Severe anaemia is a major cause of morbidity and mortality in African children. The aetiology is multi-factorial, but interventions have often targeted only one or a few causal factors, with limited success.We assessed the contribution of different pathophysiological mechanisms  to severe anaemia in Malawian children in whom etiological factors have been described previously. More complex associations between etiological factors and the mechanisms were explored using structural equation modelling. In 235 children with severe anaemia (haemoglobin<3.2 mMol/L [5.0 g/dl]) studied, RCPF, haemolysis and blood loss were found in 48.1%, 21.7% and 6.9%, respectively. The RCPF figure increased to 86% when a less stringent definition of RCPF was applied. RCPF was the most common mechanism in each of the major etiological subgroups (39.7–59.7%). Multiple aetiologies were common in children with severe anaemia. In the final model, nutritional and infectious factors, including malaria, were directly or indirectly associated with RCPF, but not with haemolysis.RCPF was the most common pathway leading to severe anaemia, from a variety of etiological factors, often found in combination. Unlike haemolysis or blood loss, RCPF is a defect that is likely to persist to a significant degree unless all of its contributing aetiologies are corrected. This provides a further explanation for the limited success of the single factor interventions that have commonly been applied to the prevention or treatment of severe anaemia. Our findings underline the need for a package of measures directed against all of the local aetiologies of this often fatal paediatric syndrome. Severe anaemia (haemoglobin concentration <3.2 mMol/L [<5.0 g/dl]) is a common cause of morbidity and mortality in African children mechanisms may lead to a reduced number of circulating red cells. In African children, mechanisms known to contribute to severe anaemia include haemolysis, acute or chronic blood loss, and red cell production failure (RCPF) etiological factors and some single aetiologies may affect more than one mechanism The pathogenesis of anaemia is complex because several distinct Despite the size of the problem, severe anaemia has received little research attention and its pathophysiology is still poorly understood To do so we studied a group of rural and urban Malawian children with severe anaemia. The aetiologies associated with anaemia in these children have been reported previously This study formed part of a large severe anaemia research programme conducted in southern Malawi at Queen Elizabeth Central hospital in Blantyre (urban site) and Chikwawa District hospital . Between July 2002 and July 2004 consecutive children with a primary diagnosis of severe anaemia were recruited. The etiologic factors and agents associated with anaemia in these cases have been reported elsewhere Children were eligible for enrolment if their blood haemoglobin concentration was less than 3.2 mMol/L, they were aged 6–60 months and had not received a blood transfusion within the preceding month. Informed consent was obtained from a parent or guardian. The study was approved by the ethics committees of the College of Medicine, University of Malawi and the Liverpool School of Tropical Medicine, United Kingdom.Procedures for admission and patient management have been described in detail elsewhere Laboratory tests, crucial to patient management, were performed within 24 hours, and sample aliquots were stored at −80°C for later analysis. Whole blood haemoglobin and plasma haemoglobin concentrations were measured using the HaemoCue system® . A full blood count, including absolute reticulocyte count, was performed by Coulter counter analyzer® . Bone marrow aspirates were used for microscopic evaluation and for measurement of the red cell precursor fractions on a FACS-Calibur flow-cytometer . Wedged spread films were air-dried, fixed in methanol and stained with May-Grüwald-Giemsa and for iron . Bone marrow differential count was done by an experienced laboratory technician (RV). Iron stained bone marrow slides were graded according to Gale's criteria for iron content in stroma, macrophages and red cell precursors Plasmodium falciparum asexual parasites were counted against 200 white blood cells and parasite density was calculated from the total white cell count. Stool samples were examined for helminths using the Kato-Katz method and polymerase chain reaction (PCR) C-reactive protein, erythropoietin, haptoglobin, bilirubin, ferritin, folate and vitamin B12 concentrations were analyzed in heparin plasma on a Roche p800 system . Serum vitamin A (retinol) and soluble transferrin receptor (sTfR) were measured using high performance liquid chromatography and enzyme linked immunosorbant assay  respectively A bone marrow or venous blood sample (1–2 ml) was inoculated into BACTEC Myco/F-Lytic culture vials and incubated in a BACTEC 9050 automated system for 56 days Anaemia mechanisms: The criteria used to define the three severe anaemia mechanisms are given in Etiological factors: Important etiological factors in the study population were defined by multivariate analysis and included: malaria, HIV, bacteraemia, hookworm infection and iron, vitamin B12 and vitamin A deficiency Dyserythropoiesis: was defined by the following nuclear features in bone marrow smears: (a) multi-nuclearity; (b) karyorrhexis; (c) intercellular chromatin bridging; and (d) incomplete mitoses. Dyserythropoiesis was expressed quantitatively as the percentage of RBC precursors fulfilling at least one of these criteria4.Nucleated red cells (NRC): were defined as all mononuclear bone marrow cells positively stained with LDS-751 (a DNA dye) and expressing CD235a (Glycoprotein A) on flow-cytometric analysis.Red cell production failure (RCPF): was defined as a whole blood haemoglobin <3.2 mMol/L (5.0 g/dL) and Reticulocytes <50,000/uL.Malaria infection: was defined as the presence of asexual Plasmodium falciparum (Pf) parasites on a blood film; Parasitaemia in excess of 10,000 asexual parasites/µL was referred to as ‘Pf>104/ul’.Iron deficiency: In our study population, a sTfR/log ferritin-index of more than 5.6 was used to define iron deficiency. This index (and cut off) has been shown to be the best predictor of bone marrow iron status irrespective of the presence of infection with a sensitivity of 70%, specificity of 75%) Gastro-intestinal blood loss: was not measured directly. A hookworm load of more then 1000 ova per gram of stool was used as substitute marker.This is primarily a study of the diversity of mechanisms of severe anaemia, the identification of which includes an assessment of bone marrow appearances. The primary statistical analysis is thus purely descriptive. The characteristics of the study cohort are summarized using frequency counts/percentages for categorical factors and mean values (with their standard deviations) for continuous factors. The prevalence of each of the four pathophysiological mechanisms is summarized using frequency counts/percentages, as are the markers and etiological factors for each of the mechanisms .The associations between etiological factors and mechanisms were explored using Structural Equation Modelling (SEM). SEM is a complex but increasingly popular data-analytical technique, the strengths and limitations of which are well documented 18.In the initial SEM model, all three “group1” etiological factors were considered to be associated with all four “stage 2” factors, and all four “stage 2” factors were considered to be associated with both mechanisms examined (red cell production failure and haemolysis). Relationships found to be statistically non-significant were removed by backward elimination. The arrows indicate the directions of the hypothesized relationships, statistically significant at the conventional alpha  = 0.05 level. The figures adjoining each arrow are the standardized regression coefficients from the SEM model; these indicate the  strength of the relationships and can be interpreted similarly to conventional correlation coefficients. The goodness-of-fit of the final SEM was determined using the RMSEA (root mean square error of approximation) statistic – a value of RMSEA <0.05 indicates an close model fit and a value <0.08 indicates a reasonable model fitAll analyses were carried out using the STATA 9 , SPSS 14 and AMOS 7.0  statistical computer packages.The study was approved by the ethics committees of the College of Medicine, University of Malawi and the Liverpool School of Tropical Medicine, United Kingdom.Three-hundred-and-eighty-one children with severe anaemia (patients), 205 (53.8%) from Blantyre (urban) and 176 (46.2%) from Chikwawa , were enrolled over a two year period (2002-04). The median age of the children was 16.5 (range 6-59.6) months and 46.7% (n = 178) were boys . Of thesRed cell production failure (RCPF) was identified in 113 (48.1%) of 235 children with severe anaemia. Of those with RCPF, 24 (21.3%) also had other mechanisms . By a leHaemolysis was found in 51 out of 235 patients (21.7%), of whom 17 (33.3%) also had criteria for RCPF and/or blood loss . Of the Evidence of blood loss was found in 17 patients (7.2%): this was through the urinary tract in 5 (2.1%) and the gastro-intestinal tract in 12 (5.1%).10, the most common etiological factors were found to be vitamin A deficiency, malaria infection, iron and vitamin B12 deficiency in 92.3, 59.5, 46.6 and 30.4 percent of patients, respectively Among all previously identified important etiological factors of severe anaemiaInfections  associated with anaemia occurred in 211/275 (76.4%) of the severe anaemia patients evaluated of whom 77.7% (n = 164) had one infection, an additional 18.0% (38) had two and 8.5% (9) three infections. A single