Severe malaria is difficult to differentiate from other forms of malaria or other infections with similar symptoms. Any parameter associated to malaria-attributable severe disease could help to improve severe malaria diagnosis.This study assessed the relation between erythropoietin (EPO) and malaria-attributable severe disease in an area of Mozambique with moderate malaria transmission. 211 children <5 years, recruited at Manhiça District Hospital or in the surrounding villages, were included in one of the following groups: severe malaria , hospital malaria without severity , uncomplicated malaria , invasive bacterial infection without malaria parasites  and healthy community controls . Malaria was diagnosed by microscopy and IBI by blood/cerebrospinal fluid culture.Mean EPO concentration in the control group was 20.95 U/l (SD = 2.96 U/l). Values in this group were lower when compared to each of the clinical groups . In the 3 malaria groups, values increased with severity . The IBI group [mean = 101.75 U/l (SD = 4.12 U/l)] presented lower values than the SM one (p = 0.002). In spite of the differences, values overlapped between study groups and EPO levels were only associated to hemoglobin. Hemoglobin means of the clinical groups were 93.98 g/dl (SD = 14.77 g/dl) for UM, 75.96 g/dl (SD = 16.48 g/dl) for HM, 64.34 g/dl (SD = 22.99 g/dl) for SM and 75.67 g/dl (SD = 16.58 g/dl) for IBI.Although EPO levels increase according to malaria severity and are higher in severe malaria than in bacteremia, the utility of EPO to distinguish malaria-attributable severe disease is limited due to the overlap of values between the study groups and the main role of hemoglobin in the expression of EPO. Plasmodium in blood can easily be determined by microscopy or rapid diagnostic tests (RDTs), but even when Plasmodium is observed, attribution of symptomatology to malaria is not straightforward. In areas that are hyper- or holo-endemic for malaria, children are exposed to repeated infective bites during infancy and early childhood, progressively acquiring immunity against the disease and developing clinical tolerance to malaria parasites An optimal management of malaria needs a correct diagnosis and a good assessment of severity. Presence of Additionally, the broad diversity of pathophysiological pathways, leading to the different syndromic presentations associated to severe malaria, further hinders diagnosis. Severe malaria in African children encompasses at least three main clinical syndromes that can occur alone or in combination: severe malarial anemia, cerebral malaria and respiratory distress , a glycoprotein hormone principally produced by the kidney’s peritubular capillary endothelial cells in response to hypoxia, is crucial for sustained proliferation and differentiation of erythroid cells Assuming that hypoxia-related acidosis is the cardinal sign of severe malaria and that EPO is the key molecule in the adaptation to hypoxia; we hypothesized that EPO levels could be related to malaria-attributable severe disease among children with different degrees of malaria severity and children with no-malarial causes of severe disease (bacteremia), in an area of Mozambique with moderate malaria transmission.Centro de Investigação em Saúde da Manhiça (CISM) and the Manhiça District Hospital (MDH), the referral health facility for Manhiça District, a rural malaria-endemic area of Southern Mozambique.This study was conducted at the 2 and approximately 80,000 inhabitants (18% children <5 years) Since 1996, CISM has been running a continuous Demographic Surveillance System (DSS) and a morbidity surveillance platform at the MDH. The DSS covers 500 kmP. falciparumMalaria transmission, perennial but with substantial seasonality, is mainly due to P. falciparum and anemia show a considerable overlap in the spatial and temporal distribution patterns. In a community cross-sectional survey among healthy children, the prevalence of anemia at the end of the rainy season was twice as high than at the end of the dry season (34.4% versus 17.9%), suggesting that malaria is an important contributor to anemia P. falciparum density and packed cell volume was found In Manhiça, Between 2001 and 2006, community-acquired bacteremia accounted for 8% of all pediatric admissions and 21% of all in-hospital deaths Hospital Clínic de Barcelona.Children included in this analysis were recruited as part of a larger study designed to explore the use of certain cytokines/other proteins to differentiate malaria, bacterial and viral infections among children <5 years old seen at MDH. Recruitment of the larger study went from September 2006 to May 2009 and included, among other patients, children with different degrees of malaria severity or invasive bacterial infection. Data presented here belong to those children from the larger study fulfilling the group definitions of the present analysis and with plasma samples available to perform EPO measurements. Written informed consent was obtained from all participant parents/legal guardians involved in the study. The study was approved by the Mozambican National Bioethics Committee and Institutional Review Board of the Children included in this analysis were classified into five groups: severe malaria (SM), hospital malaria (HM), uncomplicated malaria (UM), invasive bacterial infection (IBI) and healthy control (C). SM group was further stratified in severe malarial anemia (SMA), cerebral malaria (CM) and mixed severe malaria (MSM) subgroups. We used the HM group to describe an intermediate severity group in malaria, including those children judged to need hospitalization but without the criteria needed to fulfill the severe malaria case definition. This non-standard classification, also used in previous malaria vaccine trials Standardized questionnaires with demographic and clinical data were completed for recruited children together with the following samples: i) finger prick blood smears for malaria microscopy; ii) 1–3 ml of venous blood for bacterial culture; iii) 1.5 ml of venous blood in EDTA for full blood count, EPO, bilirubin and LDH measurements. Blood culture was not performed for the control group. Cerebrospinal fluid (CSF) was only collected from hospitalized children with suspicion of meningitis. Children in the inpatient groups residing in the DSS area were offered voluntary HIV counseling and testing. Additional written informed consent and finger prick were needed for this purpose.P. falciparum in blood , a negative blood and CSF culture (if available) and, at least, one of the following criteria: severe anemia (hematocrit <15% or Hb <5g/dl), deep coma (Blantyre coma score ≤2), prostration (inability to sit unaided or look for mother’s breast/feed in children who cannot yet sit), hypoglycemia (<2.2 mmol/l), repeated convulsions (≥2 reported episodes in the 24 hours prior to admission) or respiratory distress (deep breathing or indrawing). Among SM group, children with only severe anemia were classified in the severe malarial anemia (SMA) subgroup and those with only neurological impairment including prostration, convulsions or/and coma in the cerebral malaria (CM) one. Finally, mixed severe malaria (MSM) subgroup included those SM cases with SMA or/and CM accompanied with any other criteria of SM and those SM cases with hypoglycemia or/and respiratory distress.Severe malaria (SM) was defined as the presence of fever (axillary temperature ≥37.5°C) or reported history of fever in the preceding 24 hours in children admitted with P. falciparum in blood , a negative blood and CSF culture (if available) and not fulfilling inclusion criteria for severe malaria.Hospital malaria (HM) was defined as the presence of fever or history of fever in the preceding 24 hours in children admitted with P. falciparum in blood  and a negative blood culture in children seen as outpatients with no clinical criteria for hospital admission.Uncomplicated malaria (UM) was defined as the presence of fever or history of fever in the preceding 24 hours, P. falciparum in blood. Potential contaminants  were not included in this group.Invasive bacterial infection (IBI) was defined in children admitted with respiratory distress (chest indrawing and/or deep breathing) as the presence of bacteria isolated from blood and/or CSF and no P. falciparum parasites in blood.Controls (C) included healthy children from the community with no history of fever or detectable fever at recruitment, no report of having received medical assistance/treatment during the last 30 days, no signs or symptoms of illness and no P. falciparum parasites were detected by microscope observation of thick and thin Giemsa-stained blood films as described elsewhere Sysmex KX21 hematology analyzer . Plasma was obtained by blood centrifugation . Plasma LDH and total-bilirubin (conjugated plus unconjugated) were measured using a Vitros DT60 II biochemical analyser . Remaining plasma was stored at −20°C and sent to the Hospital Clínic de Barcelona, where EPO quantification was performed using Quantikine human Erythropoietin  immunoanalysis with a limit of detection of 0.6 U/ml.Full blood counts were performed using a Determine  and Unigold . For children <18 months positive by both RDT and for those cases of discordant results between RDT, HIV-1 infection was confirmed using antigen DNA-PCR Roche HIV-1 DNA test .HIV testing was done using two RDT: Fox Pro version 2.6  and discrepancies between two entries were resolved referring to the original forms. Statistical analyses were performed using STATA version 11 .Data were double entered using Proportions were compared using Chi-square test. EPO and bilirubin values were logarithmically transformed to perform the analysis. In the case of malaria groups, test for trend was used to assess lineal increase of different parameters with severity. ANOVA test was performed to evaluate differences among distributions of EPO, hemoglobin, bilirubin and