% in 2004. A temporary increase between 2004 and 2008 was followed by a progressive decline to 7.2% in 2012, which is more than 10-fold decrease since 1992. In Mpapayu (from 1998), the highest prevalence was 81.5% in 1999 and it decreased to 25% in 2004. After a slight increase in 2008, a steady decline followed, reaching <5% from 2011 onwards. Bed net usage was high in both villages from 1999 to 2004 (≥88%) but it decreased between 2008 and 2012 . After adjusting for the effects of bed nets, age, fever and year of study, the risk of P. falciparum infections decreased significantly by ≥97% in both villages between 1999 and 2012 (p < 0.001). The prevalence of splenomegaly (>40% to <1%) and gametocytes (23% to <1%) also decreased in both villages.In Magoda, the prevalence of A remarkable decline in the burden of malaria occurred between 1992 and 2012 and the initial decline (1992 – 2004) was most likely due to deployment of interventions, such as bed nets, and better services through research activities. Apart from changes of drug policies, the steady decline observed from 2008 occurred when bed net coverage was low suggesting that other factors contributed to the most recent pattern. These results suggest that continued monitoring is required to determine causes of the changing malaria epidemiology and also to monitor the progress towards maintaining low malaria transmission and reaching related millennium development goals. Malaria has been a major cause of morbidity and mortality in tropical areas particularly in sub-Saharan Africa (SSA), and was estimated to cause 300 – 350 million cases and 0.5 - 1 million deaths annually between 2000 and 2007 , Zambia In Tanzania, malaria has until the mid-2000s been responsible for over 18 million cases, 100,000 deaths/year and accounting for over 43% of outpatient cases, 30% of all hospital admissions and 40% of deaths occurring at health facilities . DespiteThe data used in the WHO report  were derApart from the mentioned national surveys, the observed decline in the burden of malaria in Tanzania is based on few detailed studies -28 and tA long-term presence in the two rural villages of Muheza district enabled the present study to assess the long-term trends of malaria burden over a period of 20 years (1992 – 2012). In these villages, which were until recently holo/hyper-endemic to malaria, different inventions  have been deployed, reflecting changing local demands and national policies. Their potential contribution to the observed trends of decreasing malaria is discussed, while the remarkable changes of parasite prevalence are given in more details, including differences between the two nearby villages.This study utilized assembled data collected during repeated cross-section surveys (CSS) conducted between September 1992 and June 2012 in the village of Magoda and between 1997 and June 2012 in the neighbouring village of Mpapayu both in Muheza district in Tanga region, North-eastern Tanzania. The villages were involved in malaria and filariasis studies as described elsewhere -40. Curr®  trial was the first study to be implemented in Magoda in 1993–94 to test the prophylactic effects of weekly administration of the drug on parasite infections, anaemia and splenomegaly [® to under-fives and those aged 5–9 years, respectively and followed-up weekly to determine their general condition and malaria infection status [After an initial screening of parasite prevalence of the entire population of Magoda village in 1992, and based on the promising results from the Gambia , a Malopnomegaly . Children status . Becausen status , patientn status .® trial, and the mobile clinic was continued till November 2004 to provide medical services to the community. The mobile services and malaria monitoring were extended to the neighbouring village of Mpapaya from 1997. During the visits by the mobile clinic, suspected cases of malaria had blood smears taken, and treatment was given on clinical grounds using either SP or AQ, because blood smears were only examined after returning to the research laboratory, for practical reasons. Patients not symptomatically treated but with malaria parasites in blood smears, were given antimalarials on the following day after microscopy results were obtained. Furthermore, repeated in vivo drug efficacy trials were carried out and involved patients with uncomplicated malaria (confirmed by microscopy) in 1994–1996 [Subsequently, a mobile clinic, visiting Magoda village twice weekly was introduced in 1994 to estimate if a rebound of malaria may arise after termination of the Maloprim994–1996 -45, 1997994–1996  and 1998994–1996  testing 994–1996 . In Dece994–1996  until 20994–1996 ; and rapP. falciparum infections. The CSS involved individuals aged between six months and 20 years. The surveys were conducted during (April/June) or after (July/September) the long rain season between September 1992 and June 2012 as described elsewhere [For this paper, complete data sets with personal details which were recorded on a morbidity questionnaire including age, sex and residence, parasite infection status for asexual and sexual stages, haemoglobin (Hb) levels, splenomegaly and ITN use and insecticide impregnation status were available for CSS conducted between 1999 and 2012 . Data collected in 1999–2004 were managed using Epi-Info  and Microsoft Excel, and later transferred to STATA version 11  for analysis. The analysis involved comparison of different variables such as parasite positivity, parasite density, gametocyte carriage, gametocyte density, splenomegaly, mean Hb, anaemia and ITN use in order to test for the