infection (isolated) was relatively uncommon (22.6%) among the HIV infected patients, but common among the malaria, hookworm and bacteraemia infected patients, in whom 60.0, 52.2 and 37.1% were isolated infection, respectively .4/uL), the equivalent proportions were 36.6% and 17.1%.RCPF was also the most important mechanism in the sub-groups of children with a single infection. Only in the six children with bacteraemia was haemolysis more commonly found. In the children with a positive malaria slide, RCPF and haemolysis were found in 61 (42.1%) and 26 (17.9%) and in the sub-group with a high malaria parasite count  of 0.053 (0.038-0.068), iron deficiency, vitamin B12 deficiency and malaria were directly and significantly associated with RCPF . Associations between RCPF and HIV, hookworm and vitamin A deficiency were indirect, through the iron and vitamin B12 deficiency and malaria respectively. No significant associations were found between the etiological factors and haemolysis  and the etiological factors that operated through these mechanisms  – predominates as the final pathway leading to anaemia. This was true when using stringent criteria for RCPF  and became even more apparent when the cut-off for reticulocyte count was put at <150,000/uL  The dominance of RCPF was found in all etiological sub-groups, with surprisingly little variation between the etiological factors. Of additional importance was the observation that in the majority of children with severe anaemia multiple aetiologies  were found, each with the potential to cause a RCPF on its own. In such circumstances, RCPF may not be corrected or even ameliorated unless all of its contributing aetiologies are removed. This finding may explain why interventions directed against one or two aetiologies only to treat or prevent severe anaemia may be limited in their success 4/l. In many respects, it is the relationships that were found to be non-significant and hence removed from the SEM that justify the use of this complex mathematical methodology in this context. With conventional multi-stage regression models, all three of the “stage1” etiological factors  would have to be assumed to be influencing (in some way) all four of the “stage 2” factors . However, the SEM shows clearly that this is not the case. Furthermore, the strengths of the relationships were estimated taking into account the possibility that etiological factors can influence more than one mechanism simultaneously, which is not easily done using conventional regression methods The Structural Equation Model underlines both the importance of RCPF and the complexity of the syndrome, and indicates the apparently minor role of haemolysis in the patients we studied. There were no significant associations between haemolysis and any of the aetiologies, including malaria. This was the case whether malaria was defined as any Parasitaemia or a parasite density more then 108.A limitation of a study of this kind is that data can be collected at one time point only, in a disease process that may have lasted for a long time with markers fluctuating from day to day. We have tried to limit this effect by using a large sample size. Another limitation is the difficulty of quantifying haemolysis and gastro-intestinal blood loss. The kinetics of free haemoglobin in plasma are not well known, and 1 g/dl of free haemoglobin is sufficient to saturate circulating haptoglobin, limiting the value of haptoglobin as quantitative marker of haemolysis These limitations may, in theory have caused some underestimation of blood loss and overestimation of haemolysis. However, they are unlikely to have affected the main findings, importance of RCPF, of our study and their implications for the control of severe anaemia in Malawi and similar settings. The syndromic approach that we have adopted allows a profile of paediatric severe anaemia to be developed, based on a description of both aetiologies and mechanisms for a particular population. In the case of the population we have studied, this profile indicates that although aetiologies are often multiple, the final pathogenetic pathway leading to severe anaemia is most commonly bone marrow failure. Any one of the aetiologies may on its own be sufficient to sustain this failure of red cell production. The next step will be to confirm our findings in other settings and to investigate causality between etiological factors, proposed mechanisms and severe anaemia. This may be followed by the design of a package of preventive and therapeutic measures (which may vary between different settings in Africa), aimed at reducing the burden of this common, often fatal, but neglected syndrome.


Background. Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. Methods and Results. EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/μL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. Conclusion. Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.  DM subjStudies suggest that MPs produced via activating stimuli have different protein expression patterns from those produced via apoptosis; consequently analysis of EMP phenotypic profile may provide clinically useful information on the status of the endothelium. The ratio of CD62E+/CD31+ EMP populations, rather than their absolute numbers, has been described as a criterion for distinguishing activation versus apoptosis. A ratio ≥10 identifies activation while ratio ≤1.0 identifies apoptosis. Our data suggest that apoptosis is an important mechanism for EMP release in the DM population.The release of EMPs into circulation makes their levels closely associated with the degree of vascular damage. To identify independent predictors for EMP elevation, we performed a multivariate linear regression analysis including age, gender, BMI, HTN, plasma cholesterol level, glucose, and EMP phenotypes as independent variables. BMI levels tended to be independently associated with CD31+ EMPs; however, this relationship was not significant. There was a positive correlation between IL-1 levels and the number of CD 144+ EMPs . There was no significant difference in IL-1 levels between the diabetic and nondiabetic group.μm in size and often platelets, MP aggregates, or apoptotic bodies) were excluded by our gating protocol. Circulating EMPs are associated with the presence of high-risk angiographic lesions, including eccentric type II, multiple irregular, and lesions with thrombi; however we found no significant relation of EMP levels with the presence or severity of CAD [We demonstrated that DM is associated with increased levels of circulating EMPs. This corroborates previous studies where levels of EMPs were associated with microalbuminuria and microvascular complications in patients with type I diabetes, suggesting that EMPs could be a marker of diabetes-associated endothelial dysfunction , 19. EMPy of CAD . Multivay of CAD . One of the earliest events in atherosclerosis is CAM-mediated adhesion of circulating monocytes to intact activated endothelial cells , 23. DesThe immunophenotype of MPs depends on whether they are released by cell activation or by apoptotic stimulus . CellulaAs biomarkers, EMPs allow access to a typically inaccessible tissue, the endothelium. This study found that EMPs were independently associated with the presence of DM and reflect immunophenotypes important to the atherothombotic process. Therefore, EMPs may be a useful surrogate marker for evaluating endothelial dysfunction and/or injury. There is a paucity of information regarding the change in endothelial function over time as it relates to diabetic control and atherosclerotic burden. To help circumvent these problems studies utilizing serial sampling should be performed in diabetic patients thereby allowing each patient to serve as his own biological control. In addition, the storage and freezing of plasma samples might affect the relative levels of EMPs and the reliability of the results compared to those obtained from freshly isolated preparations. Consequently, protocols need to be optimized for the clinical setting where flow cytometry cannot always be performed on the same day as specimen collection. Caution should also be taken when extrapolating laboratory experiments from the results of clinical studies, because a given EMP type may be present with a variety of compositions and biological behavioral patterns in vitro and in vivo. Atherosclerosis is a diffuse, multisystem, and chronic inflammatory disease. Therefore, it is essential to assess total vulnerability burden in subjects with a high likelihood of developing cardiac events . A quant


