The interaction between malaria and under-nutrition is not well elucidated in Ethiopia. The objective of this study was to assess the magnitude of under-nutrition and its correlation with malaria among under-five children in south-west Ethiopia..This cross-sectional study was undertaken during March–February, 2009 as part of the baseline assessment of a cluster randomized trial around Gilgel Gibe Hydroelectric dam, south-west Ethiopia. A total of 2410 under-five children were included for anthropometric measurement and blood investigation for the diagnosis of malaria and anemia. The nutritional status of children was determined using the International Reference Population defined by the U.S National Center for Health Statistics (NCHS). Blood film was used to identify malaria parasite and haemoglobin concentration was determined by Hemo Cue analyzer Significant proportion (40.4%) of under-five children were stunted (height-for-age<−2SD). The prevalence of under-weight was 34.2%. One third and one tenth of the children had anemia and malaria parasite respectively. Older children were more likely to have under-nutrition. There was no association between malaria and under-nutrition. Children who had malaria parasite were 1.5 times more likely to become anaemic compare to children who had no malaria parasite, .In this study, there is no association between malaria and under-nutrition. Children who have malaria are more likely to be anaemic. Malaria prevention and control program should consider nutrition interventions particularly anemia. Malaria and under-nutrition are the two major causes of childhood mortality in sub-Saharan Africa The relationship between malaria and under-nutrition is debatable. Although a number of observations have indicated a deleterious effect of malaria on nutritional status Results on the relationship between malaria and stunting are conflicting. Stunting was the risk factors of malaria in Gambia and Kenya In Ethiopia, where malaria and malnutrition are the major public health problems, little is known about the interaction between the two diseases. The objective of this study was to assess the effect of malaria parasite on the nutritional status of under-five children who are at risk of malaria around Gilgel Gibe hydroelectric dam, south-west Ethiopia.Kebeles (lowest administration unit in Ethiopia) which are located around the reservoir of Gilgel Gibe hydroelectric dam. In the ten Kebeles, there are 52 Gots (villages), 55,000 population and 10800 households.The study was conducted in Gilgel Gibe Field Research Center (GGFRC). This research site is selected since malaria is the major health problem in the area due to ecological disruption Gots (11 interventions and 11 controls) were selected and at least two ITN were distributed to each household in all Gots (villages). All of the heads of the households in the intervention villages were trained about the proper use of Insecticide-treated Nets (ITN). The proper use of ITN has been properly monitored in each household by trained village residents. The trained village residents also monitor the occurrence of malaria in each household in the intervention and control villages. To evaluate the effect of the intervention, mass blood investigation for the diagnosis of malaria and anaemia among all under-five children and pregnant women in the 22 study Gots has been undertaken three times a year. As part of the baseline survey, mass blood investigation and anthropometric measurements was done among 2410 under-five children in the study Gots.This cross-sectional study was undertaken as part of the baseline assessment of a cluster randomized trial. The objective of the trial was to assess the effect of tailored training of the heads of the households on the use of long lasting treated nets (ITN) on the burden of malaria in vulnerable groups. Detail description of the methods of the trial is given elsewhere Weight was measured using UNICEF electronic scale  and height was measured using stadiometer . The nutritional status of children was determined using the International Reference Population defined by the U.S National Center for Health Statistics (NCHS) and Centres for Disease Control and Prevention HemoCue analyzer in the field . Anaemia and moderately severe anaemia were defined as Hb concentrations below <11.0 mg/dL and <7.0 mg/dL, respectively. Malaria was defined as any asexual parasitemia detected on a thick or thin blood smear.For the diagnosis of malaria and anemia, a drop of blood from a finger prick was taken from the children. For malaria parasite identification, thick and thin films were prepared in the field and stained with Giemsa in Jimma Specialized Hospital. Each slide was read by experienced laboratory technicians. Absence of malaria parasite in 200 high power ocular fields of the thick film was considered as negative. Haemoglobin (Hb) concentration was determined using Data were entered into computer, edited, cleaned, and analyzed using SPSS-12 software. To calculate the anthropometric indices, the data was exported to Epi Info 2000 software . Bivariate analysis was done to see the association between socio-demographic variables and malaria with under-nutrition. To control the effect of confounding variables, stepwise logistic regression was done.The study has got ethical clearance from Jimma University and the WHO ethical committee. Written consent was obtained from caretakers of under-five children. Patients with anaemia, under-nutrition and malaria were given treatment by the health extension workers or the nearby health centres.Nearly equal number of male (50.7%) and female (49.3%) children participated in the study. Infants (age less than one year) constituted 21% of the total children and one third of the children were above 47 months. The mean monthly family income of the children's family was 1232 Ethiopian Birr (ETHB) (SD±833). More than half of the family earned monthly income of above 1000 ETHB Table 1.Significant proportion (40.4%) of the children were stunted (height-for-age<−2SD) and 18% of them were severely stunted. The prevalence of under-weight was 34.2%. One hundred and twenty two children (5.1%) were wasted (weight-for-height<−2SD). The prevalence of anemia was 32.4% and one tenth of the children had malaria parasite Table 2.There was no statistically significant association between malaria parasite and under-weight, . After controlling for the effect of potential confounding variables, children above one years of age were more likely to become under-weight as compared to infants. Sex, birth order and family income did not have statistically significant association with under-weight Table 3.As the age of children increased, the prevalence of stunting increased. Compared to boys, girls were less likely to be stunted . The other independent variables such as presence of malaria parasite, family income and birth order were not correlated with stunting Table 4.Children in the age group of 12–23 months were 2.2 times more likely to develop wasting than infants,. There was no statistically significant association between malaria, sex, income and birth order of the children with wasting Table 5.Children who had malaria parasite were 1.5 times more likely to become anaemic as compare to children who had no malaria parasite, . Children above 12 months of age were less likely to become anaemic as compared to infants Table 6.We have assessed the magnitude of under-nutrition, anemia and malaria and the interaction of malaria with under-nutrition and anaemia using a large sample size in under-five children at risk of malaria. The magnitude of stunting and under-weight in this study is almost similar to the findings of the 2005 Ethiopian Demographic Health Survey which revealed that 47% of under-five children were stunted and 38% were under-weight In previous literatures, higher family size One third of the under-five children in this study had anemia which is comparable to the finding of Wolde et al in North Ethiopia Although the study is the first of its kind in Ethiopia to assess the interaction of malaria and malnutrition in under-five children at risk of malaria, it has several limitations. First, we didn't assess level of micronutrients which might have impact on malaria morbidity. Second, several behavioural factors of malnutrition were not assessed. Third, cause effect relationship of under-nutrition and malaria could not be established.In conclusion, under-nutrition and malaria are very common in under-five children around Gilgel Gibe Hydroelectric dam. Malaria was not associated with under-nutrition but strongly correlated with anemia. The Ministry of Health in collaboration with other partners should design nutritional and malaria intervention strategies for under-five children at risk of malaria. Children with malaria should be screened and treated for anemia.


