Iron supplementation is employed to treat post-malarial anaemia in environments where iron deficiency is common. Malaria induces an intense inflammatory reaction that stalls reticulo-endothelial macrophagal iron recycling from haemolysed red blood cells and inhibits oral iron absorption, but the magnitude and duration of these effects are unclear.57Fe and 58Fe on days 1 and 15 of supplementation respectively. 57Fe and58Fe incorporation were significantly reduced (8% vs. 28%: p<0.001 and 14% vs. 26%: p = 0.045) in the malaria vs. non-malaria groups. There was a significantly greater haemoglobin response in the malaria group at both day 15 (p = 0.001) and 30 (p<0.000) with a regression analysis estimated greater change in haemoglobin of 7.2 g/l (s.e. 2.0) and 10.1 g/l (s.e. 2.5) respectively.We examined the red blood cell incorporation of oral administered stable isotopes of iron and compared incorporation between age matched 18 to 36 months old children with either anaemia post-malaria (n = 37) or presumed iron deficiency anaemia alone (n = 36). All children were supplemented for 30 days with 2 mg/kg elemental iron as liquid iron sulphate and administered Post-malaria anaemia is associated with a better haemoglobin recovery despite a significant depressant effect on oral iron incorporation which may indicate that early erythropoetic iron need is met by iron recycling rather than oral iron. Supplemental iron administration is of questionable utility within 2 weeks of clinical malaria in children with mild or moderate anaemia. In endemic countries malaria produces a flux of iron from the haemolysis of red blood cells in infants and young children many of whom are already iron deficient. The relative contributions of malaria and iron deficiency to post-malaria anaemia are often unclear, however iron supplementation combined with effective anti-malarial therapy is commonly employed and has been shown to be an effective strategy for the management of post-malarial anaemia Haemolysis, haemoglobin recycling and iron flux are central to the pathophysiology of malaria and post-malarial anaemia. Haemoglobin is recycled predominately through reticulo-endothelial macrophages to the bone marrow to fulfill erythropoetic iron need. Inflammatory insults however inhibit the release of iron from macrophages We examined and compared the red cell incorporation of orally administered stable isotopes of iron and the haemoglobin response to 30 days of iron supplementation in children anaemic after malaria and in those with non-malaria anaemia.In The Gambia 76% of children have moderate (<110 g/l) and 15% severe anaemia (<70 g/l) at the end of the malaria season th day after diagnosis when treatment was completed. Employing haemoglobin at the time of diagnosis as a baseline when examining the erythropoetic response after malaria is problematic as haemoglobin can continue to drop during and after malaria treatment th centile) and severe anaemia (<60 g/l) were excluded from the study. The study physician saw all children on days 1, 15 and 30 and documented any clinical symptoms.Anaemic children (haemoglobin <110 g/l) were recruited from the MRC Keneba clinic during the malaria season of 2003. Children between 18 and 36 months of age with anaemia associated with uncomplicated malaria were recruited from children who presented with a fever and were found to have a peripheral parasitemia of greater than 500 parasites per microliter on Field's staining of a thick blood film. This was later confirmed with Giemsa staining. These children were treated with a standard 3 day course of chloroquine (10 mg/kg/day) and fansidar (½ tablet if less than 15 kg body weight once) and were recruited on the 457Fe (3.9 mg on day1) and 58Fe (1.3 mg on day15) early in the morning after at least a 2 hour supervised fast. The iron doses were drawn up by needle and syringe from their respective solutions. The exact weight  of the needle and syringe was detailed before and after introduction of the isotope into 20 ml of orange juice and used to calculate the dose of isotope administered to each child. Thereafter each child was administered a crushed 50 mg vitamin C tablet in an additional 20 ml of orange juice. All children were observed for 2 hours after isotope administration and any spillage or vomiting was recorded. From day 2 all children were supplemented daily by a field-worker with 2 mg/kg elemental iron as liquid iron glycine sulphate  until day 30 when a final venous sample was taken. No vitamin C was given with the liquid iron glycine sulphate.All children had a venous blood sample taken on day1 and day15 and thereafter received Haemoglobin (Hb) (Medonic CA 530 haemoglobinometer) and zinc protoporphyrin (ZnPP)  were measured in The Gambia within 8 hours of sampling and each sample was assessed for the presence of malaria parasites as described above. Frozen plasma was transported to the UK for the analysis of ferritin (Imx ferritin assay based on Microparticle Enzyme Immunoassay (MEIA) technology, Abbot Laboratories), soluble transferrin receptors (STfR) (R&D systems ELISA), erythropoetin (Epo) (R&D systems Quantikine IVD ELISA) and C reactive protein (CRP)(Dade Dimension particle enhanced turbidimetric immunoassay).Iron-57 and 58 were converted to an aqueous solution of iron sulphate as previously described The clinic of the MRC Keneba field-station has provided free medical care for local villages for over 25 years. Detailed medical records of growth and morbidity are maintained for all children under 3 years of age. The nearest government facility is 20 km away and it is very unusual for a child from the local villages to receive treatment outside of the MRC Keneba clinic. The parents/guardians of children were approached after