LDH levels between the study groups. Linear regression models were estimated to evaluate the difference of EPO levels adjusted by age, sex, malnutrition, hemoglobin and days of fever prior to visit/admission. Univariate linear regression was used to explore the relation between EPO/bilirubin/LDH and Hb. P-values ≤0.05 were considered significant.Streptococcus pneumoniae (n = 22), Haemophilus influenzae B (n = 6), Escherichia coli (n = 4) Staphylococcus aureus (n = 3), Salmonella spp (n = 2), Streptococus spp (n = 1) and Klebsiella pneumoniae (n = 1). No bacterial co-infection was found.This analysis is based on 211 children <5 years recruited between September 2006 and May 2009, 132 of which were admitted to hospital. Distribution of the children according to study groups was: SM (n = 44), HM (n = 49), UM (n = 47), IBI (n = 39) and C (n = 32). Children in the SM group presented at least one of the following criteria: respiratory distress (n = 29), severe anemia (n = 16), prostration (n = 14), convulsions (n = 9), deep coma (n = 2) and hypoglycemia (n = 1). Among these children, 8 corresponded to the SMA subgroup, 6 to the CM and 30 to the MSM. The following bacteria were isolated in the IBI group: Nine of the 124 admitted children with outcome data died in-hospital. All fatalities occurred in the IBI group, which had an associated case fatality rate of 28% (9/32). Severe anemia was more prevalent in the SM group when compared to the other groups . Demographic and clinical features of the study groups are shown in Although values overlapped between study groups, differences in the distribution of EPO levels were observed (p<0.001) . Mean EPWhen stratifying SM group in SMA, CM and MSM, differences on EPO levels were observed among the subgroups  , with meThe association of EPO levels with demographic and clinical parameters in the clinical groups is illustrated in After adjusting EPO levels by age, sex, malnutrition, hemoglobin and days of fever prior to visit/admission in the multivariate analysis, EPO only remained associated to Hb concentration . Identical results were observed when repeating the analysis stratifying SM in SMA, CM and MSM subgroups (data not shown). When evaluating the role of the clinical group in the association between EPO and Hb no interaction was observed (data not shown).As shown in Although levels of LDH were higher in the SM group compared to the IBI group, values between the two groups overlapped  . Mean LDIn the present study, we have assessed the relation between EPO and malaria-attributable severe disease among children with malaria  or severe infection . The main findings are that in our setting, despite EPO levels increase according to malaria severity and the SM group presents higher levels of EPO than the IBI group, the utility of EPO to distinguish malaria-attributable severe disease is limited due to the overlap of values between the study groups and the main role of hemoglobin in the expression of EPO.According to the results, SM cases presented the highest levels of EPO among the study groups and EPO levels increased progressively with malaria severity. Despite being IBI the group with the most severe cases in the study , EPO levels were higher in the SM group when compared to the IBI one. These observations may apparently suggest that EPO could be useful to distinguish malaria-attributable severe disease. However, this conclusion cannot be assumed from this study as the results show that the differences observed on EPO levels depend only on the Hb levels and not on the group (even when considering the different forms of SM separately). In consequence, the visible pattern of EPO distribution among the study groups should vary according to the degree of anemia. This observation contrasts with data from Kenya, where EPO levels were found 3.5 times higher in children with mild malarial anemia than in those without malaria and mild to moderate anemia Although the previous results lack of practicality in terms of improving clinical diagnosis of SM, they can however contribute to understand the pathophysiological processes underlying the expression of EPO. Despite EPO being produced in response to hypoxia and different factors (not only hemolysis) may trigger its expression Besides the hypoxia due to anemia, it has been described that in severe malaria the oxygen delivery to tissues is poorer than in sepsis as indicated by the acidosis and the much higher ratios of serum lactate/pyruvate Leaving aside the mechanisms that could explain the distribution of EPO levels in this study, the results indicate that the utility of EPO to distinguish malaria-attributable severe disease is limited, at least, in settings with high prevalence of anemia among ill children. From these results, we cannot state that the suspicion of SM on the basis of elevated levels of EPO could be used to accelerate provision of medical assistance in Manhiça. Nevertheless, some studies have suggested the use of biomarkers in Africa for purposes other than clinical diagnosis In conclusion, malaria parasites can easily be detected with present diagnostic tools but the differentiation of malaria-attributable severe disease remains a challenge. This study suggests that, although EPO levels increase according to malaria severity and are higher in severe malaria than in bacteremia, the utility of EPO to distinguish malaria-attributable severe disease in Manhiça is limited due to the overlap of values between study groups and the main role of hemoglobin in the expression of EPO.


Plasmodium falciparum malaria. Its pathogenesis and management are still debated. Nine cases of this syndrome occurred in 2003 at Kiremba Hospital in Burundi in children receiving multiple quinine treatments.Blackwater fever is characterized by acute intravascular hemolysis with hemoglobinuria in patients with  Plasmodium falciparum–endemic areas who take quinine irregularly. This syndrome became less frequent when chloroquine was the drug of choice for malaria from 1950 until the 1990s (Blackwater fever (BWF) is a clinical entity well known only in long-term residents in he 1990s . Isolatehe 1990s –5.The pathogenesis of BWF thus remains unclear ,6,7. ItsClinical features defining BWF are well established ,9. The sP. falciparum resistance to chloroquine developed, quinine was increasingly used in clinical practice for treating intermittent malaria infections. BWF seemed to reappear at the end of the 1990s, according to descriptions in several European clinics of imported diseases . According to patients' health cards, all had been previously treated with quinine, either parenterally or orally according to Burundi's national policy for treating severe malaria (10 mg/kg 3×/day for 7 days). Clinical and laboratory data are presented in the When BWF occurred, quinine was stopped and artemether , was administered intramuscularly in association with 3 days of corticosteroid therapy. All patients had severe anemia requiring blood transfusion according to hospital policy (hemoglobin <4.5 g/dL or <6 g/dL with accompanying dyspnea). Four patients needed l U of blood; 5 other patients needed >1 U. No deaths were recorded, and clinical outcome on discharge was satisfactory: thick smears were negative and hemoglobin levels had improved in all patients.In Burundi, chloroquine was replaced by sulfadoxine-pyrimethamine (SP) alone as firstline treatment for uncomplicated malaria in 2001. However, the rapid development of resistance to SP brought back the use of oral quinine, a drug still available in health centers as well as in hospital settings. Since November 2003, artesunate and amodiaquine have replaced SP as firstline treatment in Burundi .The result of the new treatment guidelines was a considerable reduction in the number of hospitalized malaria cases in 2004 . No cases of BWF were observed in this period. Despite changes in policy for the use of firstline antimalarial drugs, however, parenteral quinine continued to be the drug of choice for severe cases throughout this period.All 9 patients with BWF seen in 2003 (with 1 exception) lived in the area served by Kiremba Hospital and were recorded during an 11-month period. This number represents an incidence of 11.5 cases/100.000 population/year.In reviewing recent literature, we found only 1 publication on BWF involving an African population . The stuIn our study, patients were all boys admitted to the pediatric ward. No cases of oligoanuria were seen, which is not surprising in pediatric patients . At the The management of our cases included 3 components: First, treatment with parenteral (intramuscular) artemether  after stopping quinine, according to recent trends in the literature . Second,Our experience suggests the need to review the definition of BWF since the syndrome affects not only adult expatriates but also African children. All reported African children with cases of BWF had frequently received oral quinine therapy. African adults seem to be only occasionally affected. This finding suggests that BWF occurs in nonimmune persons or those who have not yet gained immunity. This statement is supported by the lack of cases in adults cured in the same hospital.To reduce hemolysis, we treated BWF with corticosteroids, even though this step is not recommended by the World Health Organization. Our reasoning was that the phenomenon could be related to immune mechanisms in quinine-sensitized erythrocytes .The influence of quinine seems to be an important factor in the pathogenesis of BWF. Other amino-alcohol drugs such as mefloquine or halofantrine have never been used intensively in Africa, principally because they are expensive. When policy changes lead to less use of oral quinine, BWF syndrome tends to disappear. Further similar reports from other areas in the African continent that would confirm our findings could have important implications on national policies for treating malaria in African children.