differences across years and between the two villages. Categorical data were compared using chi-square test while continuous variables were tested using Students t-test. Logistic regression analysis was used to assess the effects of different interventions (using data collected from 1999 to 2012), such as deployment and distribution of ITNs, anti-malarial drug trials and changes of malaria treatment policy  on the risk of being infected with malaria parasites after adjusting for age and fever status (if study participants had fever or not). The different interventions which had specific point of deployment such as use of ITNs were coded as binary variables and interventions which covered a particular period were similarly labeled with reference to the duration of such interventions; and these variables were used in logistic regression analysis. Non-normal continuous variables such as parasite density were log transformed to normality. Linear regression models were used in modeling the relationship between Hb concentration and parasite density as response variables against explanatory variables. P-value <0.05 was considered significant.P. falciparum infections obtained in those surveys. For the nine CSS conducted in both villages between 1999 and 2012, a total of 6,050 individuals aged 0–19 years (mean age = 7.8 ± SD 4.7 years) were enrolled  were conducted in Magoda from 1992 to 2012, and in Mpapayu village from 1998 to 2012 and involved individuals aged <20 years, complete data  were only available for CSS done between 1999 and 2012  , followed by a steep and steady decline to 7.2% in 2012. In the village of Mpapayu, the highest prevalence of P. falciparum infections was 81.5% in 1999 and it decreased significantly to 24.9% in 2004 (p < 0.001), followed by a slight increase in 2008, and a steady decline to less than 5% from 2011 onwards  while for the CSS conducted in 2004 and 2008 – 2012, the prevalence was significantly higher in Magoda than Mpapayu village (p ≤ 0.018) . When the analysis was performed by categorizing study participants into three age groups , the prevalence of P. falciparum infections in both villages (Magoda and Mpapayu) decreased in all groups across the years with the lowest prevalence observed in 2012. However, the peak parasite prevalence which was observed in children aged 5–9 years up to 2004 shifted to participants aged 10–19 years from 2008 in Magoda and from 2009 onwards in Mpapayu  and 2000 (p = 0.043), while it was significantly higher in Magoda in 2004 (p = 0.040), 2008 (p < 0.001) and 2011 (p = 0.034). After adjusting for the effect of the year of survey, the decline in the prevalence of In Magoda, the geometric mean parasite density was similar in the surveys conducted between 1999 and 2004 while a significant increase occurred between 2004 and 2008 (p = 0.004); followed by similar parasite density in 2008–2011, and a significant decline in 2012 (p < 0.001). For Mpapayu, the parasite density was similar in 1999 – 2000, followed by a significant decline between 2001 and 2004 (p = 0.008) and a significant increase which was observed between 2004 and 2008 (p = 0.041). The parasite density was similar for the surveys conducted between 2008 and 2011 in Mpapayu, followed by a significant decline in 2012 (p = 0.004) as also observed in Magoda.The age-specific geometric mean parasite density was generally higher in under-fives than other age groups and ranged from 107 to 2,093 asexual parasites/μl in Magoda, and 0 to 2,062 asexual parasites/μl in Mpapayu  were available in under-fives only for the CSS conducted from 1998 to 2000, and between 2008 and 2012. For CSS conducted between 1998 and 2000 in both villages, linear regression model adjusted for the effect of age, gender and parasite density showed that the mean Hb was increasing across the year by 0.18 (95% CI: 0.04 - 0.32), p = 0.011, where the baseline mean Hb concentration was 10.20 g/dl (95% CI: 9.97 -10.41). Between 2008 and 2012, there was an insignificant decrease in mean Hb by 0.03 (95% CI = -0.09 - 0.02), p = 0.237).Vs 2008; χ2 = 156.7, p < 0.001). In Magoda village, the prevalence of splenomegaly was similar for under-fives and those aged ≥5 years (p > 0.168), except in 2004 when it was significantly higher in under-fives (p = 0.001) . The prevalence of splenomegaly among under-fives was significantly higher in Mpapayu compared to Magoda in 1999 (p = 0.006) and 2000 (p < 0.001) only, while in those aged ≥5 years, the prevalence was higher in Mpapayu in 2000 only (p < 0.001). In all age groups, the highest prevalence of splenomegaly (52.0%) was observed in Mpapayu village in 2001.The prevalence of splenomegaly in all age groups was higher in the CSS conducted before 2004 with significantly lower prevalence in the CSS conducted from 2008 onwards . When the two villages were compared, the prevalence was significantly higher in Mpapayu than Magoda in 1999 (p < 0.001), and in Magoda than Mpapayu in 2004 (p = 0.017)  was higher in the CSS of 1999 – 2004 than those of 2008–2012, with usage >88% in Magoda between 1999 and 2004 and >90% in Mpapayu between 2001 and 2004  including repeated cross-sectional studies were conducted in the villages of Magoda (since 1992) and Mpapayu (since 1997) in Muheza district of north-eastern Tanzania and have enabled long-term observations of trends in malaria for over 15–20 years. The studies also showed the possible impact of the deployed interventions at the community level and changing national malaria control policies. These villages and other parts of Muheza have been reported to have high burden of malaria, as well as high levels of