attendance at the MRC Keneba clinic for either routine monitoring or after emergency treatment for malaria. A field-worker not attached to the clinic team explained the study procedure by reading out an information sheet in Mandinka. Individual informed consent was obtained and confirmed by signature/thumbprint. All children were provided with an insecticide treated bed net on entry to the study if they did not already sleep under one. The study was approved by the MRC/Gambian Government Joint Ethics Committee and the The Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals in Houston, USA.e stable isotope incorporation at day 1 and 15 for the malaria and non-malaria groups. A multilevel model was utilized to assess the effects of whz, age, haemoglobin, CRP and iron sufficiency markers on isotope incorporation independent of day of administration.Based on variance data from previous similar studies a sample size of 38 per group was calculated. We aimed for 90% power to demonstrate a significant difference (5%) in isotope incorporation between children with malaria (estimated mean (s.d.) incorporation 20%+/−15%) and children without malaria (estimated mean (s.d.) incorporation 35%+/−25%). Microsoft Access (2000) was used for data recording and Epi-Info (v6.04) for generation of nutritional z scores. All data was dual entered with a validation performed between the two entry records and against the hard copy of the data. Differences between groups were compared using t-tests for quantitative variables (or transformed variables) with approximately normal distributions and chi-squared tests for categorized variables (SPSS 11.5.0). The strength of trends was measured by Spearman correlations. A linear regression model of iron absorption, adjusting for haemoglobin at day1, was used to compare differences between post-malaria anaemia and non-malaria anaemia groups and to estimate the marginal means of logThere was no significant difference in age or whz score between children with post-malarial anaemia (n = 37) and those with non-malaria anaemia (n = 36) . The malIn the well, non-malaria anaemic group iron deficiency could be defined retrospectively on the basis of cut-offs in at least 2 of 3 iron sufficiency markers i.e. STfR >8.5 mg/l Analyses of differences in haemoglobin, iron sufficiency marker measurements and stable isotope incorporation were adjusted for differences in day1 haemoglobin between groups. There was a significantly greater haemoglobin response  in the m57Fe incorporation on day 1 was significantly lower in the malaria group , non-malaria: 26.6% (s.e. 1.15), P<0.001) and remained lower on day15 for 58Fe incorportion , non-malaria: 24.0% (s.e.1.17), P = 0.116). When adjusted for differences in day 1 haemoglobin both 57Fe and 58Fe incorporation were significantly reduced between groups . The estimated marginal mean of 57Fe incorporation in the malaria group on day 1 was 8.3% (95% CI: 6.2–11.3) versus 27.9% (95% CI: 20.6–37.9) in the non malaria anaemic group. On day 15 the estimated marginal mean of 58Fe incorporation was 14.4% (95% CI: 9.7–21.1) in the malaria group versus 25.9% (95% CI: 17.5–38.4) in the non-malaria anaemic group. When those children that developed malaria during the study were excluded  day 15 58Fe incorporation remained lower in the post malaria anaemia group .In a crude analysis, not adjusted for differences in day 1 haemoglobin, red blood cell 57Fe incorporation on day1 and 58Fe incorporation on day15 in the non-malaria group suggesting no evident effect of 14 days prior iron supplementation on 58Fe incorporation.In the presence of malaria only ferritin  and crp  correlated with percentage incorporation and not haemoglobin, whz or other iron markers . FerritiData from days 1 and 15 from subjects with non-malarial anaemia was combined to enhance the power to examine influences on iron stable isotope incorporation other than malaria . CRP wasIncreasing malnutrition was associated with less isotope incorporation per kg bodyweight. There was a significant difference in isotope incorporation between those with a CRP measurement greater than or less than 10 mg/l (mean (s.d.) 16 (11.5) vs 34.7 (17.7), p<0.001) and between those with a whz score greater than or less than −1 whz (43.2 (17.2) vs 26.6 (17.8), p = 0.001) .A standard clinical response to severe post-malarial anaemia is to supplement with iron and it is a commonly employed therapeutic strategy in sub-Saharan Africa where endemic iron deficiency often co-exists. This study was designed to assess the effect of malaria on the red blood cell incorporation of iron supplements given to treat post-malarial anaemia. As this study is one of the largest iron stable isotope incorporation studies carried out it also allows us to look at the factors affecting iron incorporation in rural African children.We have demonstrated a significantly greater haemoglobin recovery in those children with post-malarial anaemia despite an initially reduced capacity to incorporate oral supplemental iron. The reduced incorporation of the oral stable isotope of iron was most marked at day 1 when the children had completed therapy for malaria and were clinically well and apyrexial. By day 15 the incorporation had improved and there was only a marginally significant difference from the non-malaria group. The anaemia of malaria results from the haemolysis of both infected and uninfected red blood cells, an inflammatory mediated effect on iron redistribution to reticulo-endothelial macrophages, and an impaired bone marrow response Acute clinical malaria has been associated with a reduction in STfR measurement It is not possible from this data to determine whether pre-existing iron sufficiency differed between the two groups at