Despite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease.Plasmodium vivax infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe P. vivax infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease.Active and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of Different clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease. Plasmodium vivax infection has been considered for a long time a benign and self-limited disease, mainly when compared to the burden of Plasmodium falciparum infection in African countries [P. vivax is responsible for up to 400 million infections each year, representing the most widespread Plasmodium species [Plasmodium vivax accounts for the majority of malaria cases within the Brazilian Amazon [P. vivax malaria have been rare, and documented almost exclusively by case reports or small case series [P. vivax infection represent a global health menace which needs focused efforts to its resolution.ountries . Neverth species . Plasmodn Amazon , and then Amazon . Historie series -8. Recene series -11. Sevee series . TogetheP. vivax clinical syndromes documented include important thrombocytopaenia [P. vivax infection, data regarding immunological and inflammatory patterns are scarce. In the present report, inflammatory and immunological status of both mild and severe vivax malaria cases were compared in order to explore immunopathological events in this disease.Major severe topaenia ,14, ceretopaenia ,16, and topaenia ,17, hepatopaenia ,19 dysfutopaenia -22. DespP. falciparum infection in the Brazilian Amazon is 23.7% [Plasmodium malariae case detection reaches 10% in Rondônia [A study investigating determinant factors for vivax malaria severity was performed during 2007 in Buritis , a recent urbanized municipality of the Rondônia State, in the south-western part of Brazilian Amazon. Within this region, malaria transmission is unstable, with increased number of cases being detected annually between April to September, and the risk of infection is high , with anRondônia .Active and passive malaria case detections were performed. These included home visits in areas of high disease transmission, and study of individuals who seek care at the diagnostic centers of Brazilian National Foundation of Health (FUNASA), responsible for malaria control in the country. In addition, patients admitted to the Buritis municipal Hospital  presenting clinical signs of mild or complicated malaria were also included in the study. All individuals from fifteen to seventy years, of both sexes, who had been residing in the endemic area for more than six months, were invited to be included in the study. Exclusion criteria were: documented or strong clinical suspecting of viral hepatitis , chronic alcoholism, HIV infection, yellow fever, dengue, leptospirosis, tuberculosis, Hansen's disease, visceral leishmaniasis, documented or referred cancer and/or other chronic degenerative disease, sickle cell trait, and the use of hepatotoxic and immunessupressive drugs. All participants or legal responsible gave written informed consent before entering the study. This study was approved by the Ethical Committee of the São Lucas University, Rondônia, Brazil, for the human subject protocol.P. malariae infection and 16 people infected with P. falciparum (uncomplicated forms) were identified and excluded from the study. Hence, all the volunteers selected were negative for P. falciparum and/or P. malariae infection by both microscopic examination and nested PCR.Individuals were examined and interviewed and their blood samples (20 mL) were collected for serological experiments. In hospitalized participants, two venous blood collections were performed: one at the hospital admission and other seven days after malaria treatment initiation. All malaria diagnoses were performed using two methods. First, patients were screened by thick smear examination using field microscopy and the parasitaemia (parasites/uL) was quantified in positive cases. Further, nested PCR was performed in all whole blood samples to confirm the diagnosis. Two individuals presenting P. vivax infection and remained without fever (axillary temperature >37.8°C) and/or chills, sweats, strong headaches, myalgia, nausea, vomiting, jaundice, asthenia, and arthralgia for 30 days were considered asymptomatic P. vivax-infected cases. Cases showing positive parasitological tests in the presence of any symptom listed above were classified as symptomatic infections. Patients presenting any sign of acute severe organ dysfunction [A total of 219 individuals enrolled in the study. All positive cases were followed for 30 days for the evaluation of malaria symptoms. Individuals who were positive for function  were confunction . Study iThe molecular diagnosis of malaria infection was performed in all subjects using the nested PCR technique described previously ,27, withPlasma levels of IL-10, IFN-gamma, and TNF were measured using the Cytometric Bead Array - CBA  according to the manufacturer's protocol, with all samples running in a single assay. The flow cytometric assay was performed and analysed by a single operator, and standard curves were derived from cytokine standards.Plasma measurements of creatinin, aspartate amino-transferase (AST), alanine amino-transaminase (ALT), total and direct bilirubins, haemoglobin, and CRP were made at the clinical laboratory of Faculdade São Lucas, at the Pharmacy School  and at the Laboratório LPC .Plasmodium presenting malaria-related symptoms (n = 195) or asymptomatic infection (n = 202) composed this sample. In addition, this group was very similar to the one in 2007 with regard to age, gender, time of residence in endemic area and referred previous malaria episodes (data not shown). Optimal threshold plasma values of AST, ALT, total bilirubin, fibrinogen, CRP, and parasitaemia able to discriminate asymptomatic from symptomatic malaria infection were calculated using the Receiver Operator Characteristics (ROC) curves  score was created to standardize a reproducible evaluation of severity in malaria cases. This score was developed by analysing data from another study conducted in 2006 with a sample size of 580 individuals from the Buritis Municipality, Rondônia State, Brazil. This group was composed of non-infected individuals n = 183) and those infected with 83 and ths Figure . Furthers Figure . AdditioData were analyzed using the GraphPad Prism 5.0 (GraphPad Software Inc.). For the ordinal variables, differences between groups were calculated using the non-parametric Kruskal-Wallis test with Dunn's multiple comparisons or trend analysis. The chi-square test was used to compare differences in categorized variables. The correlations were assessed using the Spearman test. Non-linear curve fit was also plotted to illustrate the general trend of the correlations. The statistical analyses used are illustrated in each figure or table. Differences presenting P ≤ 0.05 were considered statistically significant.P. vivax infection were older, had experienced more previous malaria episodes and presented lower parasitaemia than had symptomatic cases . As previously described ,28, indies Table . Patienton Table . Moreove8. As preThe further step was to assess whether the spectrum of vivax malaria clinical presentation could be associated with laboratory parameters of organ dysfunction. In a primary analysis, strong linear trends were identified regarding increasing plasma levels of CRP, serum creatinine, bilirubins and the graduation of disease severity Table . These dP. vivax paroxysms [Furthermore, a possible link between the differences in clinical presentation and laboratory parameters of organ damage and specific patterns of immune responses or inflammatory mediators profile was evaluated. Plasma TNF, which is related to aroxysms , was higaroxysms  and disearoxysms . In the In thirteen patients, who clinically recovered out of nineteen with severe vivax infection, there was an important reduction in the levels of all laboratory parameters of organ damage screened, including plasma CRP (P = 0.002; Figure These investigations suggest that different clinical presentations of vivax malaria infection are strongly associated with a potent activation of pro-inflammatory responses and cytokine imbalance. These results are of utmost importance to improve current knowledge about physiopathological concepts of this serious, widespread disease.The authors declare that they have no competing interests.Wrote the paper: BBA and ARF; Performed the field study and clinical examinations: BBA, SMSN and LMAC; Performed the laboratory experiments and data analysis: BBA and JC; Participated in the design of the study and helped with the manuscript: LMAC and AB; Coordinated the study helped to draft the manuscript: MBN. All authors have read and approved the final version of the manuscript.


Plasmodium vivax, initially mistaken for neonatal sepsis, is described. The correct diagnosis was accidentally done, as congenital malaria had been missed in the initial differential diagnosis.Although malaria in pregnancy can cause very significant neonatal morbidity, congenital malaria is a very rare condition in both endemic and non-endemic areas. A case of congenital malaria by Vivax malaria is the leading species in congenital infections in Europe. This condition should be included in the differential diagnosis of neonatal sepsis even if the mother has no proven malarial episodes during the gestational period. Plasmodium falciparum and Plasmodium vivax infections can cause adverse pregnancy outcomes, including maternal anaemia, low birth-weight due to preterm delivery and foetal growth restriction [P. falciparum [Plasmodium malariae and P. vivax [Both triction . Pregnantriction . On the triction . In endelciparum . In EuroP. vivax , associaP. vivax .P. vivax is presented and its differential diagnosis with other forms of neonatal sepsis are discussed.A case of congenital malaria by A 22-day-old female infant delivered from a Pakistani mother was referred to the Department of Neonatology and Neonatal Intensive Care, Spedali Civili, Brescia, for intermittent fever of one week duration with picks above 39 C°. The mother had a spontaneous, eutocic delivery at 40 week gestation with no abnormal events during the pregnancy period. The newborn birth weight was 2750 gr. She had mixte feeding and, up to three days before admission, she had been in good health.On admission, the infant was in bad general conditions. Physical examination revealed paleness, whining cry, liver and spleen enlargement. Breathing and cardiac functions were normal. Laboratory evaluation showed mild anaemia (haemoglobin Hb: 12.3 g/dl), normal red and white blood cell counts  and severe thrombocytopaenia with platelets (PLT) 14.000/cmm. Other relevant results included mild hyper-bilirubinaemia: 4.14 mg/dl, hyper-transaminasaemia: aspartate aminotransferase 494 U/l and elevated C-reactive protein (CRP) at 73.2 mg/dl (n.v. <5 mg/dl). The remaining biochemistry parameters and metabolic values were within normal limits. Blood and urine cultures were negative. A clinical diagnosis of neonatal sepsis was done and antibiotic treatment consisting of intravenous ampicillin and gentamicin was started.P. vivax trophozoites and few schizonts, with a parasitaemia of 2%. The diagnosis of vivax malaria was eventually confirmed by polymerase chain reaction (PCR).Twenty-four hour after admission, by accidental examination of the peripheral blood to determine the differential white blood cell count, haemoparasites were noted within red blood cells. A thick and thin blood film revealed The infant was administered oral chloroquine 10 mg/kg the first and the second day followed by 5 mg/kg the third day. Four days after treatment peripheral parasitaemia was completely cleared, the white blood cell count increased up to 13.180/mmc, and the CRP fell down