resistance to different anti-malarial drugs -47. Trea® trial , and CQ ® trial .P. falciparum infections occurred between 1997 and 2004 (Magoda) and 1998–2004 (Mpapayu) when more interventions were being implemented, further changes were also observed between 2008 and 2012 in both villages. The peak prevalence of malaria shifted from children aged 5–9 to those aged 10–19 years old in 2008–2012, a trend also observed in the highlands of the neighbouring district of Korogwe which generally have low malaria transmission [Overall, the study reported here observed a significant decline of malaria burden over the study period. Although the major decline in the prevalence of smission . The preThe decline in prevalence observed before 1999 could possibly be due to better services provided by the research team while the changes observed between 1999 and 2004 could also be attributed to drug trials, which were implemented from 1994 -47, and Presence of research team in the villages, deployment of a mobile clinic and other services provided by village health workers would also possibly reduce malaria burden through prompt diagnosis and treatment and referral to Muheza district hospital for free services, when needed. Furthermore, the records collected between 1992 and 2004 showed that the number of deaths declined significantly and cerebral malaria virtually disappeared from Magoda and Mpapayu village . Similar studies conducted in the neighbouring district of Korogwe also showed that improved health services at the community level through deployment and use of village health workers under the supervision of trained medical personnel was associated with a significant reduction in the burden of malaria ,27. AlthP. falciparum infections dropped to <10% in Magoda and <5% in Mpapayu from 2011. Of the recent interventions, AL was introduced as first-line drug for treatment of uncomplicated malaria in January 2007and free ITNs were distributed in Magoda in 2009 through the US President Malaria Initiative (US PMI), followed by long-lasting nets in 2012 . However, the coverage and usage of bed nets remained below 74%, i.e. far below the levels attained by 2004. It is thought-provoking that coverage and use of ITNs were much higher before 2004, when ITNs were distributed by the project compared to the most recent coverage under the national universal campaigns. Factors other than ITNs and deployment of AL could possibly be driving the most recent changes as also reported by Okiro et al.[In the CSS conducted in 2008 – 2012, the prevalence of ro et al.. These iThe decline in other indices of malaria transmission intensity such as the prevalence of splenomegaly and gametocytes indicate that these areas formerly known as holo/hyper-endemic have now become low malaria transmission settings within very few years as also recently shown in the neighbouring district of Korogwe ,27. The P. falciparum gametocytes observed in the CSS done between 2008 and 2012 could partly be attributed to the effects of ACT together with ITNs which reduce malaria transmission. Previous studies have shown that artemisinins reduce the parasite biomass drastically and thus reducing the rate of development and gametocyte carriage [The significant decline in the prevalence of carriage . Such efAnopheles gambiae s.s. have been replaced with the zoophilic Anopheles arabiensis[Although vector population studies have not been done in the study villages in recent years, studies conducted in the neighbouring villages in Muheza and Tanga districts showed that the density of malaria vectors has progressively declined leading to low transmission in this part of the country . Furtherrabiensis. These sThis study showed a marked decline of malaria burden in the study area between 1992 and 2012, initially between 1992 and 1996, and a further decline from 2008 to 2012. The decline in the initial period (1992 – 2004) was associated with the deployment of different interventions such as a weekly mobile clinic, bed nets and other research activities. However, the dramatic and steady decline observed from 2008 occurred when bed net coverage was much lower, suggesting that other factors such as introduction of ACT could also have contributed to the most recent changes. A shift of parasite prevalence from younger to older children/adolescents and increasing parasite density until 2011 imply that the transmission intensity has significantly declined leading to delayed development and low immunity among residents of these villages, making these areas which were holo/hyperendemic to become epidemics-prone or unstable malaria transmission. Further monitoring of malaria burden in these and other communities will potentially reveal future trends of malaria transmission and identify other factors which could be responsible for these significant changes of malaria epidemiology in order to appropriately devise new control strategies. The significant reduction in transmission implies the need for strengthening of the routine surveillance system and introduction of stratification to ensure that the fewer cases are captured and treated and apply focused intervention. These results also provide evidence that could be used in monitoring the progress of health related millennium development goals (MDGs).The authors declare that they have no competing interests.DSI, MML, MA and ICB conceived of and designed the study; DSI, MDS, BPM and MML conducted the field work and supervised the laboratory analyses. BPM and DSI participated in data management and analysis. DSI, MA, MML and ICB wrote the manuscript. All authors read and approved the manuscript.