With the dramatic drop in the transmission of malaria in Sri Lanka in recent years, the country entered the malaria pre-elimination stage in 2008. Assessing the community prevalence of hidden malaria parasites following several years of extremely low transmission is central to the process of complete elimination. The existence of a parasite reservoir in a population free from clinical manifestations, would influence the strategy for surveillance and control towards complete elimination.Plasmodium falciparum and Plasmodium vivax, simultaneously.The prevalence of hidden parasite reservoirs in two historically malaria endemic districts, Anuradhapura and Kurunegala, previously considered as high malaria transmission areas in Sri Lanka, where peaks of transmission follow the rainy seasons was assessed. Blood samples of non-febrile individuals aged five to 55 years were collected from randomly selected areas in the two districts at community level and a questionnaire was used to collect demographic information and movement of the participants. A simple, highly sensitive nested PCR was carried out to detect both In total, 3,023 individuals from 101 villages participated from both districts comprising mostly adults between the ages 19-55 years. Out of these, only about 1.4% of them (n = 19) could recall having had malaria during the past five years. Analysis of a subset of samples (n = 1322) from the two districts using PCR showed that none of the participants had hidden parasites.A reservoir of hidden parasites is unlikely to be a major concern or a barrier to the ongoing malaria elimination efforts in Sri Lanka. However, as very low numbers of indigenous cases are still recorded, an island-wide assessment and in particular, continued alertness and follow up action are still needed. The findings of this study indicate that any future assessments should be based on an adaptive sampling approach, involving prompt sampling of all subjects within a specified radius, whenever a malaria case is identified in a given focus. Dwindling malaria case counts have placed Sri Lanka in a unique situation to plan towards a pre-elimination programme. Over the past 10 years the number of malaria cases registered at government hospitals have dropped significantly, where cases peaked at 264,549 in 1999 and reduced to 591 in 2006 and then to 196 cases in 2007 .Sri Lanka has now entered the malaria pre-elimination stage together with ten other countries including Mexico, Iran, Azerbaijan, Georgia, Kyrgyzstan, Tajikistan, Turkey, Uzbekistan, DPR Korea . EliminaPlasmodium falciparum = 1 and Plasmodium vivax = 11) and 14  from Anuradhapura and Kurunegala for 2007, respectively , 1 μL of extracted DNA (or 1 μL of outer PCR product) and H2O. The PCR conditions for the outer PCR were: Initial activation of Tempase enzyme at 94°C for 15 min, followed by 30 cycles of 94°C for 1 min, 58°C for 2 min and 65°C for 2 min and finally 58°C for 2 min and 65°C for 5 min. For the nested PCR: 94°C for 15 min, followed by 30 cycles of 94°C for 30 sec, 57°C for 1 min and 65°C for 1 min and finally 58°C for 2 min and 65°C for 5 min. All PCR reactions were run in Eppendorf 96-well PCR plates with foil sealing  minimizing contamination risks and evaporation. Positive P. falciparum and P. vivax isolates as well as negative controls, extracted DNA from persons living in Denmark with no history of malaria were included in all PCR reactions, both to ensure a successful PCR and to check for contamination. All samples were run on 1.5% ethidium bromide-containing agarose gels and visualised under UV-light. In case of doubt, for instance if mixed species infections were suspected, the original protocol for species identification using P. falciparum or P. vivax specific primers and PCR conditions were used as described in reference [Genomic DNA was extracted from the blood samples using a chelex-100 method. A simple nested PCR was developed to diagnose both P. falciparum laboratory isolate FCR3. The FCR3 isolate was grown to 5,800 parasites/μl and diluted in full blood to 1000, 100, 10, 5, 2.5, 1.25, 0.6, 0.3 and 0.15 parasites/μl. This dilution series was dotted onto Whatman filter paper #3 and the blood spots were extracted similarly to the samples by use of the chelex-100 method.The sensitivity of the nested PCR was evaluated by measuring the threshold of sensitivity on a dilution series of Subsets of samples were selected using computer generated randomised tables and analysed. First, 25% was selected from the initial sample and then another 25% from the remaining sample was analysed. Since all 1,322 samples tested turned out negative rest of the sample was not analysed.In total, 3,023 participants from 101 villages constituting of mostly adults between the ages 19-55 years  of blood samples revealed that none of the samples were positive for The results show that there are no hidden parasites in the population analysed. According to the AMC (Sri Lanka), for 2007 only 12 positive cases  were reported from the Anuradhapura District  while 14 cases were reported from the Kurunegala District . Of the 12 cases in the Anuradhapura District, 11 cases were indigenous while the remainder had contracted the disease during visits to other malarious districts within Sri Lanka, specifically to Trincomalee . Only a small percentage (1.4%) of the population tested had microscopically or RDT confirmed malaria during the past five years. In Sri Lanka, microscopically confirmed blood smears or rapid diagnostic kits are standard diagnostic procedures adopted before the prescription of malaria drugs to patients. Since the medical services at government hospitals are supported by qualified staff, in general, medication is initiated only after a blood test and most patients appear to prefer this approach [P. falciparum and P. vivax parasite infections simultaneously in blood samples. The sensitivity of the nested PCR was assessed by determining the threshold of positivity on a dilution series of P. falciparum laboratory isolate, FCR3. The method could detect 1.25 parasites/μl, which is roughly 30 parasites in a 25 μl blood spot. When using the original primers for detecting P. falciparum only, as described in reference [P. vivax infections as when using nested PCR [At a pre-elimination phase, it is important to determine the occurrence of hidden parasite reservoirs by using sensitive and robust diagnostic tools. The nested PCR method used in this study was a simple, relatively cheap and sensitive method capable of identifying eference  similar sted PCR . Howeversted PCR  and thisP. vivax can form undetected hypnozoites, there is a risk that such a reservoir persists in the indigenous population. Nevertheless, the contribution of these dormant stages in causing outbreaks is limited in Sri Lanka under the present policy of co-administering primaquine in combined with the first line anti-malarial drug in the treatment of uncomplicated cases.The method used here was a sensitive diagnostic tool to test the blood stages of the parasite, and to the best of our knowledge there are no tests to detect the liver stages of the parasite. Since P. vivax) from Anuradhapura district was diagnosed  by AMC and treated. Unfortunately, the sample was lost for verification by our PCR method. However, 12 blood samples that were collected from the neighbourhood were all negative. The approaches to active case detection involve screening family members and neighbours of new cases, screening targeted individuals near (perhaps within 1 km) new cases, periodic perhaps monthly screening of targeted communities where residual transmission and continuing cases are reported [The samples collected for this study used a random method in selecting the study areas with the aim of assessing community prevalence. This sampling method picked out 60% 3/5) and 50% (4/8) of high prevalence DSDs from Anuradhapura and Kurunegala districts, respectively . However, with the lack of parasites identified from this approach and the overall very low community reported history of malaria, the future design of assessments to identify hidden parasites could be changed to focus on villages where a case of malaria had been reported within e.g. the past three months and in a broader age group. This approach would of course not address the objective set for this study but would still provide public health relevant information. Whether a different method of sampling where selection of geographical areas with a history of high transmission would have given different results, remains to be investigated. However, it is doubtful, if such a study were to change the current conclusion drastically, given such low case counts even in the high transmission areas. During the study period only one indigenous malaria case , which transmits the infection to mosquitoes, but do not affect pre-erythrocytic development. Hence, a critical part of malaria elimination is assessing the prevalence of hidden parasites. However, even extremely low levels of circulating parasites in the population or the introduction of parasites by individuals migrating into Sri Lanka may still generate an outbreak with the potential for large scale epidemics if the vector population reaches very high levels. Therefore, vector surveillance, especially following the reduced efforts of indoor residual spraying, is an important aspect to be dealt with side by side with the detection of hidden parasites. This study documents an approach that can be extended to a national scale that will enable the collection of comprehensive data sets for future planning and policy formulation.Aiming at elimination of malaria further implies the need for effective surveillance strategies to monitor progress. In the case of Sri Lanka, historical evidence shows that malaria incidence can fluctuate markedly. Thus, a well focused system of surveillance that will identify emerging malaria outbreaks and tools in place for prompt treatment is required although the case rates are dwindling. Further, the current knowledge on parasite resistance to anti-malarials  in the cBased on this analysis, the reservoirs of hidden parasites may not be a major concern or barrier to elimination efforts, but calls for an island-wide assessment and continued alertness, in particular, in areas that were affected by the civil war. Additionally, using an adaptive sampling approach for prompt screening of family members and neighbors of new cases can be carried out to confirm any malaria hotspots  before iThe authors declare that they have no competing interests.RSR acquired and analysed data, carried out the lab work and wrote the manuscript. MA contributed to study design and interpretation of data, supervised DNA work, revised the manuscript for important intellectual content. PHA and FK contributed to concept development and study design, edited and revised the manuscript critically for content. All authors read and approved the final manuscript.