to 10.5 mg/dl.The infant was discharged on day five with normal red and white blood cell counts, haemoglobin value, platelets count, biochemical profile, and normal inflammatory index (CRP < 5 mg/dl). No recrudescence was seen after six months of follow up.The history of the mother was re-evaluated. She was a primigravida. She reported one clinical malaria attack in August 2008 (before becoming pregnant) during a short stay in Pakistan to visit relatives and friends. She reportedly received treatment with sulphadoxine-pyrimethamine. In May 2009, when she was at 32 week gestation she had been admitted to hospital in Italy for fever, anaemia  and thrombocytopaenia (PLT 97.000/mmc). She received a diagnosis of urinary tract infection, was treated with amoxicillin/clavulanic acid, and she was discharged after four days of hospital stay.The mother was re-evaluated at the light of the newborn diagnosis. She had no clinical symptoms of malaria and her blood smear thick film was negative for malaria parasites. No antimalarial drugs for either the haematic or liver stages were administered.This case shows that the diagnosis of congenital malaria should be considered as an important differential diagnosis of neonatal sepsis in infants who are born from mothers coming from malaria endemic countries with or without a history of malarial disease during pregnancy. The epidemiological concept of malaria exposure is of obvious paramount importance in this context. However, a proven history of malaria episodes of the mother during the gestational period is not essential: the episode may be mild enough not to require medical attention, or, as it most likely occurred in the case described, may go undiagnosed during a careless diagnostic procedure.Postulated mechanisms for congenital transmission of malaria parasites include maternal transfusion into the foetal circulation either at the time of delivery or during pregnancy, direct penetration through the chorionic villi, or penetration through premature separation of placenta . The remP. vivax malaria is reported to be more common in primigravidae than in multigravidae and is usually associated with mild maternal anaemia and increased risk of low birthweight [Congenital malaria can occur despite the absence of any evidence of active malaria infection of the mother during pregnancy. It can be speculated that the mother had a recrudescence episode of vivax malaria during the third trimester, which was mild, resolved spontaneously, and remained undiagnosed. Consistently with the case reported here, thweight . Moreovethweight . In thisthweight ,9.Diagnosis is simple with microscopic examination of blood films, though a clinical suspicion of the disease is required to start an appropriate diagnostic procedure. This report underlines that diagnosis can be achieved by accidental observation of parasites in a blood film, even when the diagnosis had not been previously suspected.The clinical features of neonatal malaria include anaemia (77%), fever (74%), liver and spleen enlargement (68%), poor feeding/lethargy/irritability and jaundice -12. Sevevivax malaria requires a blood schizonticide, like chloroquine, whereas primaquine is unnecessary as in congenital malaria there is no hepatic stage of the parasite.The treatment of congenital P. vivax than to P. falciparum in non-endemic countries is well established [P. vivax malaria, which allows for more maternal episodes to go undiagnosed and untreated. A potential additional determinant is represented by the contraindication during pregnancy of drugs that can eradicate the liver stage of the parasite, thus increasing the likelihood of late relapses.The higher prevalence of congenital malaria due to ablished ,5. The mP. vivax congenital malaria is a rare condition that should be included in the differential diagnosis of neonatal sepsis in newborns at epidemiological risk. More studies are needed to assess the mechanism of maternal transmission of P. vivax malaria, and its incidence in both endemic and non-endemic countries.In conclusions, The authors declare that they have no competing interests.DPV provided clinical care for the case and wrote the manuscriptGM, MA, and CF provided clinical parasitological consultation and revised the manuscriptSV provided neonatological consultation and revised the manuscriptRA was responsible for diagnostic investigation and revised the manuscriptAll authors read and approved the final manuscript.


Plasmodium falciparum blood stages is mainly antibody mediated [P. falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion [Immunological protection against mediated ,2. Varia evasion . In dist evasion .msp1 and msp2 genotyping. The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group.In the current study, we investigated in distinct clinical groups of malaria patients, the antibody response specifically directed against VSA expressed by parasites isolated from a given clinical presentation, and particularly isolates obtained from cerebral malaria (CM) patients. Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults , patients presenting uncomplicated malaria , cerebral malaria , or pregnancy-associated malaria . Isolates were tested for their clonality by UM antibody levels were higher than VSACM antibody levels (Figure CM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar during the infection and one month later (Figure The levels of clonality were similar in isolates from all clinical origins. In healthy adults and children presenting UM, VSAs Figure . In bothr Figure .CM epitopes was shown but does not necessarily involve prevalent epitopes. Prevalence is more probably due to a fine balance between transmission intensity, antibody repertoire and environmental factors.The existence of shared VSA


Plasmodium falciparum infection are asymptomatic. This study aim to characterize the clinical presentation of malaria in African pregnant women and to evaluate the adequacy of case management based on clinical complaints.There is a widespread notion, based on limited information, that in areas of stable malaria transmission most pregnant women with A hospital-based descriptive study between August 2003 and November 2005 was conducted at the maternity clinic of a rural hospital in Mozambique. All women attending the maternity clinic were invited to participate. A total of 2,330 women made 3,437 eligible visits, 3129 were analysed, the remainder were excluded because diagnostic results were unavailable or they were repeat visits. Women gave a standardized clinical history and had a medical exam. Malaria parasitaemia and haematocrit in capillary blood was determined for all women with signs or symptoms compatible with malaria including: presence and history of fever, arthromyalgias, headache, history of convulsions and pallor. Outcome measure was association of malaria symptoms or signs with positive blood slide for malaria parasitaemia.In 77.4% of visits pregnant women had symptoms suggestive of malaria; 23% (708/3129) were in the first trimester. Malaria parasitaemia was confirmed in 26.9% (842/3129) of visits. Headache, arthromyalgias and history of fever were the most common symptoms  presented, but their positive predictive values for malaria parasitaemia were low .Symptoms suggestive of malaria were very frequent among pregnant women attending a rural maternity clinic in an area of stable malaria transmission. However, less than a third of them were parasitaemic. In the absence of microscopy or rapid diagnostic tests, a large proportion of women, including those in the first trimester of gestation, would be unnecessarily receiving antimalarial drugs, often those with unknown safety profiles for pregnancy. Accessibility to malaria diagnostic tools needs to be improved for pregnant women and drugs with a safety profile in all gestational ages are urgently needed. Plasmodium falciparum is predominant [Each year 25 million African women become pregnant in malaria endemic areas . In mostdominant ,4. Malardominant . Despitedominant . Howeverdominant ,11. ThisAnother widely accepted assumption is that women rarely present with malaria-related clinical complaints during the first trimester of pregnancy. As chloroquine (CQ) is being phased out to treat uncomplicated malaria, little attention has been paid to ensuring the availability of antimalarial drugs that could be safely administered during this critical period of pregnancy. The current recommendation for the treatment of malaria episodes in the first trimester is oral quinine for seven days . HoweverA hospital-based descriptive study was carried out in rural Mozambique to document the frequency that pregnant women present with clinical complaints suggestive of malaria and to assess their parasitological confirmation. The study aimed to characterize the clinical presentation of malaria in African pregnant women and to understand the extent that current case management based on clinical complaints is an adequate strategy in pregnancy, especially in the context of new, less safe and more expensive antimalarial drugs.The study was conducted in Manhiça , a rural area in southern Mozambique. The characteristics of the area have been described in detail elsewhere . The areP. falciparum. Anopheles funestus is the main vector, and the estimated entomological inoculation rate for 2002 was 38 infective bites per person per year [Perennial malaria transmission with pronounced seasonality is mostly attributable to per year . In Manhper year . Previouper year . Accordiper year .The current Mozambican policy for malaria prevention in pregnancy relies on ITN use and, more recently, intermittent preventive treatment (IPTp) with SP. However, at the time of the study, none of these measures were recommended yet. Malaria control during pregnancy relied solely on presumptive treatment of clinical episodes with CQ or SP in the first and subsequent trimesters for uncomplicated malaria, respectively, and with parenteral quinine for severe malaria.The study protocol was approved by the National Mozambican Ethics Committee and the Hospital Clinic of Barcelona Ethics Review Committee.P. falciparum parasitaemia and haematocrit was collected if women reported at least one pre-defined clinical criteria suggestive of malaria. The clinical criteria included: axillary temperature ≥ 37.5°C, reported history of fever in the last 24 hours, pallor, arthromyalgias, headache and/or history of convulsions. Project staff was available 24 hours a day to identify women and to ensure adequate documentation and clinical management. Women with a blood slide positive for malaria were treated with SP plus CQ in cases of non-complicated malaria . Oral quinine for 7 days was given as the second line treatment. Women with complicated malaria were admitted to the maternity ward and treated with parenteral quinine followed by SP. Anaemia was treated following national guidelines with oral ferrous sulphate and folic acid for one month.Between August 2003 and November 2005, a morbidity surveillance system was established at the MHC maternity clinic, as a passive case detection system, for all women  attending this clinic with clinical complaints . During the visit, after verbal informed consent was obtained, the maternity staff recorded the clinical and demographic information onto a standardized questionnaire. Health staff recorded information on age, parity, gestational age, axillary temperature, and signs and symptoms focused on malaria. A capillary blood sample for quantifying P. falciparum asexual stages according to standard, quality-controlled procedures [Duplicate thick and thin blood films were Giemsa-stained and read to determine parasite species and density of ocedures . The hae2 or Fisher's exact test. To evaluate the association between the presence or history of fever, headache, arthromyalgias, convulsions, pallor, age, parity, gestational