baseline as malaria (or the inflammation associated with it) directly affects both STfR 57Fe incorporation in post-malaria anaemia did not correlate with haemoglobin or iron sufficiency markers except ferritin. Ferritin was most likely a marker of the overwhelming influence of malaria-associated inflammation on iron incorporation as ferritin is an acute phase reactant.Iron absorption is very difficult to measure in the field and red cell incorporation offers an approximate guide to absorption as well as flux of iron to erythropoetic cells. We employed this method in children with malaria as the best available tool to assess the utility of early iron supplementation in the presence of inflammation and iron sequestration. Stable isotope iron incorporation was reduced initially after malaria and the block most likely occurred during absorption through the small intestinal mucosa. This may have been either a cytokine/hepcidin mediated effect and/or possibly resulted from iron loading of the reticuloendothelial macrophages due to malaria. Il-1 and IL-6 regulate hepcidin transcription in mouse models 57Fe and 58Fe incorporation in the non-malaria group allows for the assessment of influences on iron incorporation in this population other than malaria. Incorporation was variable and only inflammation, whz score and ferritin were independently associated and not age or haemoglobin. We excluded severely malnourished children (>−3 whz scores) from this study but still found that weight-for-height z-score was positively correlated with iron incorporation and this effect was independent of inflammation. This finding is important as it suggests that poorer whz score is associated with a declining ability to incorporate supplemental iron. Previous smaller studies Examining the combined Iron deficiency is common in areas of malaria endemicity and supplementation of young children improves iron status Distinguishing iron deficiency from iron seqestration immediately after clinical malaria is very difficult even with access to laboratory markers of iron status and erythropoesis and is a common diagnostic dilemma faced by doctors and nurses throughout malaria endemic areas. Supplemental iron is important to children with severe post-malaria anaemia if there is a likelihood of pre-existing iron deficiency. However we have shown that the utility of supplemental iron given in the first two weeks after a malarial episode to childen with mild/moderate anaemia is questionable irrespective of pre-existing iron deficiency. It is likely that the liberation of sequestered iron is a major source for erythropoesis in the early stages of recovery from malaria induced anaemia and re-assessing haemoglobin and/or iron status two weeks after a malaria episode may well better indicate the need for supplemental iron at a time when it is more likely to be incorporated.


To the Editor: Malaria is a disease caused by parasites of the genus Plasmodium. The infection is transmitted to humans through the bites of female mosquitoes of the genus Anopheles. Four species of Plasmodium are pathogenic to humans, and each leads to different clinical features: P. falciparum causes severe malaria with undulating high fever ; P. malariae, P. vivax, and P. ovale cause less severe clinical courses of disease with the manifestations of malaria quartana  and malaria tertiana . Literary evidence for malaria infection dates back to the early Greek period when Hippocrates described the typical undulating fever , a Middle Kingdom tomb in Thebes West , and various tomb complexes in Thebes West, which were built and used between the Middle and New Kingdom until the Late Period . All samples were first tested for Plasmodium spp. DNA by using the heminested PCR for the 18S rDNA primer targets usually used for malaria identification   for P. falciparum (In this second set of experiments, 2 of the 91 ancient Egyptian samples tested positive for the 134-bp fragment of the pfcrt region of lciparum . The speP. falciparum histidine-rich protein-2 antigen, doubts as to the specificity of those tests have been raised.Previously, immunologic tests have been used to investigate the presence and incidence of malaria in ancient Egyptian mummies (P. falciparum aDNA in Egyptian mummy samples, thereby proving a specific infection by falciparum malaria in ancient Egypt. With respect to the infection incidence, our molecular analysis suggests a more realistic frequency than had been previously suggested by paleoimmunologic methods. Consequently, the aDNA analysis is superior with respect to the reaction specificity, so that the latter should not further be used for that purpose.Our study unambiguously identified This report adds another infectious disease to the spectrum of paleomicrobiology in ancient Egypt, thereby further explaining the previously postulated influence of infectious diseases on the low life expectancy for ancient Egyptian populations (