Malaria, schistosomiasis and soil transmitted helminth infections (STH) are important parasitic infections in Sub-Saharan Africa where a significant proportion of people are exposed to co-infections of more than one parasite. In Tanzania, these infections are a major public health problem particularly in school and pre-school children. The current study investigated malaria and helminth co-infections and anaemia in school and pre-school children in Magu district, Tanzania.Schistosoma mansoni and STH infections using Kato Katz technique. Urine samples were examined for Schistosoma haematobium using the urine filtration method. Blood samples were examined for malaria parasites and haemoglobin concentrations using the Giemsa stain and Haemoque methods, respectively.School and pre-school children were enrolled in a cross-sectional study. Stool samples were examined for P. falciparum infection). Malaria parasites were significantly more prevalent in hookworm infected children than in hookworm free children (p = 0.046). However, this association was non-significant on multivariate logistic regression analysis . Malaria parasite density decreased with increasing infection intensity of S. mansoni and with increasing number of co-infecting helminth species. Anaemia prevalence was 34.4% and was significantly associated with malaria infection, S. haematobium infection and with multiple parasite infections. Whereas S. mansoni infection was a significant predictor of malaria parasite density, P. falciparum and S. haematobium infections were significant predictors of anaemia.Out of 1,546 children examined, 1,079 (69.8%) were infected with one or more parasites. Malaria-helminth co-infections were observed in 276 children (60% of all children with P. falciparum, S. mansoni and S. haematobium infections increase the risk of lower Hb levels and anaemia, which in turn calls for integrated disease control interventions. The associations between malaria and helminth infections detected in this study need further investigation.These findings suggest that multiple parasite infections are common in school and pre-school children in Magu district. Concurrent  P. falciparum occurs throughout the country with varying levels of endemicity. Stable and perennial transmission occurs in the warm humid coastal regions and around the great Lakes Schistosoma mansoni and Schistosoma haematobium while the major STH infections are caused by the hookworms Necator americanus and Ancylostoma duodenale, Ascaris lumbricoides and Trichuris trichiuraTrichuris trichiura and Ascaris lumbricoides) share not only the areas in which they occur, but also the human hosts P. falciparum malaria than helminth free individuals T. trichiura infections are associated with anaemia due to blood and iron loss into the intestinal tract while S. mansoni and S. haematobium infections cause blood loss in faeces and urine, respectively Malaria, schistosomiasis and soil transmitted helminth infections (STH) are the most important parasitic infections in Sub-Saharan Africa, where a significant proportion of the populations including school children are exposed to these infections 2 of which 1725 km2 (56.1%) is covered by Lake Victoria waters. Mean temperature ranges from 18°C to 20°C during the rainy season and 26°C to 30°C during the dry season. Rainfall is bimodal with the short rains between October to December and heavy rains between March and May. Mean annual rainfall ranges from 700 m to 1000 mm. In 2003, the district had a population of 416,113 people of whom 202,077 (48.6%) were males The study was conducted in Magu district, North-Western Tanzania. Magu district lies between 2°10′ and 2°50′ South of Equator and 33° and 34° East of Greenwich. It has an area of 3075 kmS. mansoni, S. haematobium, soil-transmitted helminthiasis and P. falciparum and those found with ailments not targeted by the project were treated free of charge according to national guidelines. Study identification numbers were used instead of children names and information collected was kept confidential. Feedback to the study population in the form of dissemination workshops was conducted during the course of the study.The study was approved by the Medical Research Coordination Committee (MRCC) of the National Institute for Medical Research (NIMR), Tanzania (Reference No. NIMR/HQ/R.8a/Vol. IX/355). Before commencement of the study, the research team conducted meetings with leaders, teachers and community members of all selected villages. During these meetings, the objectives of the study including the study procedures to be followed, samples to be taken, study benefits and potential risks and discomforts were explained. Informed consent for all children who participated in the study was sought from parents and legal guardians after they have been clearly informed about the study. Parental consent given from parents/guardians was written. Children were also requested to give assent and were informed of their right to refuse to participate in the study and to withdraw at any time during the study without jeopardizing their right of access to other health services. Invasive procedures such as collection of blood samples were fully explained to parents and children and were carried out using sterile disposable materials. All children found infected with any of the parasites S. mansoni and intestinal helminths  using the Kato Katz method S. mansoni and intestinal helminths were expressed as the mean eggs per gram of faeces (epg) of the two samples (four smears). Urine samples were examined for S. haematobium eggs in 10 ml of urine according to the nucleopore filtration method Children were provided with plastic containers and requested to bring stool and urine samples on two consecutive days at about 10:00am in the morning. Stool samples were examined for S. mansoni and hookworm infections, eggs per 10 ml of urine for S. haematobium and parasites per microlitre of blood for P. falciparum based on positive samples only. The student's t-test and one way analysis of variance (ANOVA) was used to compare geometric mean parasite counts and mean Hb concentrations where two or more than two groups were compared, respectively. For parasite counts, the student's t-test and ANOVA were performed on log transformed data of positive samples only whereas for Hb concentrations the students t-test and ANOVA were performed for all samples examined on original scale. The Chi-square test was used to compare proportions and to test for association between malaria prevalence, anaemia prevalence and prevalence of helminth infections between exposure groups. In the multivariate analysis, presence or absence of infection or anaemia was compared among schools, age groups, sexes and other infections using logistic regression analysis fitted as a generalized linear model with a logit link function and adjusting for possible clustering among siblings. All predictors were initially tested for significance separately and then jointly in a multi-variable model. Except for box plots which were drawn using STATA version 10, all other graphs were drawn using MS-Excel software. Tests were considered statistically significant at p<0.05.Data were double entered into Dbase V software  and analyzed using STATA Version 10 . Parasite counts were normalized by log transformation, averaged and then back transformed to the original scale. Infection intensities were calculated as geometric mean of eggs per gram of faeces for A total of 1615 school and pre-school children were examined. Pre-school children were 372 or 23% of all children examined. Children where complete information was available were included in the analysis (1546) of whom 759 (49.1%) were boys. Overall mean age was 7 years.P. falciparum, S. mansoni, S. haematobium, hookworm and T. trichiura. S. mansoni infections were generally light to moderate with only 59 children (9.6%) being heavily infected (epg≥400). Whereas 94 children (3.8%) had heavy S. haematobium infections (≥50eggs/10 ml of urine), all hookworm infections were light (epg<2000). Three children (0.2%) were infected with T. Trichiura and Ascaris lumbricoides infections were absent. S. mansoni infection was the most prevalent parasite followed by P. falciparum and S. haematobium. Hookworm was the least prevalent parasite. The prevalence of individual parasite species and the respective infection intensities are shown in Out of the 1546 children included in the analysis, 1079 (69.8%) were infected with at least one of the parasites S. mansoni, S. haematobium and hookworm infections differed significantly across age groups (p<0.001) whereby older children (6–8 years and 9–13 years) had higher prevalence of infection compared to younger children (3–5 years). Likewise, the infection intensity of S. mansoni and hookworm differed significantly across age groups (p<0.001) whereby children in higher age groups had higher parasite loads compared to children in the lower age group. The prevalence and infection intensity of S. mansoni, S. haematobium and hookworm also differed significantly across schools (p>0.001). Malaria prevalence varied considerably among schools (p<0.001) being highest in Mwamayombo and Milambi compared to other schools. Younger children had higher malaria parasite density compared older children (p<0.001).The prevalence of S. mansoni infections occurred as single as well as a multiple species infection in almost equal proportions . P. falciparum, S. haematobium and hookworms infections occurred more frequently as multiple species infections than single species infections. Out of the 1079 infected children, 430 (39.9%) harboured more than one parasite species. Overall, 2 = 51.07, p<0.001).P. falciparum infected children was 60% (276/460). The most common parasite combinations were P. falciparum and S. mansoni (27.2%), P. falciparum and S. haematobium (10.2%), P. falciparum and hookworm (7.4%), P. falciparum, S. mansoni and S. haematobium (7%), P.falciparum, S.mansoni and hookworm (6.5%) and P. falciparum, S. haematobium and hookworm (3.0%). Malaria and helminth co-infections occurred more frequently in older children (9–13 years) compared to younger children (3–5 and 6–8 years) and the difference was significant . Malaria parasites were significantly more prevalent in hookworm infected children than in hookworm free children (35.1% vs 28.8%) . However, this association turned to be non-significant when multivariate logistic regression analysis was performed while adjusting for other confounding factors . Children with hookworm infection were more likely to be infected with S. haematobium  compared to children who were not infected with hookworm. Further, children infected with hookworm were also likely to be infected with S. mansoni  compared to children who were not infected with hookworm. The prevalence of malaria parasites tended to increase with increasing number of co-infecting helminth species. The prevalence of malaria parasites was 29%, 35% and 41.2% in children harbouring one, two and three helminth specie, respectively, compared to 28.3% in helminth free children. However, the difference was not significant .The prevalence of helminth co-infections among S. mansoni infection while Except for hookworm infection, malaria parasite density was negatively correlated with helminth infections . S. mansoni. Geometric mean malaria parasite density for children without S. mansoni infection was 745 (95% CI 633–879) and was significantly higher compared to 551 (95% CI 434–700) and 399 (95% CI 297–534) for children with light and moderate to heavy S. mansoni infection, respectively  .Malaria