age, number of previous malaria episodes and season with parasitaemia, multivariate logistic regression models were used. Covariates were selected using a stepwise methodology with a p value of 0.10 for removal and 0.05 for addition to the model. The stratum specific significance tests were yielded by Wald test. Missing values were coded as such and excluded from analysis.Double data entry, validation and cleaning were done using Microsoft Visual FoxPro 5.0, and statistical analysis was performed using STATA.8.2 . Differences between proportions were compared using the XP. falciparum parasitaemia of any density and signs and/or symptoms suggestive of malaria , but without severe anaemia, history of convulsions or high-density parasitaemia. For the purpose of the analysis, the duration of any single malaria episode was estimated as 28 days, in order to distinguish between visits on the same episode and a new one. Moderate anaemia in the pregnant woman was defined as a PCV (packed cell volume) between 21% and 32%. Severe anaemia was defined as a PCV between 16% and 20%, and very severe anaemia as a PCV lower than 16%.A malaria episode was defined as a During the study period 4957 visits were made to the maternity clinic Figure . Of themDemographic characteristics of visits to the maternity clinic are shown in Table P. falciparum parasitaemia and anaemia in the 3,129 visits analysed. Malaria parasites were found in 26.9%  of the visits while overall anaemia was observed in 56.1% . Severe or very severe anaemia were found in 3.1%  of the visits.Table The frequency of visits to the maternity clinic is shown in Table In both the univariate and multivariate analyses, younger age, primigravidae, second and third trimester of gestational age and wet season were all statistically significantly associated with an increased risk of clinical malaria Table . Having P. falciparum parasitaemia. Generally, all signs and symptom had low predictive values (less than 40%) with the exception of current fever (PPV 50% 95% CI 45–56%). However, the prevalence of this sign was low (12%). The association of signs and symptoms with malaria parasitaemia did not vary when the analysis was done by gestational age (data not shown). Combining arthromyalgias, headache and fever or history of fever did not improve the positive predictive value of parasitaemia (PPV 35% 95%CI 33–37%).Table In this rural area of Mozambique the majority (77.4%) of pregnant women attending the maternity clinic with clinical complaints had signs or symptoms suggestive of malaria. However, less than a third (26.9%) of cases had microscopically confirmed parasitaemia. Nearly a quarter of the clinic visits with an illness indicative of malaria were made by women in their first trimester of pregnancy. These findings are relevant given the limited resources dedicated to healthcare in rural Africa, because the majority of pregnant women do not have access to parasitological diagnosis, and they usually receive presumptive treatment, leading to unnecessary drug exposure.Primigravidae women made fewer visits to the maternity clinic than other parities. This may be due to sociological factors such as awareness of the risks of being sick during pregnancy or lower social capacity for decision-making. There were more women attending the clinic during the rainy season, suggesting that malaria has a role in maternal morbidity in this area.P. falciparum parasitaemia, while first trimester was associated with a reduced risk [One out of four women visited the clinic more than once during the same pregnancy, indicating that morbidity may be concentrated in a specific group of individuals. This has been observed in children in relation to malaria . Nearly ced risk ,19-22.Information about the clinical presentation of malaria episodes during pregnancy in stable transmission areas is surprisingly limited or incomplete ,23. MostIn contrast, these findings suggest the opposite. All clinical signs and symptoms evaluated had low positive predictive values. Only the presence of fever had a PPV of 50% (95% CI 45–56%), however its prevalence among women was very low. The majority of women presenting a sign or symptom suggestive of malaria did not have parasitaemia, and clinical complaints had a relatively low value in guiding the treatment of malaria in the absence of parasitological diagnosis. The simultaneous occurrence of the most frequently presented signs and symptoms  did not substantially increase the PPV  for malaria parasitaemia. This study was focused in women who presented with predefined symptoms, and thus it was not possible to calculate their sensitivity and specificity. This may be seen as a limitation of the study. However, the aim was to describe the clinical presentation of malaria parasitaemia in pregnant women attending the maternity clinic because they have clinical complaints. Asymptomatic pregnant women although may also be parasitaemic, are unlikely to attend a health facility except for antenatal routine control visits.Although presumptive malaria treatment has been recommended by WHO as a malaria control measure in endemic areas ,28, thisDue to the absence of safe alternatives to failing drugs, case management of uncomplicated malaria during pregnancy poses one of the greatest challenges to malaria control. Increasing the accessibility to diagnostic tools is needed to identify positive cases and to avoid unnecessary administration of drugs. The use of rapid diagnostic tests (RDTs) is one option to reduce overtreatment in areas where microscopy facilities are absent. RDTs are being increasingly promoted, however, their implementation is still limited by their cost, and they will have distribution and life span issues that need consideration. As we improve the tools for malaria control, finding antimalarials that are safe for pregnant women with no restriction by gestational age remains a public health priority.The study was conceived by CM and this paper drafted by CM and AB. It was conducted by AB and CM with substantial contributions from CR, BS, IM and ES. Data analyses were conducted by LB, and supervised by SS and JA. PA and SM participated in the overall running of the study, and contributed to the interpretation of data and by giving critical revision of the final draft. All authors read and approved the final version.


In a malaria-endemic area of Africa, rabies was an important cause of fatal central nervous system infection, responsible for 14 (10.5%) of 133 cases. Four patients had unusual clinical manifestations, and rabies was only diagnosed postmortem. Three (11.5%) of 26 fatal cases were originally attributed to cerebral malaria. Lyssavirus, family Rhabdoviridae) and occasionally by other related enzootic viruses  infections and cerebral malaria in Malawian children, we investigated fatal cases for rabies virus.Cerebral malaria is a common cause of death in African children. It is diagnosed clinically in comatose patients with acute P. falciparum malaria), for 3 years beginning March 2002, we enrolled children (2 months to 15 years) with suspected CNS infection into a study. Suspected CNS infection was defined as a fever or history of fever and at least 1 of the following ; neck stiffness; photophobia; Kernig sign; tense fontanelle; focal neurologic signs; convulsions. Children were excluded if they had a simple febrile convulsion .At the Queen Elizabeth Central Hospital, Blantyre, Malawi  or aerophobia (a similar response to blowing air across the cheek) were defined clinically as rabies encephalitis. Children with a CSF count of 5–1,000 leukocytes/μL or features of septicemia were treated with broad-spectrum antimicrobial agents. Children with malaria parasitemia were treated with parenteral quinine. For fatal cases, permission was sought for autopsy or supraorbital needle biopsy. Samples of human brain tissue were analyzed for rabies virus by using the fluorescent antibody test  of 789 children died, including 58 (17%) of those with a diagnosis of cerebral malaria and all 10 children who had clinical features of rabies. Consent to perform autopsies or postmortem examination of needle-aspirate samples was requested from the relatives of 82 of the 133 children who died. Twenty-nine (35.4% of those requested) gave consent; 23 for autopsy and 6 for needle samples. This number included 26 children with a diagnosis of cerebral malaria, 2 with a clinical diagnosis of rabies, and 1 with suspected meningitis. Six of these 29 patients who died were positive for rabies virus, including the 2 with typical rabies manifestations (patients 9 and 10), 1 diagnosed with meningitis (patient 12), and 3 who had had a diagnosis of cerebral malaria . Thus, overall 14 (10.5%) of the 133 fatal cases were rabies: 10 diagnosed clinically and 4 that were not diagnosed until postmortem material was studied virologically. Three (11.5%) of 26 patients who died with diagnoses of cerebral malaria and for whom postmortem material was examined actually had rabies encephalitis. Although the clinical manifestations of these 3 children were indistinguishable from those of children with cerebral malaria, histologic examination showed that none of these 3 patients had sequestration of parasitized erythrocytes in cerebral tissue . None of these children, nor the 1 with a diagnosis of meningitis, had hydrophobia or aerophobia, which are characteristic of furious rabies; nor did they have limb pain or paresthesia, which are often reported. The disease progression did have unusual features, however. Patient 6 became comatose, flaccid, and areflexic. Patient 11 had periodic episodes of limb shaking; an electroencephalogram during these events showed generalized slow waves but no seizure activity. Patient 12 behaved oddly when a bag of intravenous fluid was set up, but he was not hydrophobic and readily drank water. None of these 4 children had an obvious history of exposure to a rabid animal, although detailed questioning after admission showed possible exposures  on the basis of history of exposure to a rabid animal and initial clinical manifestations . These ixposures .To determine whether the virus isolates in our study were genotype 1 rabies virus, representative of viruses circulating locally, or from other lyssavirus genotypes that also circulate in Africa , we performed a phylogenetic analysis. This analysis included our clinical isolates, rabies viruses we isolated from rabid animals during the course of the study, and other representative strains. We generated consensus trees and bootstrap values as described previously  of 133 children who died from suspected CNS infections had rabies. For 10 of these children with hydrophobia, aerophobia, or both, rabies was diagnosed clinically , but rabies in 4 children with no hydrophobia or aerophobia was not diagnosed until brain material was examined. In 3 of these 4 children, cerebral malaria had been diagnosed. Hypotonia and areflexia developed in 2 children, which might have been a clue that rabies was involved, but these conditions have also been reported in cerebral malaria (Ours was a hospital-based study. The number of rabies cases in the community is likely to be much higher. In some parts of Africa, up to 100 rabies cases are estimated to occur for each 1 officially reported (In summary, rabies is an important cause of death in children in Malawi, including some for whom cerebral malaria had been diagnosed. Although these patients had unusual disease progression for cerebral malaria, only by examining postmortem material was the diagnosis of rabies made. Rabies virus should be included in the list of pathogens to consider before diagnosing cerebral malaria, especially in fatal cases with hypotonia and areflexia. Attempts should be made to get tissue for diagnosis, e.g., a nuchal skin biopsy, saliva, or CSF antemortem, or a brain sample postmortem.