Longitudinal cohort studies are important to describe the dynamics of naturally acquired antibody response profiles to defined Plasmodium falciparum malaria antigens relative to clinical malaria episodes. In children under 7 years of age in The Gambia, serum IgG responses were measured to P. falciparum merozoite antigens AMA1, EBA175, MSP119, MSP2 and crude schizont extract, over a 10-month period. Persistence of antibody responses was measured in 152 children during the dry season when there was virtually no malaria transmission, and 103 children were monitored for new episodes of clinical malaria during the subsequent wet season when transmission occurred. Children who experienced clinical malaria had lower antibody levels at the start of the study than those who remained free from malaria. Associations between dry season antibody persistence and subsequent wet season antibody levels suggested robust immunological memory responses. Mean antibody levels to all antigens were elevated by the end of the wet season in children who experienced clinical malaria; each of these children had a boosted antibody response to at least one antigen. In all children, antibody avidities were lower against MSP2 than other antigens, a difference that did not change throughout the study period or in relation to clinical malaria episodes. Plasmodium falciparum infection remains a significant cause of mortality among children in malaria endemic countries. High antibody levels are generally associated with subsequent reduced susceptibility to clinical malaria  was collected at enrolment (day 0), for serum and thick blood smear preparation, and finger prick (∼300 μL) blood samples were requested every 2 weeks.P. falciparum positive, the therapy recommended in 2004 was provided, consisting of oral chloroquine (10 mg/kg daily for 3 days) with sulphadoxine-pyrimethamine (1/2 tablet per 10 kg). After each clinical malaria episode, a follow-up finger prick blood sample for serum and thick smear was requested within 3 weeks of treatment.At the end of the 12-week dry season study, all participants were provided with bed-nets and deltamethrin insecticide treatment for use throughout the subsequent wet season. Approximately 1 month later, during the wet season, written informed consent was sought for participation in a follow-up study of malaria and antibody responses. A finger prick sample for serum and thick blood smear preparation was collected early during the wet season in July and at the end of the wet season in December. Children were visited by field workers every week between these two surveys, and if they had fever (axillary temperature ≥37·5°C) or reported history of fever within the previous 48 h, a finger prick blood sample was taken and tested immediately for malaria using a rapid diagnostic test . A thick blood smear was taken for confirmatory laboratory analysis, and the remaining volume was collected for serum. If either the RDT or thick blood smear were P. falciparum. Finger prick blood samples for antibody analysis were collected in serum separator tubes  and stored on ice during transfer from the field to the laboratory. On arrival at the laboratory, tubes were centrifuged at 1100 g for 5 min, and sera removed for storage at −80°C prior to use in antibody assays.Thick blood smears and RDTs were performed directly from the finger prick samples. Giemsa-stained thick blood smears were read by two experienced microscopists examining 100 high-powered fields (×1000 magnification) to determine the presence or absence of detectable 19 and MSP2  were used. The AMA1 antigen was the full-length ectodomain, expressed in Escherichia coli, representing amino acids 83–531 of the P. falciparum strain 3D7 AMA1  at 490 nm, following previously described methods . Brieflym concentrations was used with a pool of positive control sera, collected from nine adults from the Brefet region of The Gambia, who had high ELISA OD antibody reactivities to all antigens  at 1/5000. After four further washes, 100 μL substrate solution was added , and incubated for 15 min before adding 50 μL 2 m sulphuric acid and determining OD at 490 nm. To generate relative antibody avidity indices for test sera, the ratio of ELISA OD for sera incubated in the presence of 0·5 m guanidine thiocyanate, and OD without guanidine incubation, were calculated for AMA1, MSP119, MSP2A and MSP2B antigens.To assess antibody avidity, chaotropic anion dissociation of antibody–antigen interactions with 100 μL guanidine thiocyanate at 4, 3, 2, 1, 0·5 and 0·1 antigens . A singlEstimates of the persistence of dry season antibody responses were made previously for children with samples at five or more time points, from day 0 until the end of the 12-week dry season study period. Dry season ELISA absorbance OD values were transformed into standardized units using a standard curve. For comparison with wet season ELISA ODs, persistence of dry season antibody response was determined by a single parameter: the lowest antibody level estimated during the dry season as a proportion of the antibody level at day 0 .t-test for parametric data or the Wilcoxon rank sum (Mann–Whitney) test for nonparametric data  was used. Linear regression was used to test associations between continuous dependent and independent variables. For normally distributed data, unadjusted Pearson’s R2 coefficient (slope between x and y) and P values are reported. For non-normally distributed data, Spearman’s Rho, and P values are reported. For children with clinical malaria, comparisons were made between proportions with and without antibody increases >0·5 OD ELISA units to individual antigens .To examine differences in antibodies between groups, the two sample P. falciparum parasites were detected in blood smears from 49 (48%) of the children early during the dry season, from six (6%) of the children early during the wet season and from 30 (29%) of the children at the end of the wet season. In total, during the wet season, P. falciparum parasites were detected in blood smears of 46 (45%) of the children.Written informed consent was given for 152 children to participate throughout the dry season study. Of this total, 138 participants completed the 12-week dry season survey, and 124 children provided samples at five or more time points from day 0 until the end of the dry season; these samples were analysed for persistence of antibody response . After aThirty four (33%) of the children were determined to have experienced an episode of clinical malaria. These children had parasite positive thick smear or RDT with fever, or reported history of fever within the previous 48 h, and were treated for clinical infection. There were 21 children with an episode of clinical malaria from whom a blood sample was collected at the time of diagnosis and another sample collected within 18 days after the start of treatment for clinical infection, allowing antibody profiles to be examined in detail for these children .n= 14, male n= 20).The mean age of children at the start of the dry season was 3·97 years (SD 1·53). There was no significant age difference between children who experienced an episode of clinical malaria during the wet season  and those who did not . The cohort was composed of 43 females and 60 males, and episodes of clinical malaria were not associated with gender  than pre-season . The trend was similar for the individual recombinant antigens, although these were not significant  antibody levels (mean ELISA OD = 0·805) to nificant .Longer persistence of antibodies during the dry season was significantly associated with higher end of wet season (mean day 285) antibody levels for all antigens except MSP2 . The datVery diverse longitudinal antibody profiles were observed in children who experienced clinical malaria. Individual antibody profiles are shown for 21 children who had clinical malaria during the wet season and from whom a follow-up sample was obtained within 18 days post-clinical episode . Some chThe individual profiles indicate that peak antibody levels usually occurred during or immediately following clinical infections. Such rapid responses co-incident with malaria episodes suggest robust immune memory. Antibody levels were often reduced from peak levels by the end of the wet season, but were generally higher than those observed at the beginning of the wet season. Some individuals  showed increases in antibody levels to all antigens tested, whereas others  showed increases in antibody levels only to particular antigens .19 and schizont extract (n= 34) had increases of >0·5 ELISA OD in one or more of the antigens during the wet season. extract . DespiteThe quality of antibody response was considered in terms of antibody avidity to individual recombinant antigens for samples with antibody levels >0·5 ELISA OD units. Avidities were neither examined for schizont extract, as this is a heterogeneous crude antigen preparation, nor for EBA175, as relatively few children had high antibody levels to this antigen.19 and AMA1 (19 (∼0·8), followed by AMA1 (∼0·6) and avidity indices to MSP2 antigens were markedly lower (∼0·1 to 0·2) . There were positive associations between antibody levels and antibody avidity index at all time points for AMA1  and MSP119 , but not for MSP2 antigens. There were no consistent associations between antibody avidity measures and age or persistence of antibody response. There were also no observed correlations between antibody avidity and presence of parasite infection, or occurrence of clinical malaria.At all time points, lower antibody avidities were observed to both allelic forms of MSP2, compared with MSP1and AMA1 . The val to 0·2) . AvidityP. falciparum merozoite antigens were observed in many children during the wet season when malaria transmission occurred. Rapid increases occurred around the time of clinical infection in all children who had a clinical malaria episode for at least one of the tested antigens, indicating memory responses that were boosted during the infections. Following treatment and resolution of clinical malaria, antibodies to specific antigens can commonly be detected for months, although the levels decline (In this cohort study in The Gambia, increases in serum IgG antibody levels to  decline ,8. A pre decline . ChildreP. falciparum antigens in the absence of persistent infection were previously associated with age of children and differences between malaria antigens (Rates of dry season antibody decline to antigens . Antibodantigens . It is p19 in this population. There is evidence that MSP2 antigens promote an IgG3 skewed antibody response compared with other malaria antigens (P. chabaudi murine vaccine study showed no differences between antibody avidities to AMA1 or MSP1 conformation-dependent and conformation-independent epitopes (19 and schizont extract antigens during the dry season amongst children in this cohort (19. It is unknown whether the differences in MSP2 antibody avidities observed in this study relate to a particular structure of the MSP2 antigens within the tandem repeat sequences, or the polymorphic nature of some of these repeat sequences (Antibodies with low avidities are considered to be produced mainly as a result of non-germinal centre B cell development, but excessive stimulation of B cells and maintenance of large numbers of somatically mutated B cells could also be associated with low avidity antibodies in response to malaria antigens . Increasantigens . While Is cohort . Taken tequences . Examina