Plasmodium falciparum.Cerebral malaria is a diffuse encephalopathy associated with seizures and status epilepticus which can occur in up to one-third of patients with severe malaria, particularly that caused by Plasmodium vivax malaria  complicated by seizures and symptoms of diffuse meningoencephalitis. Two patients had predominantly meningeal signs, while in the third patient the features were purely of encephalitis All cases were treated with artesunate. Usually, cerebral malaria is caused by P. falciparum, and rarely, cerebral malaria is a presenting complication or occurs during the course of P. vivax infection.In this article, we report three cases of  P. falciparum infection. However, there are infrequent reports of cerebral malaria associated with P. vivax infection. To our knowledge, only 45 cases of central nervous system P. vivax malaria are reported in the scientific literature since 1920, about half of these cases have occurred in children.[1Cerebral malaria is usually secondary to P. vivax.Here we report three rare cases of cerebral malaria caused by Three male patients, each aged over 18 years, presented with high-grade intermittent fever of more than four days duration. The fever was associated with chills and rigors. All the three patients had presented with altered consciousness There was history of generalized tonic clonic convulsions prior to admission, in each case. Two of them were severely dehydrated. Their capillary blood sugar level was normal at presentation.P. vivax. Antigen test for P. vivax was positive, while that for P. falciparum was negative in all cases. Their cerebrospinal fluid and electroencephalogram (EEG) findings were unremarkable. The patients were treated with supportive therapy and intravenous Artesunate in the recommended dose. Repeat blood smears after two days of therapy showed clearance of the parasites. All the three patients were discharged from ICU in a clinically stable condition and were advised to take Primaquine for 14 days. Follow-up evaluation after one month showed no residual neurological deficit.In all cases, routine blood counts, liver function tests, and serum electrolytes, serum urea, and serum creatinine were within normal limits. Peripheral blood smear revealed trophozoites of P. falciparum malaria is unusual in P. vivax infections. Any patient infected with P. viva who exhibits severe malaria is presumed to be suffering from mixed infection.[P. vivax infection can also present as cerebral malaria. Clinical data provided by Kochar et al, indicates that P. vivax can cause both sequestration-related and nonsequestration-related complications of severe malaria, all of which are commonly associated with P. falciparum infections.[P. vivax cerebral malaria. The presenting features were of acute febrile encephalopathy, convulsions, and coma. Focal neurological signs were observed in one patient. Ozen et al,[P. vivax malaria that presented with status epilepticus. Some experts also suggest that cerebral malaria subjects might have an underlying seizure disorder and those seizures are precipitated by the high fever associated with the disease.Organ dysfunction characteristic of nfection. However,fections. The exacfections. studied en et al, have rec


We conducted this study to determine the prognostic significance of the platelet count in children with cerebral malaria. We studied children with cerebral malaria admitted to the pediatric research ward at Queen Elizabeth Central Hospital, Malawi. We analyzed 1,811 children with cerebral malaria and compared them with 521 children with bacterial meningitis. There was a significant difference in platelet counts between children with cerebral malaria and those with meningitis. Among children with cerebral malaria, there was no relationship between the platelet count and either the admission coma score or the eventual outcome. Those with malarial retinopathy were more thrombocytopenic than those without. Our results suggest that the platelet count is not prognostic in Malawian children with cerebral malaria. N = 110) were 6.3  times more likely to die than those with a platelet count of above 100,000/μL. In a much larger study in coastal Kenya, Ladhani and colleaguesThrombocytopenia is common in all forms of malaria.In an autopsy study in Blantyre, Malawi, 7 out of 31 children with an illness that fulfilled the WHO clinical definition of cerebral malaria proved at post mortem to have died of a cause other than malaria.6Plasmodium falciparum infection. Because the presence of retinopathy strengthens the diagnosis of this syndrome, we have examined the relationship between the circulating platelet count and the severity and outcome of cerebral malaria in Malawian children with and without malarial retinopathy.Cerebral malaria is a common life-threatening complication of P. falciparum asexual parasites in the thick blood film, and in whom no explanation for coma other than malaria could be found. On admission, every child underwent detailed clinical assessment; from 1997 onward this included direct and indirect ophthalmoscopy. The age range of patients studied was 2 months to 171 months.The study was conducted in children admitted to the pediatric research ward at the main referral hospital, Queen Elizabeth Central Hospital in Blantyre, Malawi, between 1995 and June 2007. For the purpose of this study, we retrospectively analyzed only children who were admitted with a Blantyre coma score of 2 or less, who had As a comparison group, we analyzed the platelet counts of children admitted during the same period with a diagnosis of bacterial meningitis. Bacterial meningitis was diagnosed on the basis of the following cerebrospinal fluid (CSF) findings: either a pathogenic bacterium seen on gram stain or cultured within 7 days; or a cell count greater than 100/μL containing a predominance of polymorphonuclear leukocytes.Blood samples were collected in EDTA for immediate analysis by Coulter Counter. Thick blood films were stained with Fields stain and examined under oil-immersion. Asexual parasitemia was quantified by counting against white cells (thick film) and calculating density/μL from the total white cell count.A lumbar puncture was done in all children except those in whom it was considered to be clinically dangerous to perform. Blood cultures were done in all patients and the blood glucose level was measured (repeatedly as indicated) in every case. Chest x-rays were done only when a special indication was present. In some patients with focal neurological signs a computed tomography (CT) scan was done