parasite densities tended to decrease with increasing infection intensity of  p<0.01) . Figure 2 = 15.58, p<0.001) and S. haematobium infection . For P. falciparum and S. haematobium infections, mean Hb levels decreased significantly with increasing infection intensities (p<0.01). Children who were not infected with any parasite had the highest mean Hb levels  and hence the lowest prevalence of anaemia (27.2%). Except for P. falciparum and hookworm co-infections, children who were infected with more than one parasite species tended to have lower mean haemoglobin levels and hence higher prevalence of anaemia compared to children infected with one parasite. The highest prevalence of anaemia (60%) was observed in children co-infected with three parasites P. falciparum, S. mansoni and S. haematobium  were anaemic and only 16 (1.0%) were severely anaemic. Overall mean Hb concentration was 123.7 (95% CI 123.0–124.4). The prevalence of anaemia was significantly associated with malaria infection  and/or dyserythropoiesis S. mansoni, S. haematobium and hookworm infections cause anaemia through chronic blood loss S. haematobium infections were predictors of anaemia, a finding which indicates that in addition to the known effect of single parasite species on anaemia, multiple parasite infections can interact to enhance the risk of anaemia. Interestingly, the highest prevalence of anaemia (60%) was observed in children concurrently infected with P. falciparum, S. mansoni and S. haematobium, and in children concurrently infected with P. falciparum and S. haematobium (52.3%). Anaemia was also more prevalent in children concurrently infected with three or four parasites compared to those with only one or no parasite infection. These observations demonstrate a possible synergistic interaction of P. falciparum, S. mansoni and S. haematobium and multiple parasite infections as the aetiology of anaemia. Limitations of the current study in elucidating associations between malaria and helminth co-infections include the lack of information on household, socio-economic status and environmental factors which have been shown to influence occurrence of co-infections by other studies Anaemia was prevalent in the study area though at a relatively low level compared to what was reported by the study of Lwambo P. falciparum, S. mansoni and S. haematobium infections increase the risk of lower Hb levels and anaemia which in turn calls for integrated disease control interventions. The associations between malaria and helminth infections detected in this study were not conclusive and hence needs further investigation.Overall, results of this study have demonstrated that malaria, schistosomiasis and STH infections are prevalent in school and pre-school children in Magu district and that polyparasitism is also very common. These findings also suggest that concurrent


Plasmodium falciparum and Plasmodium vivax as the dominant species.The WHO has reported that RDT and microscopy-confirmed malaria cases have declined in recent years. However, it is still unclear if this reflects a real decrease in incidence in Bangladesh, as particularly the hilly and forested areas of the Chittagong Hill Tract (CHT) Districts report more than 80% of all cases and deaths. surveillance and epidemiological data on malaria from the CHT are limited; existing data report A cross-sectional survey was conducted in the District of Bandarban, the southernmost of the three Hill Tracts Districts, to collect district-wide malaria prevalence data from one of the regions with the highest malaria endemicity in Bangladesh. A multistage cluster sampling technique was used to collect blood samples from febrile and afebrile participants and malaria microscopy and standardized nested PCR for diagnosis were performed. Demographic data, vital signs and splenomegaly were recorded.Plasmodium falciparum mono-infections accounted for 58.9%, P. vivax mono-infections for 13.6%, Plasmodium malariae for 1.8%, and Plasmodium ovale for 1.4% of all positive cases. In 24.4% of all cases mixed infections were identified by PCR. The proportion of asymptomatic infections among PCR-confirmed cases was 77.0%, oligosymptomatic and symptomatic cases accounted for only 19.8 and 3.2%, respectively. Significantly (p < 0.01) more asymptomatic cases were recorded among participants older than 15 years as compared to younger participants, whereas prevalence and parasite density were significantly (p < 0.01) higher in patients younger than 15 years. Spleen rate and malaria prevalence in two to nine year olds were 18.6 and 34.6%, respectively. No significant difference in malaria prevalence and parasite density was observed between dry and rainy season.Malaria prevalence across all subdistricts in the monsoon season was 30.7% (95% CI: 28.3-33.2) and 14.2% (95% CI: 12.5-16.2) by PCR and microscopy, respectively. A large proportion of asymptomatic plasmodial infections was found which likely act as a reservoir of transmission. This has major implications for ongoing malaria control programmes that are based on the treatment of symptomatic patients. These findings highlight the need for new intervention strategies targeting asymptomatic carriers. The World Health Organization (WHO) estimated 660,000 deaths in 2011 directly attributed to malaria, approximately half of the world’s population being at risk of infection -6.Resistance to chloroquine and sulphadoxine/pyrimethamine (S/P) has been reported from Bangladesh  but untiIn 2004 the Ministry of Public Health and Family Welfare of Bangladesh revised the malaria treatment guidelines, introducing artemisinin-based combination therapy (ACT) in areas with resistance against chloroquine and S/P . HoweverPlasmodium falciparum. Plasmodium vivax and mixed infections accounted for only 5.3%and 4.5%, respectively. Highest prevalence rates were reported from the Chittagong Hill Tract (CHT) with up to 15% [et al. personal communication), which increases to 50% when using polymerase chain reaction (PCR), a considerably more sensitive diagnostic tool [In 2007 a rapid diagnostic test (RDT)-based, cross-sectional survey in 13 eastern districts of the country showed malaria to be endemic within the entire study area. Overall prevalence was reported to be approximately 4%, the majority of cases (90.2%) due to tic tool .P. falciparum infections in the southernmost district of the CHTs.The high sensitivity of PCR allows detecting subpatent infections that are frequently asymptomatic and it has been shown that those undetected infections represent a considerable fraction of overall infections and may therefore act as a reservoir for transmission -21. The Two cross-sectional surveys were performed: the first during the rainy season from August to October 2007 and the second during the dry season from December 2007 to February 2008. The study was conducted by a team from the Medical University of Vienna, Austria in collaboration with the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh . All selected communities were visited by members of the study team, prior to sample collection to inform villagers about the ongoing study. Laboratory tests on collected samples and data analysis were carried out at the Malaria Research Initiative Bandarban (MARIB) field research centre in Bandarban town. Written informed consent was obtained from all study participants or their legal representative, the study protocol was approved by the Ethical Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh .upazilas), 32 unions, 140 mouzas and 1,482 villages. Geographical multistage cluster sampling techniques in a single domain were employed . Eight villages in three subdistricts  were revisited during the second survey period (Survey II). Whenever possible the study team visited the same households as in the previous survey. Household members who declined to participate or had moved away from the area were replaced by their closest neighbouring household.Participants were interviewed and information was recorded on gender, age, ethnicity, occupation, height, weight, history of fever, number of previous episodes of malaria/fever, last date of malaria diagnosis and result of diagnosis, source and drugs used in malaria treatment, and the number of household members. Axillary temperature and pulse were measured in all participants. Three ml of venous blood was collected from participants eight years or older for malaria RDT/microscopy and 100 μl of venous blood transferred on filter paper  in duplicate. Five drops of blood  were drawn by finger or heel prick from children below eight years old.Malaria was diagnosed in the field by RDT and later confirmed by microscopy and PCR.®, Zephyr Biomedicals, India) based on the detection of P. falciparum-specific histidine-rich protein 2 (HRP2) and P. vivax-specific lactate dehydrogenase (Pv-pLDH) were employed in all participants with malaria-like symptoms [RDTs  and examined under an oil immersion  for parasite positivity and species determination. Declaring a slide positive or negative and initial species diagnosis was based on the examination of 200 fields in thick films. A slide was considered positive when at least one parasite was found. After finding the first parasite another 200 fields were completed to rule out mixed infections. If no parasite was found in 200 oil fields the slide was considered negative. Parasite density was calculated by counting the number of asexual malaria parasites per 200 white blood cells (WBCs) assuming a WBC count of 8,000/μL [Giemsa-stained blood smears were used for microscopic diagnosis of malaria following established standard operating procedures (SOPs). Thick and thin blood films were prepared, stained with Giemsa stain ; rVIV1/rVIV2 (P. vivax); rMAL1/rMAL2 ; rOVA1WC/rOVA2WC ; rOVA1/rOVA2 ; rOVA1var/rOVA2var  [PCR was performed to assess the malaria prevalence in both, symptomatic and asymptomatic participants. DNA was isolated from filter papers using a modified chelex-based technique ,27. Nesteviously -30. All llikeri) -33.Symptomatic clinical malaria cases were defined as PCR-positive individuals with documented fever (axillary temperature ≥37.5°C) and reported clinical symptoms consistent with malaria in the previous seven days. Oligo-symptomatic malaria cases were defined as PCR-positive, afebrile (axillary temperature <37.5°C) cases with a reported history of fever or illness in the previous seven days. Asymptomatic malaria cases were defined as PCR-positive cases without measurable fever (axillary temperature <37.5°C), who reported no malaria-related symptoms, and had not received treatment for malaria in the previous seven days .The spleen rate was determined for children aged 14 years and below. Spleen size was measured followingthe method established by Hackett  and clas; holo-endemic malaria: spleen and/or parasite rate rate in children two to nine years constantly over 75%, but a low spleen and/or parasite rate rate in adults.The four classes of endemic malaria were defined as following : hypo-enU-test was performed for comparing continuous data that did not conform to normal distribution. Student’s t-test was conducted to evaluate differences between quantitative variables that were normally distributed. All analyses were performed using Microsoft Excel® and VassarStats [.P < 0.05 was considered significant. Pearson’s Chi-square with Yates’ correction or Fisher’s Exact test as appropriate were used for categorical data, Mann–Whitney sarStats .A total of 1,418 individuals in 416 households from 21 villages were included in the first survey and 436 were revisited and included in the second