P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses.Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of  It is widely perceived that immunity to malaria is short-lived, rendering people susceptible to repeated malaria infections. However, there have been very few studies on “memory” responses, how the human immune system recognizes previously encountered malaria parasites. In particular, very little is known about the durability of malaria-specific B cells and antibodies. The aim of this study was to investigate the induction and maintenance of B cell memory responses to malaria parasites in a region of Thailand where people become infected with malaria, but where the levels of malaria transmission are so low that repeated infection is uncommon. From hospital records we were able to identify people who either had been infected with malaria over the past 6 years and/or had never been infected. Blood samples were collected on four separate occasions over a period of one year and analysed by microscopy and PCR for presence of malaria parasites and by ELISA and ELISPOT for anti malarial antibodies and malaria-specific memory B cells. We found that, in a significant proportion of individuals, malaria infection results in the generation of antibodies and the establishment of populations of memory B cells against malaria parasites, which were very stably maintained over time despite the lack of any evidence of malaria reinfection. Contrary to the widely held idea that memory to malaria is suboptimally induced, our data demonstrate that B cell responses to malaria can be maintained for many years after a malaria infection and indicate that there is no inherent reason why malaria vaccines should not also induce long-lasting protection against malaria. Plasmodium, P. falciparum and P. vivax, remains an important cause of mortality and morbidity in many parts of the world. Development of a vaccine against malaria has proven challenging due to the complex nature of the parasite and to the difficulty in correlating naturally-acquired immune responses with clinical immunity. While immunity against some of the severe clinical symptoms may be achieved quite rapidly, following perhaps as few as one or two infections Malaria, a parasitic disease of humans caused predominantly by two species of et al+IgD− memory B cell population and the transitional CD10+CD19+ B cell population, following an episode of acute malaria in African children but this study lacked any analysis of the specificity of B cell responses as well as any long term follow up to ascertain the duration of the response.Antibodies are a crucial component of naturally acquired protective immunity against blood stage malaria with roles that may include inhibition of merozoite invasion into new red blood cells (RBCs), blocking cytoadherence of infected RBCs (iRBCs) to endothelial cells, and enhancing phagocytic activity of monocytes and macrophages  clinical episodes of Malaria-specific humoral immune responses of 93, HIV negative Thai adults were studied . IndividP. falciparum, P. vivax or both parasite species, as well as those who recalled a previous infection and were seropositive to P. falciparum schizont extract (PfSE) but for whom hospital records could not be found, were designated as “previously malaria infected” . In this group, 21 subjects (45.7%) reported at least one episode of infection with P. falciparum, 14 (30.4%) reported at least one episode of infection with P. vivax and 6 (13.0%) reported infection with both species in the past 6 years. The frequency of malaria infections within the six years prior to recruitment varied from 1–3 episodes . Five Rural 2 subjects (10.9%) were strongly seropositive to PfSE and recalled prior malaria episodes, but no documentary evidence of these malaria episodes was found. The time since last documented malaria infections prior to recruitment varied from 4–58 (21.2±12.9) months for those known to have been infected with P. falciparum and 7–39 (20.6±10.1) months for those known to have been infected with P. vivax.Muang Na residents who had had one or more fully documented episodes of infection with Of the 76 rural subjects included at enrolment, 49 (64.5%) were seen again at 3 months, 44 (57.9%) at 6 months and 51 (67.1%) at 12 months. All city individuals were re-sampled 3 months later.P. falciparum or P. vivax - as determined by blood film examination and PCR - at any visit. However, one of 76 rural subjects demonstrated a significant increase in antibody titre during the study  suggesting that this individual may have experienced a recent malaria infection, even though they did not report being ill. The three groups did not differ significantly by age or sex.None of the subjects were infected with Antibody levels against tetanus toxoid (TT) were measured by indirect ELISA. There was no significant difference among the groups in the overall levels of antibodies to TT  but onlyRelative antibody titres to PfSE were measured, at the time of enrolment and at subsequent follow-up. Among the Rural 1 population, 4 (13.3%) individuals had antibody responses above the cut-off, indicating that they had, in fact, been exposed to malaria . Among RP. falciparum only or infected with P. vivax only  who had blood collected at the beginning and at the end of the study (n = 51), 19 (37.3%) were seropositive for PfSE at the time of recruitment and all of these remained positive 12 months later . The ind19, PfMSP-2 and PfCSP were examined by indirect ELISA. PfAMA-1, PfMSP-119 and PfMSP-2 are antigens of blood stage merozoites. PfCSP is the major surface protein on the surface of sporozoites, the infective stage of the malaria parasite. All of these antigens are key P. falciparum vaccine candidates. City naive subjects were seronegative to all P. falciparum antigens tested  group were 17 (37%), 22 (48%), 7 (15%) and 4 (9%), respectively. Again, seropositivity to individual malaria antigens was not significantly correlated (data not shown) and, overall, 30 (65%) Rural 2 subjects were seropositive to one or more P. falciparum antigens (schizont extract and/or recombinant proteins). The proportion of individuals in the Rural 2 and Rural 1 groups who were seropositive for recombinant malaria antigens was not significantly different, except that a higher proportion of Rural 2 were seropositive for PfMSP-119 .The frequencies of antibody responses to PfAMA-1, PfMSP-119 and PfMSP-2 were significantly higher among Rural 2 subjects than among Rural 1 subjects but among the Rural 2 group the levels of antibodies to individual P. falciparum antigens were not different between previously P. falciparum- and P. vivax- infected subjects (data not shown). Moreover, the titres of antibodies against P. falciparum antigens in some Rural 2 subjects were at least as high as those of the positive control of pooled adult African sera. There was no detectable antibody response to the carrier proteins used in the production of recombinant antigens (data not shown).The titres of antibodies to PfMSP-1P. vivax had antibodies to P. falciparum antigens. Of the 14 individuals with recorded P. vivax infections but no recorded P. falciparum infections, 8 (57%) had antibodies to at least one P. falciparum antigen. This is consistent with results of previous studies P. falciparum or cross-reactivity of antibodies to the two parasite species Several subjects reporting prior infection only with 19 and PfMSP-2 were significantly correlated with titres of anti-PfSE Abs (data not shown). No correlations between age and the antibody titres against individual malaria antigens were found (data not shown).As shown in P. vivax antigens, PvAMA-1, PvMSP119 and PvDBP by ELISA, all of which are P. vivax blood stage antigens and are key vaccine candidates. City naïve subjects were seronegative to all P. vivax antigens  Rural 2 subjects were seropositive to one or more P. vivax recombinant antigens. Of the 20 subjects known to have been previously infected with P. vivax , 5 (25%) were seropositive to one or more P. vivax antigens and 15 (75%) were seronegative. The proportion of subjects who were seropositive to P. vivax antigens did not differ significantly between the Rural 1 and Rural 2 groups. Similarly, no significant differences in anti-P. vivax antibody titres were observed between the Rural 1 and Rural 2 groups, although the power of this analysis was poor due to the very low numbers of seropositive subjects. Antibody responses to P. vivax antigens were not different between subjects known to have been infected with P. falciparum- and those known to have been infected with P. vivax (data not shown). Most of the subjects who were seropositive to P. vivax antigens at the time of recruitment and who had samples collected at more than one time point remained seropositive over the course of the study and there was no evidence of declining titres  since the last documented malaria episode (19 n = 12) in a repeated measurements analysis including multiple data points from the same subjects. Subjects known to be infected with P. vivax but not known to have been infected with P. falciparum were not included in this analysis in order to ensure specificity to P. falciparum.To determine the longevity of the antimalarial antibody responses, we analysed the change in concentrations of antibodies to PfSE, PfAMA-1 and PfMSP-1 episode . The hal19 antibody concentrations over time and statistically, these rates could not be distinguished from zero . These analyses suggest that antibody responses to malaria are stably maintained in this population.Mixed-effects regression models revealed very low rates of decline (converted to years) in anti- PfSE, PfAMA-1 and PfMSP-1rom zero . The besWe next enumerated memory B cells to malaria antigens and to TT using a highly sensitive ELISPOT protocol 19  Rural 2 subjects had memory B cells to one or more P. falciparum recombinant antigens.At the time of recruitment, of the 21 Rural 1 subjects whose PBMCs were available, 3 (14.2%), 3 (14.2%), and 1 (4.8%) subjects had memory B cells specific to PfAMA-1 , PfMSP-119 , and PfM19 , respect19 . A much 19  had memory B cells specific to one or more P. vivax antigens.None of the 14 Rural 1 subjects tested