when the facility was available, but this constituted a small minority of patients. Blood electrolytes were measured when judged clinically useful and when the facilities were available, but this too covered a minority of cases.All children were started on intravenous quinine 20 mg/kg (loading dose) infused over 4 hours, and then 10 mg/kg 12 hourly until parasites cleared, followed by completion of antimalarial therapy by mouth. Anemic patients were transfused when necessary, and other supportive and antimicrobial therapy was given according to the guidelines of the Pediatric Department of the College of Medicine, University of Malawi.Clinical data were analyzed using Stata 8.1 . The outcome of the illness was categorized as satisfactory  or unsatisfactory . Distributions were compared using the Mann Whitney test. Spearman's test was used to examine the correlation between parasite density and platelet counts. Proportions were compared by the Fisher's exact test. Thrombocytopenia was defined as a platelet count of less than 150,000/μL.We analyzed 1,811 children who were admitted to the Pediatric Research Ward with the diagnosis of cerebral malaria between 1995 and 2007.The age range of the children was 2 months to 14 years (median 34 months). Out of the 1,811 patients, 361 (19.9%) patients were admitted deeply unconscious with a Blantyre coma score of 0. Six hundred seventy-two (672) (37.1%) children were admitted with a Blantyre coma score of 1, and 778 (43.0%) had a coma score of 2.The 1,317 (72.7%) patients made a full recovery, 188 (10.4%) had neurological sequel, and 306 (16.9%) died. The median admission parasite count was 104,160/μL.P. falciparum asexual parasitemia on the thick blood film; blood films were negative for parasites in the remaining 439. The median platelet count in patients with bacterial meningitis was 243,000/μL We analyzed 521 children who were admitted with bacterial meningitis. The age range was 2 months to 168 months (median 13 months). Out of the 521 children, 82 patients had P < 0.0001).In 1,811 patients admitted with a diagnosis of cerebral malaria, the median platelet count was 80,000/μL. There was a highly significant difference between the cerebral malaria and meningitis groups  . The 18.7% of children in our study with bacterial meningitis had parasitemia, but the median platelet count in this group was not significantly lower than in children with meningitis who were not parasitemic. This suggests that in children admitted with acute bacterial meningitis, parasitemia may be an incidental finding not contributing to the child's disease.10In this study, we found a highly significant difference in the platelet counts between children who were admitted with a diagnosis of cerebral malaria and those with bacterial meningitis . Even among children with retinopathy, however, there was no significant difference in the platelet counts between those that had full recovery and those that either died or had neurological sequela (P = 0.3).Children with malarial retinopathy, a physical sign strongly associated with intracerebral sequestration of parasites and suggestive of a diagnosis of “true” cerebral malaria,P = 0.097). The same analysis with a platelet count cutoff point of 100,000 revealed similar results (P = 0.36). This compares with the findings of a study in 2002 by Ladhani and othersWe have shown that there was no difference in outcome between children who had a platelet count of less than 50,000 and those with a platelet count of greater than 50,000 (In conclusion, we have confirmed the prominence of thrombocytopenia in malaria. Among children with cerebral malaria, those with retinopathy are more likely to be thrombocytopenic than those without retinopathy. However, we have found no relationship between the circulating platelet count and disease severity or outcome. The platelet count is not prognostic in Malawian children with cerebral malaria.


In Spain, malaria cases are mostly due to migrants and travellers returning from endemic areas. The objective of this work was to describe the malaria cases diagnosed at the Severo Ochoa University Hospital (HUSO) in Leganés in the south of the Madrid Region from 2005 to 2008.Descriptive retrospective study performed at HUSO. Data sources are registries from the Microbiology Department and malaria cases notified to the Preventive Medicine Department. Analysed parameters were: administrative, demographical, related to the stay at the endemic country, clinical, microbiological diagnosis method, pregnancy, treatment and prophylaxis, co-infections, and days of hospital stay.Plasmodium falciparum was responsible for 94.7% of the cases. The more frequent symptoms were fever (77.2%), followed by headache and gastrointestinal symptoms (33.3%). Nine cases needed hospital admittance, a pregnant woman, three children, four VFR and an African tourist, but all evolved favourably. Chemoprophylaxis data was known from 55 patients. It was taken correctly in one case (1.8%), in five (9.1%) the prophylaxis was improper while the others 49 (89.1%) cases had not followed any anti-malarial prophylaxis.Fifty-seven patients diagnosed with malaria were studied. Case distribution per year was 13 in 2005, 15 in 2006, 15 in 2007 and 14 in 2008. Thirty-three patients were female (57.9%) and 24 male (42.1%). Mean age was 27.8 years. Most of the malaria cases were acquired in Nigeria (49.1%) and Equatorial Guinea (32.7%). 