survey. The male/female ratio was 0.84. The median age was 24 years (IQR 9–40). The distribution of the different age groups is shown in Table District-wide malaria prevalence  and 14.2% (95% CI: 12.5-16.2) by PCR and microscopy, respectively. Of the 1,418 samples collected 435  were PCR-positive. Plasmodium falciparum accounted for 58.9% , P. vivax for 13.6% , P. malariae for 1.8%  and P. ovale for 1.4%  of all malaria-positive individuals by PCR. The remaining 24.4%  were mixed infections. Plasmodium falciparum gametocytes were detected by microscopy in 11  individuals, seven of whom had only gametocytes.ce Table : MalariaWith 188 cases  the malaria prevalence in individuals under 15 years was significant higher (p < 0.01) compared to older participants . A significantly higher malaria prevalence (p = 0.015) was found in males  as compared to females .P. falciparum and 345 parasites/μl (95% CI: 246–484) for P. vivax. Parasite densities in individuals younger than 15 years were significantly higher (p < 0.01).The overall geometric mean parasite density determined by microscopy was 707 parasites/μl (95% CI: 565–883) for P. falciparum or P. vivax no difference (P > 0.05) was found between the proportion of asymptomatic and oligosymptomatic clinical cases  were classified as asymptomatic, 86  as oligosymptomatic, and 14  symptomatic cases were found. Significantly (p < 0.01) more asymptomatic cases (N = 208) were recorded among participants older than 15 years compared to younger individuals (n = 127). In individuals infected with Out of 416 households in 223  at least one member of the household tested positive for malaria, in 64 households  two people tested positive for malaria and in 56  households three or more individuals tested positive by PCR.Plasmodium falciparum was the predominant species in all subdistricts. The highest malaria prevalence was found in the eastern subdistricts Rowangchari and Thanchi, followed by Bandarban Sadar, Ruma, Naikhongchhari, and Lama. The lowest malaria prevalence was detected in Ali Kadam, which shares only a short stretch of border with Myanmar. Malaria prevalence among the seven subdistricts is shown in detail in Table P. falciparum or P. vivax. A total of 121  children tested positive by PCR for any of the four malaria species. Thirty-eight  and 48  children were malaria microscopy-positive or PCR-positive and had an enlarged spleen at the same time. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of spleen enlargement for malaria in this population are shown in Table Out of a total of 350 children aged two to nine years, 65 had a palpable spleen ; 91 of them  were slide-positive for malaria either for Plasmodium falciparum accounted for 58.6%, P. vivax for 21.6%, P. malariae for 4.3%, and P. ovale for 0.9% of all PCR malaria-positive individuals, the remaining 14.7% were mixed infections.From the 436 individuals included in the winter survey Table  only 35 Plasmodium falciparum gametocytes were detected by microscopy in four  individuals of whom only one also had asexual parasites. Malaria prevalence within all participants in the winter survey (dry season) was not significantly (p = 0.117) lower compared to the summer survey.No patient testing positive for malaria was febrile at the time of the survey, therefore 103  were classified as asymptomatic and 13  as oligosymptomatic  for P. vivax, respectively. No statistically difference (p > 0.05) was found between the parasite rate in summer and winter survey.The geometric mean parasite densities were 487 asexual parasites/μl (95% CI: 281–845) for From a total of 172 households, in 77  at least one member tested positive for malaria, in 27  households two individuals and in seven  households three or more people tested positive for malaria.In the winter survey the highest malaria prevalence by subdistrict was found in Bandarban Sadar  followed by Rowangchhari , and the lowest malaria prevalence was detected in Ruma .Out of 97 children aged two to nine years, nine  had a palpable spleen and with 30.9%  the parasite prevalence by PCR in survey II was again high but not significantly different from survey I (p = 0.584). Malaria-positive PCR and an enlarged spleen at the same time were found in four  children two to nine years old. With 9.3% the spleen rate in winter was also lower than in summer . Sensitivity, specificity, PPV and NPV of spleen enlargement for malaria in the winter survey are shown in Table Plasmodium parasites in southeastern Bangladesh.Malaria is a major public health problem in southeastern Bangladesh and remains one of the most common reasons for hospital admissions during the malaria season . Mappinget al. have previously reported for this region that environmental factors, such as proximity to forest, household density, and elevation tend to be significantly and positively correlated with malaria risk [Three major observations can be deducted from these cross-sectional surveys. First, this study demonstrates that malaria is meso-endemic in the region and is concentrated in rural communities  where thria risk . The devria risk . A spleeria risk ) and malria risk ,38.Second, malaria prevalence was significantly higher in the northern and eastern subdistricts that are located in the hilly areas bordering Myanmar and Rangamati, another endemic district, whereas endemicity was lower in western and southern subdistricts in or close to the coastal plains, which are more accessible and where access to health care facilities is better. The collected data indicate a level of malaria transmission similar to some African countries, such as Gabon , SomaliaThird, a large reservoir of asymptomatic, parasitaemic individuals that are likely to act as a source of infection, was observed in both surveys. The absence of malaria-like symptoms may be an indication of a certain level of immunity in rural communities. Although generally considered to be a typical feature of malaria in highly endemic regions in Africa, a number of studies from Brazil, Peru, Thailand, Cambodia, Myanmar, Vietnam, eastern Indonesia, and Papua New Guinea have reported the existence of asymptomatic malaria infections outside of Africa and in areas with lower endemicity ,43-49. TP. falciparum, which is believed to build up with long-term exposure to malaria and presents with lower parasite densities and fewer clinical malaria episodes in older children and adults, has been reported from endemic areas in Myanmar, eastern Indonesia and India [Naturally acquired immunity against nd India . In areand India . Malariaet al. [As previously reported by Alves et al. , asymptoLimitations of this study include all potential shortcomings of a point-prevalence study as well as the potential bias arising from the fact that only one district has been surveyed which may not be representative for all 13 malaria endemic districts in Bangladesh. Secondly, there was some overlap with the previously reported cross-sectional survey based on RDTs , which, The surveys showed that there are areas in Bangladesh with prevalence rates comparable to those found in malaria-endemic regions of tropical Africa. This study indicates, in accordance with other studies from Southeast Asia, that there is still a substantial proportion of asymptomatic, parasitaemic individuals in Bangladesh that may act as a silent reservoir for malaria transmission. This has major implications for ongoing malaria control programs in Bangladesh that are based on prevention of infection through bed nets and treatment of symptomatic patients. Particularly considering ongoing elimination efforts these findings highlight the need for new intervention strategies targeting all infections, symptomatic as well as asymptomatic, for reducing potential sources of infection and for interrupting the transmission cycle. Based on this study, it is evident that malaria remains an important public health problem in the southeastern part of Bangladesh. Further research is needed to determine the role of asymptomatic individuals for malaria transmission in the area.The authors declare that they have no competing interests.HN contributed to all steps from elaboration to the final review . PS, KT, PSw, BL, MJ supervised and carried out the surveys (three study teams worked simultaneously during the summer survey) and data collection in the field. PS, BL and KT contributed to implementation, to data entry and data analysis. PS wrote the first draft of the manuscript. BL and KT contributed to the writing of the manuscript. RH helped to design the study protocol, monitored laboratory quality and corrected the manuscript. WAK participated in the coordination of patient samples collection in the field, drafted the manuscript and helped to analyze the data. WG helped to design the study protocol and revised the final manuscript. H-PF and VEH carried out the molecular genetic studies. All authors read, approved and gave their consent to the final manuscript.


Malaria transmission was reported to have declined in some East African countries. However, a comparable trend has not been confirmed for West Africa. This study aims to assess the dynamics of parasite prevalence and malaria species distribution over time in an area of highly seasonal transmission in Burkina Faso. The aim was also to compare frequency of asymptomatic parasitaemia between wet and dry season by parasite density status and age group.During the years 2009–2012, six cross-sectional studies were performed in the rural village Bourasso in the Nouna Health District in north-west Burkina Faso. In subsequent rainy and dry seasons blood samples were collected to assess the parasite prevalence, species, density and clinical parameters. In total, 1,767 children and adults were examined and compared to a baseline collected in 2000.Plasmodium malariae infections (mono- and co-infections) decreased parallel to the overall parasite prevalence from 13.4% in 2000 to 2.1%, 4.1% and 4.7% in 2009–2011 (p <0.001). Comparing parasite-positive subjects from the rainy season versus dry season, the risk of fever was significantly reduced in the dry season adjusting for parasite density (grouped) and age group.The microscopical parasite prevalence  measured over the rainy seasons decreased significantly from 78.9% (2000) to 58.4%, 55.9% and 49.3%, respectively . The frequency of P. malariae infections (both mono-infections and co-infections). Asymptomatic parasitaemia was more frequent in the dry season even adjusting for parasite density and age group in a multivariate regression. Possible reasons for this observation include the existence of less pathogenic Plasmodium falciparum genotypes prevailing in the dry season, or the effect of a reduced incidence density during the dry season.The results of this study suggest a decline of malaria transmission over the rainy seasons between 2000 and 2009–2011 in the region of Nouna, Burkina Faso. The decreased transmission intensity was associated with lower prevalence of  Malaria is one of the major health problems in sub-Saharan Africa (SSA) with approximately 174 million cases and 655,000 deaths per year, which is >80% of cases and >90% of malaria deaths worldwide . A numbeMalaria transmission and morbidity has been reported to have declined in areas of East Africa, which is assumed to be at least partly a result of the up-scaling of interventions  and distribution of insecticide-treated bed nets (ITNs) -10. WithThe epidemiology of asymptomatic malaria in different transmission settings is attracting increasing attention, because asymptomatic individuals are still able to produce gametocytes and therefore provide the reservoir