had detectable memory B cells against PvAMA-1, and only one individual (7%) had detectable memory B cells to PvMSP-119 . However19-specific memory B cells in relation to time since their last documented malaria infection, using mixed-effects regression analysis  as described above. We found that PfAMA-1- and PfMSP-119 specific memory B cells were stably maintained over time  seronegative individuals . We therP. falciparum antigens at the time of recruitment, 7 (21%), 8 (24%) 2 (6%) and 0 (0%) had both circulating antibody and memory B cells to PfAMA-1, PfMSP-119, PfMSP-2 and PfCSP respectively . Among the 33 Rural 2 subjects for whom we had both antibody data and memory B cell responses to ectively . Four  and 2 (8%) individuals had both antibody and memory B cells against PvAMA-1 and PvMSP-119 respectively Different individuals had different patterns of antibody and memory B cell responses to the various malaria antigens. Antibodies are critical in protection against blood stage malaria infection through numerous, diverse mechanisms P. chabaudi infection leads to expansion of short-lived, immature B220+ splenic plasma cells however secondary infection is accompanied by apparently normal emergence of a larger population of fully mature , terminally-differentiated B220- plasma cells in the bone marrow One explanation for these observations might be that short-lived antibody responses are the result of induction of short-lived, but not long-lived, plasma cells following acute malaria infection. In murine models of malaria infection, primary P. falciparum and P. vivax are endemic but transmission is kept at extremely low levels by an assiduous malaria surveillance and control programme in which all detected infections are recorded and effectively treated In our study area, both Nevertheless, some of the rural village residents with no record or recollection of malaria infection in the past 6 years  appeared to have experienced malaria infections at some time in their lives as shown by seropositivity to malaria antigens in ELISA and positive B cell ELISPOTs. Given the lack of evidence for acquired protective immunity in this population, and since we found no evidence of asymptomatic malaria infections, it is unlikely that these individuals had experienced undiagnosed malaria infections and thus the presence of antibodies and B cell memory responses in this group suggests that anti-malarial seropositivity can be maintained for many years in the absence of reinfection.P. falciparum schizont extract, PfMSP-119 and PfAMA-1 was in fact higher than for tetanus. Thus, despite the fact that the anti-tetanus response is likely induced by a very potent vaccine and boosted by environmental exposure or revaccination (which is routinely given during pregnancy), humoral immune responses to tetanus do not appear to be particularly more robust than those induced by infrequent natural exposure to malaria. Moreover, frequencies of malaria-specific memory B cells in Thai adults were similar to frequencies of diphtheria-specific memory B cells in UK adults . Therefore, frequencies of malaria-specific memory B cells seem to be of the same order of magnitude as responses to commonly used vaccine antigens Overall, the prevalence and magnitude of antimalarial antibody and memory B cell responses compared favourably with the anti-tetanus responses. Although the frequencies of tetanus-specific memory B cells tended to be somewhat higher than the frequencies of malaria-specific memory B cells, the prevalence of antibodies to the 19 over periods of more than 5 years since the last known malaria infection. One reason for this discrepancy may be that previous studies have characterised the decay of the antibody response in the first few days or weeks after resolution of an acute malaria infection As the time since last detected malaria infection was known for the Rural 2 group we were able to obtain estimates of the rate of decline of both serum antibodies and circulating memory B cells. Importantly, and in marked contrast to previously published data from African children P. chabaudi mouse model that malaria infection induces long-lived antibody responses as well as memory B cells Our best estimates of the half-life of this maintenance phase of antibody responses to malaria antigens ranged from ∼5 to ∼16 years, putting them in the same range as the half-lives recently estimated for nonreplicating antigens such as tetanus or diptheria toxoids 19. Given that everyone in the study had experienced their most recent malaria infection at least 4 months before recruitment and that the genotypes of their infecting parasites are unknown, we cannot tell whether these seronegative individuals had completely failed to make a humoral response to malaria, whether they had made antibodies to polymorphic epitopes that did not cross-react with the antigens used in our assays or whether they had developed only very short-lived responses. In a previous study, in a higher malaria transmission area, very short-lived antibody responses to malaria were particularly associated with younger individuals who (presumably) had had the fewest number of malaria infections However, it is important to note that 11 subjects (24%) who were known to have been infected with malaria had no detectable circulating memory B cells and/or antibodies and less than 50% of subjects tested had either antibodies or circulating memory B cells to PfMSP-2, PfCSP, PvAMA-1 or PvMSP-1in situ. However, in the absence of prolonged antigenic stimulation, plasmablasts express another chemokine receptor CXCR4 allowing them to migrate to the bone marrow Since the pharmacological half-life of a human IgG molecule is around 21 days Immediately after malaria immunisation in humans the frequency of antigen-specific memory B cells is positively correlated with antibody titres 19 did not change during the 12 months of study; this is not surprising since there was no evidence of reinfection of any of the subjects during the follow-up period which might have driven further avidity maturation. However the avidity of anti-PfMSP-119 antibodies was significantly higher among Rural 2 subjects than among Rural 1 individuals, supporting the notion that the Rural 2 population had had more frequent exposure to malaria parasites than the Rural 1 group.High affinity antibodies are expected to play an important role in the humoral immune response. The avidity indices of antibodies against PfAMA-1 and PfMSP-1In summary, we conclude that B cell memory responses to malaria are effectively induced and maintained – in a significant proportion of individuals - in areas of low malaria transmission.  of malaria infections were confirmed from the records of the Office of Vector Borne Disease Control in the Department of Communicable Diseases Control at the Ministry of Public Health, which maintains detailed records of all malaria cases detected by active or passive case detection and during periodic population surveys as described in detail elsewhere Subjects were interviewed to ascertain their previous malaria exposure. Residents of Chiang Mai were selected on the basis that they had not travelled to, or lived in, malaria endemic areas. In Muang Na, dates and species  according to the manufacturer's protocol and subjected to nested PCR for As HIV infection may have an effect on immunological parameters, all subjects were tested for HIV infection (presence of anti-HIV antibodies by gel particle agglutination assay) at the time of recruitment and at the end of the study (3 months after recruitment for city subjects and 12 months after recruitment for other groups); subjects received pre- and post-test counselling from trained HIV counsellors and HIV-infected individuals were given access to the National Antiretroviral Programme. Data from HIV-infected subjects were excluded from the analysis.P. falciparum circumsporozoite protein (PfCSP) [(NANP)4] and P. falciparum merozoite surface protein-2 (PfMSP-2) were a gift from J.E. Tongren . The 19kDa fragments of P. falciparum and P. vivax MSP-1 (PfMSP-119 and PvMSP-119) were gifts from A. Holder  and the proteins were expressed as described P. falciparum apical membrane antigen-1 (PfAMA-1) was a gift from R.F. Anders ; the equivalent P.vivax antigen (PvAMA-1) was a gift from B. Farber and A. Thomas . P. vivax duffy binding protein (PvDBP) was a gift from L.H. Carvalho Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil). Since Thai populations are routinely vaccinated with tetanus toxoid (TT), antibody responses to TT were included as a positive control. TT was obtained from the National Institute of Biological Standards and Control . Keyhole limpet haemocyanin (KLH) was from Thermos Fisher Scientific .P. falciparum (3D7 strain were maintained in the laboratory 8 schizont-infected red blood cells (iRBC)/ml and exposed to three freeze/thaw cycles to obtain P. falciparum schizont extract (PfSE).Continuous cultures of 5 iRBC/ml for PfSE, 0.5 µg/ml for Plasmodium-derived antigens and TT) in bicarbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked with PBS containing 1% non-fat milk powder. Diluted plasma samples  were incubated in dubplicate. Plates were subsequently developed with anti-human IgG horseradish peroxidase conjugate  followed by o-phenylenediamine substrate (Sigma). The enzyme reaction was terminated with sulphuric acid (2N) and absorbance was then read at 492 nm on a Spectra MR plate reader (Dynex Technology).Plasma antibody levels were detected by indirect ELISA, as described Antibody levels were determined by comparison to a standard curve  on each plate, as described previously 4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) at RT for 5 minutes. The cells were washed twice with RPMI, resuspended in 10% human AB serum/RPMI (R10 culture medium), counted, adjusted to the required concentration and cryopreserved in 10% dimethylsulfoxide (DMSO)/foetal calf serum.PBMCs were separated from citrated blood by gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences). Contaminating erythrocytes were removed by incubating with lysis buffer , 6 µg/ml CpG 2006 (Qiagen/Operon), and 1/10,000 dilution of Staphylococcus Aureus Cowan (SAC) (Sigma), as previously described 