29.1% of the patients were immigrants who had arrived recently, and 61.8% acquired malaria when travelling to their countries of origin to visit friends and relatives (VFR). Majority of cases were diagnosed between June and September. Microscopy was positive in 39 cases (68.4%) immunochromatography in 42 (73.7%) and PCR in the 55 cases where performed. P. falciparum.Children, pregnant women and the VFR have the highest risk to present severe malaria and to need hospital admittance. Another important risk factor for acquiring malaria is incorrect prophylaxis. The first place for malaria acquisition was Nigeria and the main species causing malaria was  Approximately, 50% of the world's population, mostly that living in the world's poorest countries, is at risk of malaria. In 2006, there were an estimated 247 million malaria cases and an estimated 880,000 deaths from malaria. Most cases and deaths are in sub-Saharan Africa, mostly in children under five years of age. However, Asia, Latin America, the Middle East and parts of Europe are also affected . TravellIn 2007, international tourist arrivals grew to a new historical maximum of 900 million . In agreThe last case transmitted by vectors in Spain was diagnosed in 1961, but no cases of vector transmission have been reported since , except In Spain, 345, 342 and 338 cases were reported in 2007, 2008 and 2009 respectively. In 2007 the incidence rate (IR) was 0.8 cases/100,000 people, while in the Community of Madrid with 114 cases, IR was 1.9 cases/100,000 people, 2.4 times higher than the Spanish IR. Health areas in the Madrid Region with higher rates of incidence were those corresponding to Leganés- Fuenlabrada (southern area), and Alcalá de Henares-Torrejón de Ardoz (north-eastern area), with 8.0 and 4.0 cases per 100,000 people, respectively [The objective of the study is to describe the malaria cases diagnosed in the Severo Ochoa University Hospital (HUSO) placed in Leganés in the south of Madrid Region during the 2005-2008 period.Studied population was all patients with parasitological diagnosis for malaria between January 2005 and December 2008. Patients come from the area covered by HUSO, which included Primary Care Units of Leganés and Fuenlabrada. On 1st January 2008, Leganés population was 188,676 people, out of which 23,858 were immigrants, representing 12.7% of the total registered population . FuenlabThe study was performed as a descriptive retrospective survey performed in the HUSO. Data sources were records from the Department of Microbiology and malaria cases notified to the Preventive Medicine Department. A database was designed, completed after the retrospective review of the Microbiology registries, discharge report (for patients with admittance) and notification sheets from the Preventive Medicine Department.Plasmodium species and parasitaemia -<250 very low trophozoites/μl, 250 to 20,000 low, 20,000 to 50,000 moderate and >50,000 high-), pregnancy, treatment, co-infections , intake or not of prophylaxis and days of hospital stay in case of admittance.The main parameters analysed were as follows: administrative , demographical , data related to the stay at the endemic country , clinical , laboratory data , parasitological diagnosis method  immigrants recently arrived to Spain (less than six months of stay), 2) people having resided in Spain for more than six months, travelling to endemic areas to visit relatives and friends  and 3) people travelling to an endemic area for tourism, business, cooperation or others.Parasitological diagnosis was performed by microscopy and antigen detection (Binax NOW ICT) in the 57 cases analysed and by PCR in 55 of the cases   for quantitative values. Data were processed using SPSS 15 software.Plasmodium species.Fifty-seven patients diagnosed with malaria were studied. In two patients, diagnosis was performed at the Hospital Carlos III in Madrid, with data submitted to the HUSO because they originated from this health area. Serological and haematology data for these patients were unknown. Also, for another two patients the only known data was birth date, sex, diagnosis method used and Case distribution per year was 13 in 2005, 15 in 2006, 15 in 2007 and 14 in 2008. Twenty-four out of 57 (42.1%) were male, while 33 patients were female (57.9%), of which four were pregnant. The mean age was 27.8 years, with 11 patients under 18. Six out of 55 patients (10.9%), of whom nationality was known, were Spanish. The other 49 patients (89.1%) came from sub-Saharan Africa countries; 22 (40.0%) from Nigeria, 19 (34.5%) from Equatorial Guinea, 6 (10.9%) from Cameroon, one (1.8%) from Ivory Coast and one (1.8%) from Mali. Out of the six patients born in Spain, one was an adult tourist returning from India; three were brothers whose parents were Nigerian and the other two were the spouse of people coming from Ivory Coast and Nigeria. Out of the 49 foreign patients, 19 (38.8%) were recently arrived immigrants with residence in Spain for less than 6 months, one was resident in Equatorial Guinea (2.0%), who came as a tourist to Spain, and 29 (59.2%) had a longer resident period, between six months to 20 years. In one case, the residence time was unknown.The more numerous group of patients was the VRF 61.8%), where both the immigrants with long residence in Spain (29 cases: 52.7%) plus their spouses and children born in Spain (5 cases: 9.1%) are included, followed by the immigrants recently arrived from endemic areas (19 cases: 34.5%), and finally, a minority group for tourists .The acquisition of malaria for the 55 cases for whom the country was known was mainly Nigeria (27 cases: 49.1%) and Equatorial Guinea (18 cases: 32.7%). The rest of cases were acquired in Cameroon (6 cases: 10.9%), in Ivory Coast (2 cases: 3.6%), in Mali (1 case: 1.8%) and in India (1 case: 1.8%). The patients acquired malaria in their own country or in the family's native country. Only one Spanish adult tourist and a sub-Saharan African tourist became infected in an unrelated country , the immunochromatography test in 42 out of 57 cases (73.7%), and