for transmission ,15. The Plasmodium species are common in malaria-endemic areas and it has been proposed that interaction between different species may influence the epidemiology and clinical presentation of malaria [Plasmodium species by microscopy and PCR to monitor the variation between the different seasons over time.Infections with multiple  malaria . HoweverThe study was conducted to evaluate the levels of anti-malarial drug resistance on a molecular basis in Bourasso village, Kossi District, Burkina Faso, approximately 30 km from the district town Nouna. The village is located within the area of the Nouna Health and Demographic Surveillance System (HDSS), which included almost 80,000 individuals under constant demographic surveillance. The HDSS population was used as the sample frame for various epidemiological and clinical studies ,6. The uA random list of all households in the village was generated using the data from the HDSS. All household members of the first 50–100 households were invited to participate until the target of ~250 participants was reached (inclusion criterion above six months of age). Sample size was determined with the aim of ~50 parasite positive isolates available for drug resistance testing per cycle. For the dry season an overall parasite prevalence of ~15% (no previous data available) was assumed, which later had to be updated. The assumed parasite prevalence for the rainy season was 80% as it was reported in previous studies .Plasmodium parasites were treated with amodiaquine-artesunate. The blood sample was divided into blood for culture and serum for pharmacological analysis of anti-malarial drugs, then frozen and transported to Heidelberg for further experiments. Ethical approval was obtained by the ethical review board of Heidelberg University Hospital and the Institutional Review Board in Nouna.Written informed consent was taken from all patients prior to enrolment. Anthropometry for children was performed by skilled health care personnel. Each patient was examined by a physician including recent history of drugs taken. A blood sample (maximum 5 ml) was taken by a skilled provider. Thick and thin blood smears were prepared and parasite density counted against 200 WBC [Plasmodium species were analysed by microscopy and species-specific nested-PCR [Plasmodium falciparum was the basis for the calculated PCR prevalence.The genomic DNA was extracted from the filter papers using the Chelex-100 method . The difsted-PCR , the posGraphs were created with Sigma-Plot 11 . Statistical analysis was performed using STATA 11 program . A t-test for proportions was used to assess the significance of differences between parasite prevalences and species distributions in dry and wet season of this study. A Mann–Whitney Rank Sum Test was carried out to compare the parasite densities excluding all zero counts. To evaluate the joint influence of season, age, and parasite density on symptomatic disease/fever a logistic regression analysis was performed.During the first cross-sectional study in 2000 all inhabitants of the village Bourasso were included, in total 1,561 individuals . In the Parasite prevalence was evaluated by microscopic examination of thin and thick blood smear at four time points at the end of the rainy season  and three time points at the end of the dry season . In the survey in October 2000, 78.9% of the individuals were parasite-positive by microscopy and 92.0% by PCR. The results obtained for the years from 2009 to 2012 indicate that the overall parasite prevalence has significantly decreased , where the prevalence for In the dry season, between April 2010 and April 2012  the parasite prevalence did not show marked variation with values between 29.6% and 32.6% by microscopy and 34.6% and 39.3% by PCR.Plasmodium infections differed markedly between the hot dry season and the rainy season. The pooled estimate for the rainy seasons 2009–2011 was calculated at 55.4% (95% CI: 51.9-58.6%), as opposed to the pooled estimate from the dry seasons 2010–2012 with 30.6% .As expected the parasite prevalence for P. falciparum is the most prevalent Plasmodium species (>95% of infections) in this region. Besides P. falciparum, Plasmodium malariae  and P. ovale (0.3%) were detected in microscopy. Both these species were detected as single infections or co-infections with P. falciparum . In total 85.7% of the P. malariae or P. ovale infections were found in children less than 15 years. Most of the non-falciparum infections had low parasites densities, so that only 23.8% of the isolates had counts of more than 1,000 parasites/μl.The distribution of malaria species differed between the rain and dry season. Infections or co-infections with P. malariae infections from 13.4% in 2000 to 2.1%, 4.1% and 4.7% in 2009–2011. Comparing the estimate from 2000 with the pooled estimate from 2009-2011 the t-test for proportions is statistically significant (p <0.001). Plasmodium ovale was only present in a small number of blood smears of October 2000 (0.7%) and 2010 (1.4%) , whereas in the dry season the number of asymptomatic individuals was higher at an average of about 97%  with microscopically confirmed Symptomatic infections with high parasite density  were more likely to be observed in the rainy season, where 51.3% (59/115) of the symptomatic infections exceeded 1,000 parasites/μl Table . During The regression analysis revealed a significantly increased risk of fever in univariate logistic regression for patients in the rainy season as opposed to the dry season , age group, and cough. Other than dry season, high age and low parasite density are the best predictors for a reduced risk of fever, thus for being an asymptomatic parasite carrier. The OR for parasite density decreased in the multivariate regression compared to the univariate regression - probably due to adjusting for age as a correlate of acquired semi-immunity to malaria.The data of this study showed a significant decline in parasite prevalence between the rainy season of 2000 and 2009–2011. A marked reduction of malaria incidence was described for countries in East Africa like Tanzania or Kenya -9,23. HoThe reduced parasite prevalence over time was most pronounced in the group of children under five years of age - which might indeed be linked to the increased bed-net coverage. In children between five and 14 years of age the findings with regard to reduced parasite prevalence over time are not as pronounced, whereas the difference is significant again in older age. One hypothesis could be that this effect is associated with the age-specific role of immune mechanisms developing in populations exposed to malaria. The transmission decline if confirmed would also have an impact on acquisition of naturally acquired (semi-) immunity and consequently also ratio of symptomatic disease to asymptomatic disease ,26. ClosP. malariae decreased significantly during the study period from 2000 to 2009–2011. It was shown before in Malawi, that higher P. falciparum prevalence was linked with higher prevalence of minority species, which could be explained by the raised possibility of species co-transmission [P. malariae infections more substantially than that of P. falciparum[P. malariae infections and co-infections between the baseline from the year 2000 and the results from 2009–2012.The frequency of mixed infections with smission . Furtheralciparum. As ITNsP. malariae is low in the April surveys with only three, zero or one infected patient for the years 2010–2012. These results are supported by similar findings of one study in Burkina Faso, near Bobo-Dioulasso, about 230 km south from the study site Nouna, where prevalence for P. malariae infections was reduced by approximately half in March and May compared to October and December [P. malariae exhibits opposing seasonal fluctuation with P. falciparum[P. malariae during the dry season in those study areas.The observation, that higher transmission is associated with greater species diversity, would also be in accordance to the results of this study for the dry season, which is a low transmission scenario. Prevalence for December . Howeveralciparum, which lP. malariae in this study showed low parasite density levels <1,000 parasites/μl. It is known that the blood stages of this Plasmodium species persist in the blood at low levels for long periods; some believe even lifelong [P. malariae burden, as the microscopy detection limit is low. These results demonstrate that PCR is a more sensitive tool to monitor the minor abundant Plasmodium species. Despite the use of molecular techniques, P. ovale infections in the study were still rare and limited to the high transmission time of the rainy season. Studies in children from Tanzania and Ivory Coast P. malariae were found to reduce fever and symptomatic disease [P. malariae and P. falciparum co-infections 30% of the patients were symptomatic, while in single P. falciparum 37.5% were symptomatic. However, the difference is not significant and the number of co-infected patients is low in the study.Most of the infections and co-infections with lifelong ,32. This disease ,34. The P. falciparum genotypes prevailing in the dry season, or the effect of a reduced incidence density during the dry season. The latter hypothesis would assume that clinical disease is also a cumulative result of a high number of sequential infections.Asymptomatic parasitaemia was more frequent in the dry season even adjusting for parasite density and age group in a multivariate regression. Possible reasons for this observation include the existence of less pathogenic Plasmodium infections during the dry season, a disease outbreak was associated with new genotypes, although it is not clear if this was a result of genetic crossing in the re-emerging Anopheles vector or if the genotype was imported from outside [Evidence from Sudan suggests that chronic infections during the dry season are often multiclonal  and are  outside .The age and sex distribution of the cross-sectional surveys is comparable over the years and also compared to the initial survey, which included the whole population of the village. The proportion of male participants was slightly reduced in some of the surveys, probably due to the fact that male inhabitants were busy with agricultural work. As prevalence and other epidemiological markers are not sex-specific this should not influence the results of this study.The results of this study suggest a decline of malaria transmission over the rainy seasons between 2000 and 2009–2011 in the region of Nouna, Burkina Faso. The parasite prevalence in the dry season was higher than expected with ~ 30% by microscopy throughout the years 2010–2012.P. falciparum was associated with lower prevalence of P. malariae infections.Asymptomatic parasitaemia was more frequent in the dry season even adjusting for parasite density and age group in a multivariate regression. Furthermore, the results of the study could show that reduced parasite prevalence for The authors declare that they have no competing interests.CG participated in the data collection, carried out laboratory investigations, analysed the data, produced figures and tables, and prepared the original draft. HKA carried out laboratory investigations and interpreted the data. GC, BC, and AS coordinated the data collection, contributed to the conception and design of the study, and interpreted the data. ML and HB analysed and interpreted the data. TJ conceived and coordinated the study, analysed the data, and prepared the original draft. All authors contributed to the drafts, read and approved the final manuscript.