2 at 37°C for 5 days.Cryopreserved PBMCs were quick thawed in a 37°C water bath. The cells were washed twice with warm RPMI, resuspended in R10 culture medium and added at a concentration of 1×102. Cultured PBMCs were recovered from the 24 well culture plates, washed, transferred directly to antigen-coated ELISPOT plates and incubated for 6 hours at 37°C, 5% CO2. After 4 washes with PBS and 4 washes with PBS-T, 100 µl of Biotin-SP-conjugated donkey anti-human IgG (Jackson Immunoresearch) were added to each well and the plates were incubated overnight at 4°C. The plates were washed, 100 µl of alkaline phosphatase-streptavidin (Vector Laboratories) was added and incubated for one hour at room temperature. After three washes with PBS-T and three washes with PBS, 100 µl of 5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium - alkaline phosphatase substrate solution (Vector Laboratories) were added to each well and the reaction was allowed to proceed for 8 minutes before being stopped with distilled water. In vitro restimulated PBMCs incubated overnight with an irrelevant protein, KLH, as well as PBMCs cultured without stimulation and then incubated overnight with malaria antigens were used as negative controls. Since no malaria-specific spots were detected in city naïve individuals, this group was not be used to set a cut-off for positivity. Rather, a positive ELISPOT response was defined when spots were observed in 2 or more replicate wells and where the total number of spots in the antigen-coated wells was at least twice the number observed in the negative control wells.B cell ELISPOT assays were performed as described previously An enzyme immunoassay for determination of antibodies against malaria antigens was carried out as described above. Following the incubation step of sera with antigens, one duplicate set of sera was treated with 4.0 M guanidine dissociating solution  for 10 minutes prior to washing with PBS-T. Avidity indices were calculated as the ratio of the OD of guanidine -treated wells to the OD of the untreated wells.Pf- or Pv-derived antigens, or TT), cut-offs for positive antibody titres were calculated using a mixture model, which assumes that untransformed titres for seropositive and seronegative samples each follow a normal Gaussian distribution To determine whether an individual was seropositive for a particular antigen  and P. vivax (diamond) exposed subjects. Each symbol represents the antibody titre of one individual. Dotted lines show cut-off values calculated from a mixture model as described in Antibody responses against PfSE in (0.03 MB DOC)Click here for additional data file.


Plasmodium falciparum malaria. The data presented herein describes the experience in a malaria-endemic zone in northeastern Venezuela (state of Sucre), where a similar bias between haematocrit and haemoglobin in patients with Plasmodium vivax infection was found. In summary, the relationship between haematocrit and haemoglobin needs to be specifically evaluated according to each particular region or epidemiological setting.It has been recently reported that the standard threefold conversion from haematocrit to haemoglobin underestimates the prevalence of anaemia and low levels of haemoglobin in children living in areas endemic for  It has been a widely accepted assumption that this conversion could be used as an alternative measurement to haemoglobin in malaria studies [Plasmodium falciparum malaria is endemic, but the corresponding haemoglobin levels cannot be derived with an acceptable accuracy using the value three as a conversion factor [Plasmodium vivax infection.In agreement with Carneiro  studies . This isn factor . As a con factor ,4, showiData from one study that have measured haemoglobin and haematocrit was used to assess the reliability of the standard threefold conversion factor. Finger-prick blood samples were collected for determination of anaemia status from 120 patients with malaria aged 4–89 years-old from a prospective survey carried out between 2000 and 2002 in Carupano, Sucre, Venezuela . In thiset al [a priori covariates of age and sex. The differences between both measures were also tested in terms of means comparison using the Student's t test for one-sample, for independent-samples and paired-samples.As described by Carneiro et al , the meaet al , is compet al , adjustir2 = 0.1379, F = 18.87, P < 0.0001) , with a linear trend in the relationship so that more negative differences were seen with increasing haemoglobin levels . The differences between both measures in terms of means comparison for Student's r2 = 0.9526, F = 2372.0, P < 0.0001):Then, the relation between observed haematocrit and observed haemoglobin was modelated Figure   shown that the haemoglobin measurements were lower than corrected observed haemoglobin in 49.2% (59/120), with a linear trend in the relationship significantly more slight than the original  declare that they have no competing interests.AJRM analysed and interpreted the data, and wrote the first draft of the paper. CFP designed and co-ordinated the original studies, contributed to data interpretation and to drafting and revising the manuscript. ES, MA, MV, CP and RC were involved in data collection and critically revised the manuscript. All authors read and approved the final manuscript.


Malaria is a leading cause of disease burden in Uganda, although surprisingly few contemporary, age-stratified data exist on malaria epidemiology in the country. This report presents results from a total population survey of malaria infection and intervention coverage in a rural area of eastern Uganda, with a specific focus on how risk factors differ between demographic groups in this population.Plasmodium species infection. All permanent residents were invited to participate, with blood smears collected from 1,844 individuals aged between six months and 88 years (representing 78% of the population). Demographic, household and socio-economic characteristics were combined with environmental data using a Geographical Information System. Hierarchical models were used to explore patterns of malaria infection and identify individual, household and environmental risk factors.In 2008, a cross-sectional survey was conducted in four contiguous villages in Mulanda, sub-county in Tororo district, eastern Uganda, to investigate the epidemiology and risk factors of Plasmodium, with prevalence highest among 5-9 year olds (63.5%). Thin films from a random sample of 20% of parasite positive participants showed that 94.0% of infections were Plasmodium falciparum and 6.0% were P. malariae; no other species or mixed infections were seen. In total, 68% of households owned at least one mosquito although only 27% of school-aged children reported sleeping under a net the previous night. In multivariate analysis, infection risk was highest amongst children aged 5-9 years and remained high in older children. Risk of infection was lower for those that reported sleeping under a bed net the previous night and living more than 750 m from a rice-growing area. After accounting for clustering within compounds, there was no evidence for an association between infection prevalence and socio-economic status, and no evidence for spatial clustering.Overall, 709 individuals were infected with These findings demonstrate that mosquito net usage remains inadequate and is strongly associated with risk of malaria among school-aged children. Infection risk amongst adults is influenced by proximity to potential mosquito breeding grounds. Taken together, these findings emphasize the importance of increasing net coverage, especially among school-aged children. In Uganda, malaria remains the leading cause of morbidity and mortality, with an estimated 12 million clinical cases treated annually in the public health system alone , most re2. The sub-county was purposively chosen as having a large population, being accessible to a health centre IV and as being representative of an area of high (stable) malaria transmission [Plasmodium falciparum. A 1999 survey among 1-9 year olds in Nagongera, 15 km away from Mulanda, reported a P. falciparum prevalence of 90.6% [P. falciparum bites per year [Anopheles gambiae s.s., and to a lesser extent Anopheles funestus [The study was conducted in four villages in Mulanda sub-county, located in Tororo district, eastern Uganda in 2008. The area is characterized by dry savannah grassland interrupted by bare rocky outcrops and lower lying swamps, although natural vegetation has mostly been replaced by cultivated crops. Average daytime temperature is 27°C, with two rainy seasons (March to May and August to October); annual rainfall is 1,000-1,500 mmper year . The majfunestus . No vectfunestus , and no Between July and December 2008, a complete census and household survey of the entire sub-county was conducted by the study team in order to gather basic demographic and socio-economic information about the target population. Study personnel systematically covered the sub-county on foot to identify and enumerate all households, and invite an adult resident (18 years of age and older) from each household to participate in a brief socio-demographic survey. Using a standardized questionnaire, information was gathered on the age and gender of each resident and socio-economic characteristics including house construction, water and sanitation, and ownership of selected household assets. A resident was defined as a person who intended to sleep primarily at that location for the subsequent six months. In total, 33,167 individuals residing in 6,397 households were enumerated in the sub-county.Household locations were mapped using a hand-held eTrex global positioning system (GPS) receiver . All health care facilities , open and protected water sources and other points of interest  were also geo-located using a GPS. High resolution (0.6 m) QuickBird satellite data were obtained  and used for geo-referencing the location of roads, infrastructure and potential mosquito breeding sites. Geographic data were compiled and maps created using ArcGIS 9.2 . Nearest straight-line distances between residents' compounds and the health centre and potential mosquito breeding sites were calculated in ArcGIS.2 