Pneumonia is the major cause of mortality and morbidity in children worldwide. Procalcitonin (PCT) and C-reactive protein (CRP) are used in developed countries to differentiate between viral and bacterial causes of pneumonia. Validity of these markers needs to be further explored in Africa.We assessed the utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children <5 years hospitalized with clinical severe pneumonia (CSP) in rural Mozambique, a malaria-endemic area with high HIV prevalence. Prognostic capacity of these markers was also evaluated. Out of 835 children with CSP, 87 fulfilled definition of viral pneumonia and 89 of invasive bacterial pneumonia. In absence of malaria parasites, levels of PCT and CRP were lower in the viral group when compared to the invasive bacterial one . However, in presence of malaria parasites distribution between clinical groups overlapped . None of the two markers could predict mortality.Presence of malaria parasites should be taken into consideration, either for clinical or epidemiological purposes, if using PCT or CRP to differentiate viral from invasive bacterial pneumonia in malaria-endemic areas. Pneumonia is the major cause of morbidity and mortality in children worldwide. More than 90% of the estimated 1.6 million annual pneumonia deaths in children <5 years occur in developing countries In rural Africa, pneumonia diagnosis is mainly based on WHO's Integrated Management of Childhood Illness (IMCI), a set of highly sensitive clinical algorithms developed to guide management of most common mortality causes in children. IMCI has contributed to the reduction of pneumonia childhood mortality Plasmodium falciparum, highly prevalent among children in most of Africa, increases levels of CRP Biomarkers to differentiate viral from bacterial infections have been evaluated mostly in the developed world to support clinical diagnosis and to be applied as rapid diagnostic tests (RDT). Despite disparity in results In this study, we assessed the utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children in Manhiça, a rural malaria-endemic area of Mozambique with high HIV prevalence. Prognostic capacity of these markers was also evaluated.Centro de Investigação em Saúde da Manhiça (CISM) at the Manhiça District Hospital (MDH), the referral health facility for Manhiça District, a rural malaria-endemic area of Southern Mozambique with subtropical climate This study was conducted by the 2 and approximately 80000 inhabitants, 18% are children <5 years. Under-five mortality rate was 138.6/1000 in 2005 Since 1996, the CISM has been running continuous Demographic Surveillance System (DSS). The DSS covers 500 kmSevere pneumonia accounted for 16% of admissions to MDH and 11% of case-fatality rate among children <2 years in 2004–2006; 19% of these children presented clinical malaria The MDH is a 110 bed hospital with 36 paediatric beds. Since 1997, MDH and CISM have operated round-the-clock surveillance of all paediatric visits th September 2006 and 19th September 2007 Hospital Clínic de Barcelona (HCB).This analysis is part of a larger study designed to describe clinical and epidemiological characteristics of children <5 years admitted to the MDH with suspected clinical severe pneumonia (CSP) between 20As part of the study protocol, blood sample was collected on admission for full blood cell count and PCT/CRP determination. Nasopharyngeal aspirate (NPA) was collected to detect respiratory viruses. Blood culture and malaria determination were extended to all children with CSP. Chest-x-rays were also performed. Children residing in the DSS area were offered voluntary HIV counseling and testing. Additional written informed consent and finger prick blood were required.Pneumocystis pneumonia (PCP) or, according to parents/legal guardian, had received antibiotics before admission were excluded from the analysis.According to definitions, children with CSP were classified in two groups, viral and invasive bacterial pneumonia. Children who presented clinical suspicion of Asymptomatic healthy children <5 years from the DSS area were randomly selected to participate into this study as part of the control group. After parental/legal guardian consent, blood was collected for determination of malaria and PCT/CRP levels. No other tests were performed to this group.9/l leukocytes in blood, a negative blood culture and at least one of the following viruses isolated from NPA: respiratory syncytial virus (RSV), influenza (Flu), parainfluenza (PIV), human metapneumovirus (hMPV) and adenovirus (ADV).Viral pneumonia was defined when a child presented on admission with a normal chest x-ray or with abnormalities other than alveolar consolidation or pleural effusion Staphylococcus epidermidis, Viridans group Streptococci and Bacillus spp. (non anthracis)].Invasive bacterial pneumonia was defined when a bacteria was isolated from blood, excluding potential contaminants [P. falciparum parasites.Control group was defined as healthy children from the community with absence of fever, signs or symptoms of illness and P. falciparum parasites were detected by microscope observation of thick and thin Giemsa-stained blood films To detect RSV, Flu, PIV, hMPV and ADV from NPA two single polymerase chain reactions (PCR) and two multiplex PCRs were performed Determine®  and Unigold® . HIV-1 infection was confirmed using antigen DNA-PCR Roche HIV-1 DNA test®  for <18 months positive children and for those cases with discordant results from the two RDT.HIV testing was done using two RDT: Institut d'Investigacions Biomèdiques August Pi i Sunyer. PCT quantification was performed using automated immunoanalysis with Liaison®  or Kryptor Compact® . For CRP, immunoturbidimetric assay with ADVIA Chemistry CRP_2®  was used. Limits of detection of these techniques were 0.04 ng/ml, 0.02 ng/ml and 4 mg/l respectively.Plasma obtained from 1.5 ml of blood in EDTA  after full blood cell count was stored at -20°C until processing at HCB/Data were double entered using Fox Pro version 2.6  and analyzed using STATA version 11 . Discrepancies in data entry were resolved by referring to the original forms.Proportions were compared using Chi-square test. Distributions of both markers were evaluated by mean of Kruskall-Wallis test. Linear regression models were estimated to evaluate the difference of PCT and CRP levels, adjusted by HIV, hematocrit, oxygen saturation and malnutrition. P-value of 0.05 or lower was considered significant.Optimal cut-offs to determine viral (used as reference) and invasive bacterial pneumonia were investigated using receiver operating characteristic analyses.P. falciparum determinations were available for 176 (93%) of these children, 87 (49%) corresponding to the viral group and 89 (51%) to the invasive bacterial one. 93 viruses were detected from the 87 children in the viral group , being the most prevalent viruses in decreasing order: ADV (n = 33), RSV (n = 18), hMPV (n = 16), Flu (n = 15), PIV (n = 11). 90 bacteria were detected from the 89 children in the invasive bacterial group , being the most prevalent bacteria in decreasing order: Streptococcus pneumoniae (n = 42), Haemophilus influenzae type b (n = 16), Staphylococcus aureus (n = 7), Escherichia coli (n = 6), Salmonella spp (n = 6), others (n = 13). A total of 37 children met criteria of the control group.A total of 835 children admitted to MDH during the study period met study entry criteria, 190 (23%) fulfilled definition for viral or invasive bacterial pneumonia. Both plasma and Overall, 11% (17/157) of the children in the clinical groups with outcome data died in-hospital. All fatalities occurred in the invasive bacterial group. Comparison of descriptive and clinical features among groups is shown in P. falciparum . In the invasive bacterial group median values corresponded to 8.31 ng/ml and 21.75 ng/ml in absence and in presence of P. falciparum . In absence of P. falciparum . In the viral group median values were 0.21 ng/ml and 23.1 ng/ml in absence and in presence of lciparum , PCT dislciparum , PCT disP. falciparum . In the invasive bacterial group median values corresponded to 185.4 mg/l and 217.4 mg/l in absence and in presence of P. falciparum . In absence of P. falciparum . In the viral group median values were 18.3 mg/l and 96.8 mg/l in absence and in presence of lciparum , distriblciparum , CRP disP. falciparum due to the observed effect of malaria parasites in PCT and CRP levels.Further analyses were performed in absence of P. falciparum no differences were found in PCT and CRP distribution within viruses (p = 0.482 and p = 0.649)  or bacteria isolated . In absence of = 0.649) , online.= 0.649) , online.As shown in P. falciparum, the area under the curve (AUC) in predicting clinical group was 0.9 for PCT and 0.87 for CRP . PCT and CRP cut-offs were maximized respectively at 0.72 ng/ml  and 20.9 mg/l . Combination of both markers did not improve diagnostic profile (data not shown).In absence of P. falciparum, PCT distribution between survivors and fatalities was different . However, an important overlap of values was observed between the two groups . However, an important overlap of values was observed between the two groups .In absence of o groups . Median o groups . Median We have assessed the usefulness of PCT and CRP to differentiate viral from invasive bacterial pneumonia in rural Africa, where malaria, HIV and other co-morbidities are common. Our main finding is that PCT and CRP can not differentiate between these two clinical groups among hospitalized children with malaria parasites. However, both markers can differentiate these two groups in absence of malaria parasites and independently of their HIV status. None of the two markers could predict mortality.P. falciparum. This contrasts with data from Malawi, where authors showed no significant differences in PCT and CRP levels between children with and without malaria infection P. falciparum. We do not know the clinical group of these 13 cases. Along with our findings, studies conducted in The Gambia and Tanzania observed that malaria infection increased levels of CRP P. falciparum can rapidly be assessed with a RDT. In African malaria-endemic countries efforts are being done to establish malaria parasitological diagnosis as routine practice According to our results PCT and CRP have similar distribution among study clinical groups in presence of Strategies to reduce pneumonia burden of disease and severity include prevention through early diagnosis and selection of appropriate treatment. In a context where pneumonia plays an important role in high in-hospital mortality and where diagnostic facilities are poor, all children meeting WHO criteria for CSP should be treated empirically with parenteral antibiotics. Our study suggests that in in-hospital children without WHO criteria for parenteral antibiotics, elevated levels of PCT or CRP in absence of malaria parasites could indicate the need of antibiotics. Additionally, in absence of malaria parasites elevated levels of PCT and CRP could warn of invasive bacterial pneumonia at peripheral health posts and outpatient departments. This might respectively facilitate transfer to hospital and admission, as well as rationalization and prioritization of antibiotics administration.Haemophilus influenzae pneumonia in epidemiological and intervention studies Standardized definitions of pneumonia in epidemiological studies are still a challenge and measurement of disease burden and efficacy in vaccine trials is necessary using proxy measures. WHO and CDC jointly developed a generic protocol to measure burden of pneumonia using standardized definitions based on radiological interpretation P. falciparum. Although the study was underpowered for this outcome, no trend was observed. Similarly, PCT and CRP did not demonstrate any value in predicting death among Malawian children with signs of pneumonia or meningitis To determine prognostic markers is another daily challenge for clinicians in resource-limited settings. We also evaluated PCT and CRP as predictors of mortality among clinical groups without The major limitation of this study is related to the definitions of the clinical groups. On one hand, we expect some miss-classifications in the viral group due to undetectable incipient bacterial co-infection P. falciparum in areas with high prevalence of HIV infection. How P. falciparum parasites increase PCT and CRP levels independently of the pathogen associated to pneumonia should be considered in the application of these markers for clinical and/or epidemiological purposes.In conclusion, this study provides evidence of the potential utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children free of Figure S1P. falciparum.Distribution of procalcitonin (PCT) amd C-reactive proteinin (CRP) concentrations within viruses in absence of (1.48 MB TIF)Click here for additional data file.Figure S2P. falciparum.Distribution of procalcitonin (PCT) amd C-reactive proteinin (CRP) concentrations within bacteria in absence of (1.48 MB TIF)Click here for additional data file.