Plasmodium falciparum  also expresses parasite-derived proteins on the surface of the iRBC that enable the parasite to cytoadhere to endothelial and other intravascular cells. These RBC modifications are at the root of malaria pathogenesis and, in this ancient disease of man, have formed the epicentre of a genetic ‘battle’ between parasite and host. This review discusses some of the critical modifications of the RBC by the parasite and some of the consequences of these adaptations on disease in the human host, with an emphasis on advances in understanding of the pathogenesis of severe and cerebral malaria (CM) from recent research.Residence in the human erythrocyte is essential for the lifecycle of all Plasmodium that infect man. It is also the phase of the life cycle that causes disease. Although the red blood cell (RBC) is a highly specialized cell for its function of carrying oxygen to and carbon dioxide away from tissues, it is devoid of organelles and lacks any cellular machinery to synthesize new protein. Therefore in order to be able to survive and multiply within the RBC membrane the parasite needs to make many modifications to the infected RBC (iRBC).  Plasmodium falciparum is intracellular, with only brief direct exposure of the ‘naked’ parasite to the immune system. Several candidate vaccines have attempted to use these short periods of vulnerability to produce therapies based on surface antigens of the sporozoite and merozoite forms, with varying success. Some of the difficulty with targeting this phase of the life cycle may be explained by recent work on the proteolytic modification and mobilization of merozoite proteins  (For much of its life in the human host ll (RBC)  through ll (RBC) , with prP. knowlesi. This largely biochemical and immunological characterization was translated to P. falciparum in the mid-1980s . Molecular characterization of PfEMP1 took another 10 years and was greatly facilitated by early efforts at genome sequencing, which identified a number of large, variable open reading frames with characteristics that matched PfEMP1 (rifs) exceeding the repertoire of var genes, which encode PfEMP1.The presence of antigenically diverse proteins on the surface of the iRBC and its implications for antigenic variation were first alluded to by d PfEMP1 , the funP. falciparum disease is widely described and since Marchiafava and Bignami’s report in P. falciparum mature forms of iRBC are virtually absent from the peripheral circulation. However, the other Plasmodium species that can cause severe disease in humans, P. vivax and P. knowlesi, do not cytoadhere to the same extent, with mature forms readily seen in the peripheral circulation, nor do strains that cause murine models of severe malaria. Hence, a long raised question has been whether severe falciparum malaria can occur without sequestration and particularly whether sequestration is required for cerebral malaria (CM); the most serious complication of P. falciparum malaria, which causes coma and seizures and has a mortality of approximately 20%. This question has been elusive because the only definitive way to demonstrate sequestration has been by observing parasites in vessels from tissues from patients who have died. It is also difficult because malaria-infected patients may have other pathologies, such as co-infection with bacteria, and it can difficult to separate the effects of the different components. In this light, a significant advance in understanding malaria pathogenesis is the recognition that retinal changes, detected clinically by opthalmoscopic examination, are a sensitive and specific way of detecting sequestration in cerebral vessels  the discovery that placental malaria is caused by presentation of a specific In addition to endothelial cytoadhesion, iRBC can also increase sequestration by binding to or altering circulating host cells. The most notable example of this is the binding of iRBC to CD36 receptors on platelets that have been activated by either thrombin or tumour necrosis factor (TNF) , 2006.in vivo. Conceptually this molecule has the potential for dual binding and for bridging iRBC to endothelial cells by platelets, as implicated in the report by Platelets also are involved in another mechanism that contributes to sequestration and is thought to be important in malaria pathogenesis; platelet iRBC clumping, in which activated platelets and iRBC bind, forming a large cluster of cells that is unable to pass through narrow microvessels and so becomes sequestered. A captivating link between sequestration and platelet clumping has been proposed by In vitro, more than 20 malaria patient isolates showed binding to FKN-transfected CHO cells, and only mature forms of the parasite were able to bind.Another interesting link by which iRBC can bind to endothelial cells is endothelial membrane fractalkine/CX3CL1 (FKN) . These ‘strings’ of ulVWF unfold under flow conditions and provide a highly adhesive platform for platelets and iRBC binding, via CD36  membrane into the RBC cytoplasm. The details of this process go beyond the scope of this review, however some elegant studies have identified specific sequences in the genes encoding these proteins (known as PEXEL or HT motifs) that identify them for transport across the PV membrane  are small vesicles formed by the budding of membrane from a wide variety of different cells, they are devoid of organelles but maintain the surface receptors of their cell of origin. They have diverse physiological functions but are also involved in the pathogenesis of many conditions, including malaria. Endothelial microparticles, which are pro-coagulant and pro-adhesive have been shown to be increased in Malawian children with CM Fig. 2). Years of striving to understand parasite modifications to the RBC have led to a partial grasp of the nature of the remodelled iRBC and the mechanisms used by the parasite to achieve these alterations. These advances have assisted in the understanding of malaria pathogenesis and in the generation of vaccine candidates. From this there has been some notable progress. A picture of malaria pathogenesis involving the combination of different mechanisms is emerging and potential adjunctive therapies aimed at correcting endothelial dysfunction and at the coagulation-inflammation interface hold some promise and further research into these areas is warranted. However, to date there are still no proven effective adjunctive treatments for severe malaria and mortality remains high. On vaccine development there has also been progress gleaned from better understanding of the parasite and its interactions with the host. After many previous failures more recently some vaccine targets have shown significant protection. Nonetheless, although they do prevent disease, they are far from meeting the levels of efficacy considered acceptable for vaccines to other common infections. To assist in closing the gap in our understanding of this complex pathogen, the advent of more powerful sequencing techniques and the ability to knockout parasite genes allow more systematic assessment of the parasite genome and already have revealed that there are genes for many parasite proteins that have not been recognized previously. This technology may be invaluable in the development of effective treatments and vaccines. However the work described in this review highlights how parasite RBC adaptations are frequently only understood when in the context of the host environment with which they interact, either in disease models or in patients. Therefore translating the findings from this new technology into vaccines and treatment will require the consideration of the phenotypic effects of these genes on the iRBC, not just in isolation, but in the context of human infection and disease.


Understanding the role of local environmental risk factors for malaria in holo-endemic, poverty-stricken settings will be critical to more effectively implement- interventions aimed at eventual elimination. Household-level environmental drivers of malaria risk during the dry season were investigated in rural southern Malawi among children < five years old in two neighbouring rural Traditional Authority (TA) regions dominated by small-scale agriculture.P. falciparum infection status in bivariate analysis were included in generalized linear models, including multivariate logistic regression (MLR) and multi-level multivariate logistic regression (MLLR). Spatial clustering of RDT status, environmental factors, and Pearson residuals from MLR and MLLR were analysed using the Getis-Ord Gi* statistic.Ten villages were randomly selected from TA Sitola (n = 6) and Nsamala (n = 4). Within each village, during June to August 2011, a census was conducted of all households with children under-five and recorded their locations with a geographic position system (GPS) device. At each participating house, a nurse administered a malaria rapid diagnostic test (RDT) to children under five years of age, and a questionnaire to parents. Environmental data were collected for each house, including land cover within 50-m radius. Variables found to be significantly associated with Plasmodium infection by RDT. The MLLR modelled the statistical relationship of Plasmodium positives and household proximity to agriculture (<25-m radius), controlling for the child sex and age (in months), bed net ownership, elevation, and random effects intercepts for village and TA-level unmeasured factors. After controlling for area affects in MLLR, proximity to active agriculture remained a significant predictor of positive RDT result . Mapping of Pearson residuals from MLR showed significant clustering  predominantly within TA Sitola, while residuals from MLLR showed no such clustering.Of 390 children enrolled from six villages in Sitola (n = 162) and four villages in Nsamala (n = 228), 45.6% tested positive (n = 178) for This study provides evidence for significant, dry-season heterogeneity of malaria prevalence strongly linked to peridomestic land use, and particularly of elevated risk associated with nearby crop production. Malaria represents a critical public health issue in a severely resource-constrained country such as Malawi. Nationally, clinically diagnosed cases of malaria increased from ~3.8 million in 2006 to over six million in 2009 . Young cAlthough malaria is considered endemic throughout Malawi, spatial heterogeneity of transmission is not well characterized. Regional prevalence of fever among children under-five varies from 29% in the urban and peri-urban Blantyre to 47% in the highly rural Thyolo, while district-level incidence of under-five malaria varies similarly, from 997 per 1,000 in Machinga, to 715,636, and 542 in adjacent Zomba, Mangochi, and Balaka Districts, respectively. With as much as two-fold difference in incidence between adjacent districts, it is likely that finer scale analysis of environmental heterogeneity will show even greater geographic variability of malaria risk. Urban–rural differences in symptomatic malaria are also present, with rural area under-fives more often experiencing fever within the previous two weeks than did those from urban settings .Plasmodium transmission, while interacting with sociocultural factors commonly associated with malaria disease [Diverse environmental exposures modulate patterns of malaria risk, particularly in rural areas, including land cover, agriculture, elevation, temperature, soil moisture, topography, human and vector population densities, and construction of human dwellings . Some sp disease ,22.Plasmodium falciparum infection in children under five years of age around rural Liwonde, Malawi. These associations were investigated during the dry season when transmission was supposed to be less intense. In particular, this study’s aims included: 1) describe household-scale land cover and environmental risk factors for malaria, and evaluate associations with questionnaire-based demographic, socio-economic status (SES) and malaria knowledge, attitudes and practices (KAP); 2) identify clusters of environmental and social variation, at different spatial scales, that represent increased or decreased malaria risk; and, 3) generate new insights to aid decision-making on malaria risk management and mitigation.This study was designed to investigate relationships among local land use/land cover, social factors and recent Anopheles funestus, Anopheles gambiae sensu stricto, and Anopheles arabiensis[This