surrounding the health centre IV were selected as geographically and socio-economically representative of the sub-county, with easy accessibility and moderate population density  four villages covering an area of 7.5 kmy Figure . No popuSchistosoma mansoni, using the Kato-Katz technique.Upon enrolment a questionnaire was administered to adults and to mothers (or carers) of children to record demographic and socio-economic characteristics, medical history and mosquito net use. A clinical examination was then performed that included axillary temperature  and height and weight. A rapid diagnostic test for malaria  was performed on all participants with fever (temperature > 37.2°C) or reported history of fever in the previous 24 hours. Those with a positive test but no evidence of severe illness were diagnosed with uncomplicated malaria and treated with a 6-dose regimen of co-artemether  in accordance with national guidelines. Research participants found by the study nurses to be suffering from severe malaria or an illness not covered by the protocol were referred to the local health facility  for diagnosis and management as recommended by national guidelines. Finger-prick  blood samples were obtained to assess haemoglobin using a portable photometer  and to prepare thin and thick blood films. Individuals were also asked to provide stool samples which were examined microscopically for the eggs of intestinal nematodes and P. falciparum and 6.0% were P. malariae; no other species or mixed infections were seen.Blood slides prepared for malaria were declared negative only after examination of 100 high-powered fields by two independent microscopists. Thin films from a sub-sample of 20% of parasite positive participants were read for species identification showing that 94.0% were Plasmodium infection, and ii) Plasmodium parasite density in positive participants. However, analysis of parasite density revealed no additional insights and therefore the focus of the present analysis is on infection prevalence.All data were double-entered and cross-checked using Access (Microsoft). Statistical analyses were carried out using Stata XI software  and WinBUGs version 1.4.1 . Two outcome variables were initially analysed: i) the prevalence of Information on ownership of household assets was used to construct a wealth index for each household using principal component analysis (PCA), using the method of Filmer and Pritchett . VariablPearson's chi-squared test and Fisher's test were used to compare proportions between groups as appropriate. Potential risk factors for prevalence were initially investigated using frequentist logistic regression models. Explanatory variables significant at the 10% significance level were subsequently entered into a Bayesian multivariate logistic regression model, using a process of backwards-stepwise elimination. Between-household variation was taken into account by introducing into the model household-level random effects with an exchangeable correlation structure. Analysis employed a Bayesian Monte Carlo Markov Chain (MCMC) approach, which readily allows the development of random effects models . InferenGeoR. Monte Carlo envelopes  were used to formally assess whether the data was compatible with spatial structure, under the assumption of no correlation [In order to assess global spatial heterogeneity in infection patterns across the whole study site, semi-variograms were generated using the R module relation ,15. SemiThe study protocol was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (#2008-043) Uganda National Council of Science and Technology (#HS 476) and London School of Hygiene and Tropical Medicine Ethics Committee (#5261). Prior to the start of the study, investigators met with elected government representatives and community leaders to inform them of the study and explain the methodology. Verbal consent was obtained for the brief census survey described above. Written informed consent was obtained from all adults and from parents or guardians of minors for participation in the biomedical survey; written assent was also obtained for children aged 13-18 years.Of the 2,474 individuals from 491 households enumerated in census, 116 were no longer living in residence by time of the biomedical survey. A total of 2,358 residents living in 473 households were invited to attend the health post; 2,037 (86.4%) individuals chose to attend and could be unambiguously tied to a single household. Of these, 34 individuals were excluded from enrolment , 58 refused to provide a finger prick, 83 did not provide household socio-economic data and 19 did not provide information on bed net usage. Complete questionnaire and biomedical data was, therefore, available for 1,844 individuals from 438 households . Individual and household characteristics for participants enrolled in the study are summarized in Table Plasmodium spp. Figure Plasmodium spp. parasitaemia stratified by village. Prevalence did not significantly vary between villages : 37.8% in Mulanda Store, 36.6% in Koribudi B, 37.8% in Mulanda Ayago and 42.6% in Koribudi A. Examination of semi-variograms for household prevalence found no evidence of consistent global spatial structure, indicating that on average across the whole study site infection was randomly distributed (data not shown). Prevalence was significantly higher among males than females  and peaked among individuals aged five to nine years (63.5%), falling to 29.1% by 16 years and 12.9% in those over 30 years old  of households owned at least one mosquito net; 45.1% of which were long lasting insecticide nets (LLIN) or had been treated with an insecticide (ITN). Net ownership increased with socio-economic status, ranging from 43.2% any net/17.2% ever-treated net among households of the poorest quintile to 95.0% any net/50.0% ever-treated net among the richest quintile . However, only 9.9% of households owned at least one net per two residents, the Uganda national recommended minimum net coverage, and only 39% of participants reported having slept under a bed net the previous night. In terms of differences by age, net use was initially highest among pre-school children, sharply declining among school-aged children, before rising again across the ages 20-44 years and finally decreasing gradually in older ages; this pattern was similar among females and males , although prevalence in children aged 1-9 years is lower than that reported in 1999 by Talisunu et al for the same age group in a neighbouring population (55.0% vs. 90.6%) [Community-based epidemiological studies serve an important public health role since they provide contemporary estimates of infection prevalence and intervention coverage, and can identify factors whose manipulation could further prevent or control malaria in endemic populations. In Uganda, there are remarkable few recent studies of malaria within communities, with previous detailed descriptions provided by Onori and Jelliffe in the 1960s -20 and s. 90.6%) , it is sMosquito net use was found to be lowest among school-aged children, an observation also reported during individual studies in Tanzania -24, SoutNotable among the results was a lack of association between infection risk and socio-economic factors. This contrasts with previous studies conducted in both rural and peri-urban settings that have reported increased risk of malaria infection in children living in poor housing ,34 and wAn. gambiae, the most likely vector in this region [Infection risk was strongly associated with proximity of households to rice-growing areas, emphasizing how this habitat may provide optimal breeding and resting sites for mosquitoes. There was however little evidence of a consistent second-order spatial pattern occurring over the entire study region as shown by variography. The relationship between malaria infection and distance from potential vector breeding sites has been reported in studies in both rural ,46 and us region ,55; furts region .There are a number of policy implications arising from this study. First, given the protection afforded by sleeping under a mosquito net coupled with the current low usage among school-aged children, there is a clear need to promote net use amongst school-age children. Whilst targeting individual protection to vulnerable groups is an accepted priority, recent evidence does suggest that protecting all community members can yield enhanced benefits in terms of health and social equity . HoweverSecond, the data demonstrates a strong relationship between infection risk and proximity to rice fields, emphasizing a role for localized environmental management. There has been recent renewed interest in larval control as part of integrated malaria control strategies , with thCost-effective intervention options in malaria endemic communities need to be guided by an informed understanding of the local epidemiology of infection. These results show bed net use to be strongly associated with a decreased risk of infection in this rural Ugandan community, especially among school-aged children. Despite this, the number of individuals reporting sleeping under a bed net was inadequate, with dramatic increases in coverage required to meet current national and international targets. Findings also indicate that infection risk is influenced by proximity to potential mosquito breeding grounds, highlighting potential targets for localized environmental management. Taken together, these results underline the need for a targeted approach to integrated vector-control initiatives, further emphasizing the value of spatially explicit, community-based studies when evaluating ongoing control efforts.The authors declare that they have no competing interests.The study was designed by RLP and SB. RLP collected the data, with support from HB, SGS and SB. RWS provided scientific advice on analysis and interpretation. RLP developed the initial draft paper and all authors read, commented on and approved the final manuscript.Plasmodium spp. infection, stratified by age groupBivariate logistic regression models for . The data provided represent the preliminary statistical analysis of risk factors for Plasmodium spp. infection, for all ages and stratified by age group.Click here for file