A moderate association has been found between asymptomatic parasitaemia and undernutrition. However, additional investigation using the gold standard for asymptomatic parasitaemia confirmation, polymerase chain reaction (PCR), is needed to validate this association. Anthropometric measurements and blood samples from children less than five years of age in a rural Ghanaian community were used to determine if an association exists between chronic undernutrition and PCR-confirmed cases of asymptomatic malaria.This was a descriptive cross-sectional study of 214 children less than five years of age from a community near Kumasi, Ghana. Blood samples and anthropometric measurements from these children were collected during physical examinations conducted in January 2007 by partners of the Barekuma Collaborative Community Development Programme.Findings from the logistic model predicting the odds of asymptomatic malaria indicate that children who experienced mild, moderate or severe stunting were not more likely to have asymptomatic malaria than children who were not stunted. Children experiencing anaemia had an increased likelihood  of asymptomatic malaria. Similarly, increased spleen size, which was measured by ultrasound, was also associated with asymptomatic malaria . Fast breathing, sex of the child, and age of the child were not significantly associated with the asymptomatic malaria.No significant association between chronic undernutrition and presence of asymptomatic malaria was found. Children who experience anaemia and children who have splenomegaly are more likely to present asymptomatic malaria. Programmes aimed at addressing malaria should continue to include nutritional components, especially components that address anaemia. More than half of child mortality related to major infectious diseases is attributed at least in part to undernutrition . For manNot all research, however, agrees that malaria and undernutrition have a synergistic relationship ,12-14. FMalarial infection does not always result in symptomatic or overt disease. Children who experience malarial infection, but who do not exhibit acute symptoms, have asymptomatic parasitaemia, which is common in malaria endemic areas of sub-Saharan Africa, with some areas experiencing parasitaemia prevalence as high as 90% -34. Mostth out of the 19 countries estimated to have 90% of the cases in Africa [Plasmodium falciparum infections are estimated to account for approximately 10% of health life-years lost [This study took place in the country of Ghana, where malaria is endemic year round. With a total population of 23 million people, Ghana's health system reported more than 7 million cases of malaria during 2006 and is ranked 8n Africa ,44. Nearn Africa . Furthern Africa . Finallyars lost .Anthropometric measurements and blood samples from children less than five years of age in a rural Ghanaian community was evaluated to determine if an association exists between chronic undernutrition (i.e. stunting) and PCR-confirmed cases of asymptomatic malaria. Further, the prevalence of stunting and asymptomatic malaria in addition to other basic physical examination findings is reported.This study uses a cross-sectional design to evaluate the association between stunting and asymptomatic malaria. Data were obtained from physical examinations conducted by partners of the Barekuma Collaborative Community Development Programme (BCCDP). The BCCDP is a collaborative partnership between 20 rural Ghanaian communities near Kumasi, Ghana and researchers from the Komfo Anokye Teaching Hospital , the Kwame Nkrumah University of Science and Technology , and the University of Utah . The BCCDP, which began in 2001, uses a Community Based Participatory Research (CBPR) framework and serves approximately 25,000 people in 20 communities in a rural area approximately 25 kilometers north-west of Kumasi, the second largest city in Ghana . Over thNear the beginning of the collaboration, community partners requested assistance addressing malaria and disease surveillance in the community. Researchers began by first assessing the burden of disease in partner communities. A pilot community was identified in which to conduct physical examination of children to identify common health problems. Data collection was specifically planned to address malaria. The results were intended to be used to inform programme and policy decisions, to test the specificity and sensitivity of a rapid diagnostic test, and to understand the interaction between malaria and other health outcomes in the study area.Approximately 300 children from Adankwame and surrounding communities were brought by their guardians to the community center to receive a physical examination and to participate in the study. Notice of the study was given to all members of the communities involved and participation was voluntary. Blood samples and anthropometric measurements were available for 214 children less than five years of age.University of Utah and local Ghanaian pediatricians performed physical examinations on children in the community of Adankwame during January of 2007. Community leaders and researchers encouraged mothers to bring children to the community center to receive the free physical examination. Children were examined on successive days. The physical examination included the collection of standard examination measures, such as age, height, weight, body temperature, pulse, blood pressure, spleen size. Additionally, blood, urine, and stool samples were also collected.® Malaria tests were used on-site. Blood smears to check for malaria parasites and complete blood counts were performed at the Komfo Anokye Teaching Hospital malaria laboratory. Additional laboratory work, including malaria PCR of blood smears, was completed at the University of Utah infectious diseases laboratory.Field workers also gathered information from the child's caregiver on the recent health history of the child including data on recent febrile episodes and malaria symptoms. Binax NOWThe primary outcome variable is a positive test for asymptomatic malaria parasitaemia. While rapid diagnostic malaria tests were conducted in the field, all blood samples were tested using PCR technology to confirm results from the field. No cases of symptomatic malaria were present in the sample.Height-for-age z-score (HAZ) was the primary predictor of interest. This measure is based on the international reference standard from Centers for Disease Control and Prevention and World Health Organization. The z-score is calculated through an interpolation function that accounts for sex, age, and height. A child with an HAZ greater than -1.0 standard deviations below the mean on the international reference standard was determined to have no stunting. Mild stunting is height-for-age z-score between -1.0 and -2.0 while moderate stunting is defined as a height-for-age z-score from -2.0 through -3.0. Severe stunting is defined as a height-for-age z-score less than -3.0 standard deviations below the mean on the international reference standard. Other physical examination characteristics examined included fast breathing, anaemia, and spleen size. Fast breathing was defined as greater than 50 breaths per minute in children 2-12 months and greater than 40 breaths per minute for children 12-60 months of age. Anaemia was defined as a child with a haemoglobin count of less than 11.0 (g/dl). Lastly, spleen size was determined through the use of ultrasound.All data were entered into the computer using Microsoft Excel . Statistical analyses were conducted using SAS statistical software . Anthropometric indicators were calculated using a SAS macro designed and made available by the World Health Organization (WHO). This macro uses the WHO International Growth Reference standard. Statistical analyses include a description of the characteristics of respondents. Additionally, percentages used to describe the population and to identify associations between undernutrition and presence of malaria were evaluated using Pearson chi-square tests. Further, prevalence of stunting by age groups was calculated. The relationship between height-for-age z-score and the presence of asymptomatic malaria parasitaemia was analysed using logistic regression. Appropriate confounding variables that were available from physical examination data, such as age and sex, were retained or dropped from the models based on p values (<.1) and conceptual considerations. Regression coefficients and 95% confidence intervals were calculated for retained variables. Interaction among confounders was also examined.Research ethics approval was obtained in the US from the University of Utah and in Ghana from the Institutional Review Board of Kwame Nkrumah University of Science & Technology School of Medical Sciences and Komfo Anokye Teaching Hospital. Informed consent was obtained from all household heads or guardians of the children enrolled in the study. Participation was voluntary and participants were able to withdraw at any time. Anti-malarials were provided to children who were found to have a positive test for malaria parasitaemia at the time of the physical examination. Children found to need treatment for some other problem were referred to a partnering Ghanaian physician and entered into the Ghana health care system.Children were fairly evenly distributed in each age group with four year olds making up the largest single group at 22.9% Table . Nearly The change in HAZ scores generally decreases the older the child was at the time of assessment Figure . Median The percentage of positive cases of asymptomatic children appears to rise with age Table . For exaAdjusted results for the logistic model predicting the probability of asymptomatic indicate that children who experienced mild, moderate or severe stunting were not more likely to have asymptomatic malaria than children who experienced no stunting Table . ChildreThese findings show no significant relationship between presence of asymptomatic malaria and stunting, a measure of chronic undernutrition. Children who were mildly stunted, however, appeared to be more likely to be asymptomatic than children who were not stunted, though the difference was not significant. This trend was not found for moderate and severely stunted children. Additionally, there is a strong relationship between anaemia and asymptomatic malaria as well as between spleen size and asymptomatic malaria. Lastly, findings indicate a general decline in median HAZ over the first 24 months of life followed by a leveling off and some subsequent recovery.While other studies who have found an association between asymptomatic parasitaemia and undernutrition, no such relationship was found in the study population. There could be a number of explanations for these differences. First, several studies found a relationship between asymptomatic parasitaemia and measures of undernutrition that did not include stunting ,35,39,41These findings indicating a strong association between presence of asymptomatic malaria and anaemia are consistent with findings from other studies that have linked malaria and anaemia -57. ShanPrevious research indicates the key role the spleen plays in protection against malaria . IncreasThis study suffers from some limitations. First, a cross-sectional design was used, which cannot be used to infer causality, but should only used to identify associations. Second, the sample was a convenience sample of children, which may or may not be representative of the entire community or children from the country as a whole. Lastly, only physical examination and brief survey data were available. As a result, statistical models used for this study lack the ability to control for other potential confounders of the relationship between chronic undernutrition and asymptomatic malaria. For example, children from families with favourable economic circumstances may be less likely to be stunted in addition to having reduced exposure to malaria through the purchase and use of insecticide-treated bed nets.In conclusion, no significant association between chronic undernutrition as measured by stunting and presence of asymptomatic malaria was found. This result does not suggest that no association exists between undernutrition and clinical malaria. Rather, it appears from these findings that malaria infection is not associated with chronic undernutrition. This is not incongruent with research that indicates that undernutrition may influence the burden of disease related to malaria or that clinical malaria may impact undernutrition . AdditioThe authors declare that they have no competing interests.BTC conceived the study, performed the statistical analysis, and drafted the manuscript. SCA participated in the study design, helped develop the BCCDP, assisted in the data collection, and helped to draft the manuscript. IB participated in the data collection and revisions of the manuscript. RMM participated in the study design, assisted in analysis of data, and helped to draft the manuscript. JHA participated in the interpretation of results and the revision of the manuscript. CAP participated in the study design and helped to draft the manuscript. JBS participated in the study design and helped to draft the manuscript. TTD participated in the study design, interpreted the results, and helped to revise the manuscript. KAD participated in the study design, assisted in the data collection, and helped to draft the manuscript. DCH participated in the data collection, helped develop the BCCDP collaboration, and assisted with manuscript revisions. JS participated in the data collection, coordinated the physical examinations, and assisted with manuscript revisions. BSS assisted in the data analysis and manuscript revisions. AOA participated in the data collection, helped coordinate physical examinations, and assisted with manuscript revisions. DA participated in data collection, assisted with the coordination of physical examinations, coordinated the development of the BCCDP, and helped with manuscript revisions. All authors read and approved the final manuscript.