the PCR in the 55 cases (100%) where it was performed. In three patients, malaria diagnosis was performed more than one year after the date of return from the endemic country and in one case five months later, none of these were from Spanish origin. For these cases, PCR was the only test rendering a positive result.Plasmodium ovale infection who was diagnosed 277 days after travel return. The mean for the time lapse between the return date and the diagnosis for patients without symptoms was 112 days, with a maximum of 468 days.The mean for the time lapse between the onset of symptoms and parasitological diagnosis was 4.5 days, while the lapse between the date of return from the trip and the diagnosis was 21.5 days. A special case was a patient with Plasmodium falciparum was characterized in 54 cases (94.7%), while P. ovale was present in the other three cases. The mean parasitaemia was 32,245 trophozoites/μl of blood . Parasitaemia was very low in 12.5% of the cases, low in 46.9%, moderate in 15.6% and high in 25.0%.Mansonella perstans (5), Giardia lamblia (1), Ascaris lumbricoides (2), Trichuris trichura (2), Onchocerca volvulus (1)). Furthermore, three patients were co-infected with chronic viral diseases, one out of the 20 patients analysed was infected with HIV, one with chronic HBV and one with HIV plus HCV.Six patients showed single or multiple co-infection with other parasites , nine patients did not show any symptom (15.8%) and for the two remaining cases this data was unknown. Most frequent symptoms were fever (77.2%), headache and gastrointestinal symptoms (33.3%), followed by asthenia (15.8%). Other described symptoms included arthralgia, malaise, hepatomegaly, jaundice and anaemia.The haematological data was known in fifty-one patients. Haemoglobin (Hb) ranged between 6.9 and 16.1 g/dl, 29 patients (56.9%) had a Hb value lower than 12, while just one had a Hb lower than 7. Haematocrit (Hto) ranged between 22.1 and 45.2%, presenting a Hto lower than 37% in 34 patients (66.7%). Platelets ranged between 37,000 and 584,000 with a mean of 186,392, with 25 patients (49.0%) presenting thrombocytopaenia <150,000 platelets/μl.Nine patients needed hospital admittance, a pregnant woman, three children , four VFR and the African tourist, with a mean stay of 7.5 days (SD: 4.5). None of the patients presented any complications.P. ovale infection. Chemoprophylaxis data was known from 55 patients. It was taken correctly in one case (1.81%), whereas in five cases (9.09%) the prophylaxis was inadequate. The remaining 49 (89.1%) cases had not taken any anti-malarial prophylaxis.Treatment data was available for 14 patients, with quinine and doxycycline (57.1%) being the most frequently used, followed by quinine and clindamycin (28.6%), atovaquone/proguanil (7.1%) and chloroquine (7.1%). Treatment was completed with primaquine in the three cases with Malaria was eradicated in Spain in the early 60 s of last century. Since then, very low or null incidence has been reported until recently years, where due to increase of tourism and immigrants from malaria endemic countries, the number of reported cases has increased to near 400 per year .This study described the epidemiology of malaria in imported cases, diagnosed in the Reference Hospital of a health area in the south of Madrid, where about 15% of the population is immigrant, including a high number of natives from endemic malaria countries. This population pattern confers a special characteristic to this area, which differs from other Spanish health areas.All patients, except a Spanish tourist infected in India, acquired the infection in a sub-Saharan Africa country, in agreement with the fact that this continent is where malaria acquisition is more frequent -16. ExceP. falciparum, which shows a higher prevalence than that observed in France (80%) or in Italy (73.3%), where most cases are also imported from sub-Saharan Africa countries [Plasmodium vivax is the prevalent species, present in over 50% of the cases diagnosed [For nearly 95% of the patients in this study, the species causing malaria was ountries . Insteadiagnosed ,20.It is often reported in Spain that most of malaria imported cases were acquired in Equatorial Guinea, a small country in West Central Africa with 600,000 inhabitants and a former Spanish colony. Reports carried out in Barcelona and Valencia, two of the biggest Spanish cities, show that Equatorial Guinea was the place were more cases are acquired with 35.9% and 40% of the total cases respectively ,20. MoreMalaria is an infectious disease affecting health in a variable way. Residents in endemic areas acquire a certain degree of immunity after repeated exposure to parasite where frequency and seriousness of malarial episodes decreases with age . It is tSeveral patients 15.8%) were diagnosed without presenting any symptom. These asymptomatic patients can be an important factor in a possible autochthonous transmission of the disease. Most of the Mediterranean countries, where malaria has been eradicated, are still susceptible to its reintroduction due to the persistence of the vector  and seve% were diP. ovale infection, which was diagnosed 277 days after return. Removing this case results in an average between diagnosis and travel return of 14 days. The delay in the correct diagnosis of the diseases is directly related with the severity of the symptoms and the correct evolution of the patients.The asymptomatic patients suffer long delays in the diagnosis with an average of 112.7 days after travel return, while for the symptomatic patients the delay was 21.5 days. This value for correct diagnosis is still too high, but it was due to a The risk of complicated malaria is higher in children and pregnant women in endemic areas