study was conducted within the catchment area of Machinga District Hospital (MDH), a government-operated hospital which receives patients from villages in Balaka and Machinga Districts along the Shire River of southern Malawi. In addition to the government-operated MDH, smaller private clinics are available in the region, but are beyond the financial means of most residents. Villages in two adjacent Traditional Authorities (TAs), Nsamala pop. ~175,000) and Sitola , were studied  using lists provided by each TA chief. Because of smaller village populations, six villages were selected from TA Sitola, and four from TA Nsamala. Additionally, because TA Nsamala is partly serviced by another district hospital, the village sampling frame was limited to those villages identified by the TA chief as primarily accessing health services at MDH. To conform with Malawian culture and to maximize participation, the research team met with the heads of the two TAs and ten villages, and received permission to work within their domains. The TA and village heads then discussed the study with the residents before the arrival of researchers. Using residents suggested by the village chiefs as guides, a census was conducted of all households with children under five years within each village. For households with more than one under-five child, all children would be tested and treated for malaria if the rapid diagnostic test (RDT) was positive, but a researcher would flip a coin to randomly determine which child would be enrolled in the study. No study households had more than two age-eligible children present at the time of the interview.Recruitment and enrolment was done by the research team, which included a Malawian nurse and translator who received training in confidentiality and research ethics. Due to widespread illiteracy, the study team approached households with verbal informed consent at the time of recruitment, and used thumbprints to indicate consent of illiterate guardians. Copies of the informed consent were available in Chichewa  and in English for any participant who requested one. This study received ethical approval from the University of Michigan Health Sciences and Behavioral Sciences Institutional Review Board and the University of Malawi College of Medicine Institutional Review Board.All households with a child aged four months to five years were eligible for enrolment in the study. A Malawian nurse administered the questionnaire to a parent or guardian of each enrolled child in every participating household. The questionnaire included items on the child’s recent history of and treatment for malarial illness, as well as bed net ownership, insecticide treatment, and use. When the questionnaire respondent was the child’s mother, the nurse asked additional questions on the mother’s malaria illness, treatment and prevention methods during either her most recent or current pregnancy. After completing the questionnaire, and with parental consent, the nurse tested all children between four months and five years of age for malaria using SD Bioline Ag. Pf. RDT, and results were recorded on the questionnaire form. Children who had a positive RDT results were designated as cases, and those with negative RDT results were considered controls. The nurse administered one weight- and age-appropriate dose of artemether/lumefantrine combination therapy to children with positive RDT results, and provided necessary medication and instructions to parents for subsequent doses.While the nurse and a research team member administered the questionnaire and RDTs, two researchers conducted environmental data collection with the assistance of a Malawian translator who was familiar with local agriculture. Each participating household's location  was recorded after averaging a minimum of ten readings by a hand-held Garmin eTrek Venture HC GPS unit to ensure estimated measurement error of <5 m. House structure and materials for windows, roof, and walls were visually characterized. Using the GPS waypoint for the participating house, a member of the study team (LRT) assessed land use/land cover at ≤25 m and 26–50 m of the dwelling, treating the distances from 0–25 m and 26–50 m as discrete distance bands encircling the house. In addition, surveyors counted the number of neighbouring houses within each distance band, as there was often at least one house within 50 m of a participant’s house. Four distinct types of land use/land cover were recorded: bare earth, natural vegetation/fallow land, agriculture, and brickmaking that created large mud pits. Environmental surveyors were blinded as to the malaria status of the enrolled children, and were required to agree about location and types of land cover classification before recording information. Where multiple types of land cover were present, surveyors recorded each of the types. In addition to land cover and household characteristics, the number of neighbouring households present in each band was also recorded. The Malawian translator assisted with distinguishing between actively cultivated crops and harvested/fallow cropland; however, there were no data collected on specific types of crops .X2 and p-value. Variables found to be significantly associated with RDT status at p <0.10 were included in multivariate logistic regression (MLR). These same variables were then analysed in a generalized linear mixed model, specifically multilevel, multivariate, logistic regression (MLLR) using PROC GLIMMIX, which allows for generalized linear mixed models, including random intercepts to account for potentially correlated data due to cluster sampling strategy. As cluster selection was done for geographic units , random intercepts for these variables were included in MLLR analysis to account for group-level effects, which would likely include many unmeasured spatially mediated environmental exposures. For further confirmation that spatial autocorrelation was accounted for in the models, each model was run with the Getis-Ord Gi* z-scores as the dependent factor. All analyses were conducted using SAS 9.2.The outcome variable of interest was case or non-case status as defined by the dichotomous RDT result. Exposure variables include demographic information, bed net ownership and use, household building materials, and proximity to different types of land cover. Because the aims of this study pertained more to the effects of proximity to land cover at a finer scale than is generally assessed and also wished to minimize the number of neighbouring houses within the radius of a given participant’s house, land cover within a 25 m radius was the primary exposure of interest, but results were also calculated for the association of land cover between 25–50 m, and for the entire range of 0–50 m. Bivariate analyses were performed using simple logistic regression, the relevant Wald i* statistic in ArcGIS. Clustering of case and exposure variables was investigated at the level of the entire study area for each TA, and for each village, to assess scale-dependency of effects. In order to assess the role of geographic-dependence on cluster measurements, Pearson residuals from MLLR were joined to GPS locations in ArcGIS, and assessed for clustering with the Gi* statistic.Spatial patterns were explored through ArcGIS 10.0. Geographic data such as elevation, rivers and administrative boundaries, were provided by the Malawi National Statistical Office. GPS point data of household locations were uploaded from the Garmin eTrex Venture HC using a GPS software application . Tabular data were joined to GPS data, and all data were projected into the UTM Zone 36S projection. Clustering of cases and exposure variables was assessed using the local Getis-Ord GPlasmodium infection (“cases”). Additional study participant characteristics are in Table A total of 390 children and households were enrolled, with one child ultimately being excluded from analysis because of missing RDT status, thus analysis was conducted on 389 children Table . The numFrom bivariate Chi-square analysis, cases were found to be 4.2 times more likely than non-cases to live in TA Sitola than TA Nsamala (95% CI 2.74-6.46), and were 2.9 times more likely to live within 25 m of active dry-season agriculture crops (95% CI 1.74-4.92) Table . All hypPlasmodium infection remained relatively unchanged after expanding analysis from crude bivariate (OR = 2.93) to multilevel, multivariate logistic regression (OR = 2.80). This similarly greater risk of infection near active dry-season agriculture was observed even after having controlled for bed net ownership.Using MLR, the associations between positive RDT status and child’s age, sex, bed net ownership, proximity to agriculture (<25 m), household elevation (m), roofing, and wall materials were evaluated Table . Becausei* for all significant variables in bivariate statistical analysis. While the distributions of positive household clusters and of households within 25 m of agriculture overlap significantly, positive and negative clusters of bed net ownership were more widely distributed , nor were entomological parameters related to vector mosquitoes or people’s socio-economic status directly measured. Future research in the area should include these variables. Recent, fine-scale, satellite image data on land use/land cover were also not available. Despite the use of point averaging of household locations, GPS measurements are likely to be accurate to within only 5 m. However, even under circumstances of maximum measurement error, the land cover data remain capable of assessing differences in household-level vegetation patterns. It is further possible that land covers was incorrectly categorized in some instances, however, such errors would likely reduce the strength of observed associations, and would not be biased since researchers assessing land cover were blinded to the RDT status of enrolled children. Thus, these rigorously analysed observations can be said to accurately reflect a surprisingly strong association between dry-season cropping nearby households and malaria risk among residents. This association was observed using different analytic methods and after controlling for possible confounders. Another potential limitation of this study is the reliance on RDT for diagnosis of recent malaria, which, although highly sensitive, have been shown to have low specificity in comparison to microscopy or PCR-based diagnostics ,34. HowePlasmodium infection prevalence was still at >50%. This type of fine-scale modelling of malaria risk allows for more efficient targeting of prevention and intervention programmes. Future applications of these methods could integrate the molecular biology of mosquito vectors and other entomological variables across fine spatial scales, along with high resolution remote sensing data of land cover and other ecological and environmental parameters. Such information may be helpful in understanding both the patterns of risk at scales where transmission is occurring, and the underlying mechanisms that are giving rise to that risk.This study examined the relationships between childhood malaria and household proximity to directly observed, active, dry-season crop production on a scale of <25 m, finer than that of most previous studies. Analysis demonstrated that proximity to agriculture increases the odds of malaria in children under-five by nearly three-fold, even after controlling for age, sex, household elevation, and bed net ownership. The combination of multilevel statistical analysis and spatial analysis in GIS provides a powerful means to describe and analyse environmental risk patterns for malaria. These results suggest that measuring disease transmission parameters may be complicated in spatially heterogeneous regions, and that inferences may depend in important ways on the sampling scale. Since dry seasons are normally considered periods of relatively low malaria incidence, it is worth noting that in some villages The authors declare that they have no competing interests.LRT conceived of the study, participated in study design, collected environmental and survey data, performed the statistical and spatial analyses, and drafted the manuscript. DM participated in study design and coordination, and revised the draft manuscript. DPM participated in study design and critically revised the manuscript. MLW participated in study design, and helped draft the manuscript. All authors read and approved the final manuscript.