While the protective effects of sickle cell trait (HbAS) against severe malaria and the resulting survival advantage are well known, the impact on the physical development in young children remains unclear. This study was aimed to investigate the relationship between HbS carriage and stunting in children below two years of age in a cohort from the Ashanti Region, Ghana.1,070 children were recruited at three months of age and followed-up for 21 months with anthropometric measurements performed every three months. Incidence rate ratios with 95% confidence intervals were calculated by Poisson regression to estimate the association of β-globin genotypes with the number of malaria episodes. Odds ratios (OR) were calculated for the association between the occurrence of β-globin genotypes and/or malaria episodes and stunting. The age-dependent between-group and within-group effects for the β-globin genotypes were assessed by population-averaged models estimated by generalized estimation equation with autoregressive correlation structure.Analyses showed a significantly lower age-dependent risk of stunting  in carriers of the HbAS genotype (n = 102) in comparison to those with HbAA (n = 692). This effect was restricted to children who experienced malaria episodes during the observation period suggesting that the beneficial effect of the β-globin HbS variant on the incidence of stunting is closely linked to its protection from mild malaria episodes.The lower risk of chronic malnutrition in early childhood, mediated by protection against mild malaria episodes, may contribute to the survival advantage of HbAS carriers in areas of high malaria transmission. In addiortality . It has ortality ,9.So far, most studies on the effect of HbAS on physical development were performed in areas without malaria transmission. In the majority of these studies no significant differences between individuals with the HbAA and HbAS genotype were found ,11, whilz-score of less than -2 according to WHO (moderate and severe stunting combined) [In the frame of a trial on Intermittent Preventive Treatment in infants (IPTi), conducted from January 2003 to September 2005, a cohort of 1,070 children from a rural district in the Ashanti Region of Ghana was recruited at the age of three months and followed-up monthly until two years of age. On each visit a clinical examination was performed, a standardized medical history taken and parasite density measured. The length of each child was assessed at recruitment and subsequently in three-monthly intervals, with measurements performed on the same standardized length board for all children. Details on the study area, study group and procedures have been published elsewhere ,14. A maombined) . DNA preombined) .Statistical analyses were performed with STATA . Only children with HbAA or HbAS genotype and with at least six completed anthropometric measurements were considered for the analyses. Missing values for anthropometric data between two existing follow-up visits were interpolated by calculating the mean of the two flanking visits . P values below 0.05 were considered significant. Incidence rate ratios (IRR) with 95% confidence intervals (CI) were calculated by Poisson regression to estimate the association of β-globin genotypes with the number of malaria episodes. Odds ratios (OR) were calculated for the association between the occurrence of β-globin genotypes and/or malaria episodes and stunting. The age-dependent between-group and within-group effects for the β-globin genotypes were assessed by population-averaged models estimated by generalized estimation equation (GEE) with autoregressive correlation structure of order 5 and assumption of binomial variable distribution. Autoregression was used to account for intra-individual age-dependency of anthropometric measurements and the most appropriate autoregression order was assessed by correlation matrices. Resulting coefficients were log-transformed and given as ORs with CI to estimate the risks for stunting at three-monthly cross-sectional time-series.Out of the 1,070 children recruited, 794 children were included in the analyses as they were carriers of the HbAA or HbAS genotype and had completed at least six anthropometric measurements. A total of 692 children were HbAA homozygotes while 102 children were carriers of HbAS  were tested for effect modification but no influence on the effect of HbAS and malaria on stunting was detected. There was also no effect modification by the study-arm (IPTi vs. placebo).In addition to being protected against stunting, HbAS carriers in this study were also protected against mild malaria episodes . In order to analyze a direct effect of malaria episodes on stunting, the ORs for the risk of stunting at the end of the study period were calculated only for children who were not stunted at recruitment. Children with two or more episodes of malaria during the study period had an increased risk of becoming stunted at two years of age compared to children with zero or one episode . Other factors with reported influence on stunting  and other genetic hemoglobin disorders  have a lower risk of stunting in an area with high malaria transmission, most likely mediated through protection against mild malaria. Since stunting in early years is known to be a strong risk factor for morbidity and mortality and considering the high incidence of mild malaria, it seems probable that protection against stunting contributes essentially to the survival advantage of HbAS carriers.IPTi: Intermittent Preventive Treatment in infants; HbS: Sickle cell mutation; GEE: Generalized Estimation Equation; OR: Odds Ratio; IRR: Incidence Rate Ratio; CI: 95% Confidence Interval.The authors declare that they have no competing interests.BK collected data, performed statistical analyses and wrote the first draft of the manuscript. SE contributed to the writing of the manuscript. CK, RK and SA organized and supervised the study in Ghana and collected data. GDB critically reviewed the manuscript. CE and MA performed the laboratory work. OA was principal investigator of the study and JM designed and implemented the study, performed statistical analyses and was involved in the writing of the manuscript. All authors contributed significantly to the final version of the manuscript.


The socioeconomic and sociodemographic situation are important components for the design and assessment of malaria control measures. In malaria endemic areas, however, valid classification of socioeconomic factors is difficult due to the lack of standardized tax and income data. The objective of this study was to quantify household socioeconomic levels using principal component analyses (PCA) to a set of indicator variables and to use a classification scheme for the multivariate analysis of children < 15 years of age presented with and without malaria to an outpatient department of a rural hospital.In total, 1,496 children presenting to the hospital were examined for malaria parasites and interviewed with a standardized questionnaire. The information of eleven indicators of the family's housing situation was reduced by PCA to a socioeconomic score, which was then classified into three socioeconomic status . Their influence on the malaria occurrence was analysed together with malaria risk co-factors, such as sex, parent's educational and ethnic background, number of children living in a household, applied malaria protection measures, place of residence and age of the child and the mother.The multivariate regression analysis demonstrated that the proportion of children with malaria decreased with increasing socioeconomic status as classified by PCA (p < 0.05). Other independent factors for malaria risk were the use of malaria protection measures (p < 0.05), the place of residence (p < 0.05), and the age of the child (p < 0.05).The socioeconomic situation is significantly associated with malaria even in holoendemic rural areas where economic differences are not much pronounced. Valid classification of the socioeconomic level is crucial to be considered as confounder in intervention trials and in the planning of malaria control measures. Malaria is one of the major public health challenges subverting development in the poorest countries in the world. The direct and indirect costs of malaria are very high and the disease has played a significant role in the poor economic performance of sub-Saharan Africa. Sachs 2002) estimated, that the gross domestic product in these countries would be up to 32% greater today if malaria had been eliminated 35 years ago . In cont002 estimIn Ghana, where the study was conducted, malaria is prevalent during the entire year and accounts for about 32-42% of all outpatient admissions and for major in-patient causes of death . SociodeThe aim of the presented study was to investigate the association between the socioeconomic status of families classified with a number of indicators as a PCA-based score and their association with childhood malaria.2; the coverage population of the study hospital was 61,346 inhabitants (census data 2004) where the population ranged from 890 inhabitants in the smallest village to 15,383 in the largest.This survey was accomplished at the Child Welfare Clinic and the Pediatric Ward of the Agogo Presbyterian Hospital, Asante Akim North District (Ashanti Region) in central Ghana, West Africa. Recruitment area included 14 villages which were summarized into 4 cluster ("Greater Agogo" (Agogo city and Hwidiem), "Greater Konongo" (Konongo and Odumasi), "West of Agogo"  and "Near Street"   [The vegetation of the study area is mainly semi-deciduous forest with major vegetation types of open forest, closed forest and wooded savannah. The climate is tropical with a mean annual temperature of 26°C and two rainy seasons: a first rainy season from May to July and a second from September to November. The dry harmattan season occurs between December and April and is associated with drought conditions. The topography of the district is generally undulating and the altitude variation is 226 m between the lowest (227 m) and the highest (453 m) village included in our study. Agriculture is the predominant major occupation among people; main staple food crops produced in the district include maize, cassava, plantain, cocoyam and yam . The pri (> 90%) . EntomolP. falciparum parasitaemia count of > 0 parasites/μL was detected in the thick or thin smears. Parasite examination was done according to quality-controlled standardized procedures described elsewhere [The study was carried out between May 2007 and August 2009 (duration 26 months). Diagnostic assessments were integrated into the hospital routine. In total, 1,496 children up to 14 years of age, who visited the hospital for medical care, were included in the study. The case definition for malaria was fulfilled if the axillary temperature was ≥ 37.5°C and a lsewhere .Information on personal or family characteristics with a possible influence on malaria  and information about factors indicating the family's financial situation  was collected through interviewing a parent or the guardian who accompanied the child to the hospital using a structured interview with a questionnaire in English or if necessary in the local language, Twi. The question sheet was composed according to standard questionnaires adjusted to local requirements and appropriateness. Data from questionnaires and forms were double entered after case closed, plausibility checked, and cleaned before the database was locked. All information on participants and their parents was treated confidentially. Only children who were examined for malaria and where information about the sociodemographic and socioeconomic situation was available were included in the analysis (n = 1496).The study was approved by the Committee on Human Research, Publications, and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.Participants were allocated into one of the four village clusters described above , according to their place of residence. Additionally, all participants were classified into two ethnic groups according to their tribal background: the Akan and those who are the natives of the area and the Northeners who have a migratory background but are now permanent residents of the area. It was hypothesized that children between the ages of 1 to 5 years are at highest risk of acquiring malaria; hence we stratified for age . It was also suggested that the mother's age might be of importance for the child and its risk for malaria; all mothers were stratified and grouped to young mothers (≤ 30 years) and older mothers (> 30 years). High numbers of children living in a household were assumed as an influence factor (two groups: ≤ 4 children and > 4 children). Additionally, it was asked in the interview whether a family used protective measures such as bed nets or window-screens. Individuals with missing values on any of these variables (n = 18) were excluded from the analysis (n = 1478).To classify the family's economic status, the following socioeconomic indicator variables were considered: mother's and father's profession (employed/unemployed) and education (ability to read and write: yes/no), type of house the family is living in (cement/brick house or mud/wood house), water supply (open water source/closed water source), existence of an indoor kitchen (Yes/No), electricity (Yes/No), indoor toilet (Yes/No), use of freezing as measure of conservation (Yes/No), existence of a relative abroad who might financially support the family (Yes/No), the self-rated ability to manage with the available monthly income (difficult or not difficult) as well as the membership in the health insurance (Yes/No). All socioeconomic and sociodemographic data including information on protective measures based on self-reports of the mothers or guardians and were not confirmed by direct observations during household visits.For the sake of the multivariable analysis, a principal component analysis (PCA) was applied to those socioeconomic indicator variables, which showed relevant contributions (> 10%) to the combined socioeconomic status score factor . The facFor the PCA, missing values of distinct binary variables were replaced by the means of all summarized "0" values (asset not present) and "1" values (asset present) of this variable n = 1496) . This ap96 . ThisFor each potential risk factor of malaria, the odds ratio (OR) was calculated and the significance level was tested by the chi-square test. Adjusted ORs were estimated by multivariate logistic regression. Confounding was determined as a relative difference of 15% between crude odds ratios and odds ratios adjusted for predefined covariates without signs of effect modification. All covariables in the multivariate regression model were examined for possible effect modification by Wald tests and preference of the model with interaction by log-likelihood tests (both p < 0.05).In total, 1,496 children were examined for malaria parasites and participated in the questionnaire survey; 1,478 without missing values were included in the multivariate model. Most participants came from the region Greater Agogo (n = 871), fewer came from Greater Konongo (n = 333), Near Street (n = 229), and West of Agogo (n = 63). Most malaria cases were reported from Greater Agogo (n = 364), followed by Greater Konongo (n = 66), Near Street (n = 61), and West of Agogo (n = 21)   and evenly distributed under children with and without malaria. Likewise, the variables "house type", "income manage", "membership in a health insurance", "existence of an indoor kitchen" and "mother's and father's occupation" did not show any distinct association with malaria  was checked by using a histogram to show the distribution of socioeconomic scores. Internal coherence for our study region could be shown, suggesting appropriate and sufficient choice of asset variables.All potential risk factors, together with the newly created variable describing the socioeconomic status were included in the final logistic regression model to assess their independent effect on malaria risk Table . In the The analysis showed that, in an area of high endemicity, the proportion of malaria in children presented to a hospital is markedly influenced by the socioeconomic status of the family: children from households classified as poor had a significantly higher chance to get malaria. This is in agreement with previous reports on distinct socioeconomic risk factors for malaria -8,15,16.Socioeconomic levels might also be associated with diseases beyond malaria and may influence the proportion of malaria cases among all children seen in the hospital. This would have an indirect influence on the calculated odds ratios. However, the two symptom complexes predominant in children without malaria, namely respiratory distress and gastrointestinal symptoms, were not or only weakly associated with socioeconomic levels. The study design, which bases on a single hospital, might limit the generalization of the results to other regions. On the other hand, the focus on one hospital allowed the thorough collection of data on the clinical condition, infectious disease agents, and exact diagnosis.One problem of the determination of individual socioeconomic levels in Africa is the fact that unambiguous quantitative measures often do not exist and various proxy measures must be used as an approximation. The use of single variables as risk indicators often leads to false conclusions because they only reflect parts of the general view. In contrast, in PCAs socioeconomic indicator variables were combined to enable a quantification and classification of individual socioeconomic levels and to use the resulting score for risk analyses. The PCA showed that usage of a freezer as conservation method, which was interpreted as ownership of a freezer in a household, had the highest weight for the socioeconomic score. On the other hand, having a health insurance was the smallest compared to the other ten variables and, hence, was of a minor importance for the socioeconomic score.An advantage of the PCA is that it reduces measurement problems, such as recall bias, and that it reduces the complexity of correlated data, which can be easily collected as single indicator variables in household surveys ,22. On tApart from the socioeconomic status, sociodemographic factors were associated with malaria. As expected the malaria risk was highest in the age group of children between 1 and 5 years, compared to children below the age of 1 year  and loweAge of mothers did not influence the occurrence of malaria of their children. However, in the study group only 3% of the mothers were younger than 20 years of age, which does not represent the mother's age distribution in Ghana or other African countries. Although ethnicity was found to influence malaria risk in a study conducted in an adjacent region in Ghana  this couIn conclusion, the herein presented results show that children from poorer households are of greater risk for malaria. It is under discussion how far poverty influences the occurrence of malaria or malaria influences the occurrence of poverty. In either case, the fight against malaria has to be escorted by the fight against poverty and improvement of living standard. Moreover, the spatial variability of malaria risk might be of importance for the planning of control measures and the conduction of intervention trials.The authors declare that they have no competing interests.ACK participated in the conception, performed the statistical analysis, was involved in the interpretation of results and participated in drafting the results. NGS participated in the design, assisted in performing the statistical analysis, was involved in the interpretation of the results, and helped to draft the manuscript. WL created Case Report Forms and was responsible for data management and data preparation for analyses. UR was involved in the initial design of the study, assisted in performing the statistical analysis and participated in drafting the manuscript. JM conceived and coordinated the study, was involved in the initial design of the study and writing of the manuscript. NS organized the day-to-day work on the ground, BN and SA carried out the malaria parasite examination and contributed to the writing of the manuscript, and YAS planned, initiated the study, and reviewed manuscript. All authors have read and approved the final manuscript.Characteristics of the study group. the table shows the results of a first univariate analysis including variables which give information on personal or family characteristics with a possible influence on malaria and information about factors indicating the family's financial situation.Click here for file