and in travellers coming from a non-endemic area, who do not have a previous immunity against the parasite . People The most used treatment pattern was quinine (85.7%) in combination with doxycycline (57.1%) or clindamycin (28.6%). There is no Spanish guideline for malaria treatment and this depends of several factors, including access to the anti-malarial drugs. Most drugs, including quinine, have to be obtained through the Service of Foreign Drugs Supply from the Ministry of Health, as only atavaquone/proguanil and chloroquine are available at chemists. Treatment with artemisinin derivatives is not approved by the Spanish Drug Agency, as is the case in other countries, such as the United States.P. ovale infection. These data confirms that taking correct prophylaxis is essential to avoid malaria infection.It is estimated that only between the 12 to 43% of the travellers to endemic areas follow an anti-malarial prophylaxis, with only 3.1 to 22.8% performing a correct and complete treatment ,18,26. FP. ovale malaria.Two main conclusions can be drawn from this study. First, that children and pregnant women have the highest risk to present severe malaria and to need hospital admittance, but also the immigrant visiting his/her own country and come back have a high risk to suffer severe malaria. Second, the most important factor responsible for acquiring malaria is incorrect prophylaxis. None of the malaria positive cases took appropriate prophylaxis, except one who acquired A possible solution may be to increase the information given to the sub-Saharan immigrants, which are the highest risk group. This information must include that the semi-immunity acquired by native people noticeably diminishes after living for some months outside an endemic area and, therefore, their risk to acquire malaria increases considerably, and they specially need to take prophylaxis and to follow it correctly.The authors declare that they have no competing interests.SR, IZ and FJM designed the study and compiled data. BMM and JMR reviewed the design on the study. SR and IZ created the databases, ordered and interpreted the results and wrote the article. BMM performed the statistical analysis. JMR performed PCR at CNM. SR, IZ, JMR and FJM performed the bibliographic search and review. FJM and JMR made the final review of the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria disease in infants is due to same parasites present on the placenta at birth.Although recent reports on congenital malaria suggest that the incidence is increasing, it is difficult to determine whether the clinical disease is due to parasites acquired before delivery or as a result of contamination by maternal blood at birth. Understanding of the method of parasite acquisition is important for estimating the time incidence of congenital malaria and design of preventive measures. The aim of this study was to determine whether the first P. falciparum parasites on the placenta detected by PCR were followed up to two years and observed for malaria episodes. Paired placental and infant peripheral blood samples at first malaria episode within first three months of life were genotyped (msp2) to determine genetic relatedness. Selected amplifications from nested PCR were sequenced and compared between pairs.Babies born to mothers with P. falciparum (P < 0.001) as compared to infants from secundigravidae and multigravidae mothers during the two years of follow up. Infants from multigravidae mothers got the first P. falciparum infection earlier than those from secundigravidae and primigravidae mothers (RR = 1.43).Eighteen (19.1%) out of 95 infants who were followed up developed clinical malaria within the first three months of age. Eight pairs (60%) out of 14 pairs of sequenced placental and cord samples were genetically related while six (40%) were genetically unrelated. One pair (14.3%) out of seven pairs of sequenced placental and infants samples were genetically related. In addition, infants born from primigravidae mothers were more likely to be infected with Plasmodium falciparum malaria parasites present on the placenta as detected by PCR are more likely to result in clinical disease  in the infant during the first three months of life. However, sequencing data seem to question the validity of this likelihood. Therefore, the relationship between placental parasites and first clinical disease need to be confirmed in larger studies. About 300,000 foetal and infants deaths and 2,500 deaths of pregnant women are attributable to malaria . Poor ouCongenital malaria infection refers to diagnosis of malaria parasites in the newborn within seven days of birth, or later if there is no possibility of postpartum infection by either mosquito bite or blood transfusion. On the other hand, neonatal malaria may be defined as symptoms attributed to malaria parasites in the erythrocytes of an infant within the first twenty-eight days of life . The mecThe study was conducted at Muheza District Designated Hospital, in Muheza District, north-east Tanzania. Muheza district, (5°42' S and 38°48'E) has a human population of 278,405 and the inhabitants are mainly involved in subsistence agricultural production. The district is served by one district hospital, three health centres and 32 dispensaries. The lowland areas of the districts are areas of intense transmission [Plasmodium falciparum parasites and their respective neonates. Recruitment of the study participants was done from September 2002 to September 2005 from maternity ward at Muheza DDH and 1,022 mother-neonate pairs were screened. Mothers diagnosed with placental parasitaemia identified in a blood smear by microscopy and their respective neonates were enrolled in the study. Mothers with evidence of chronic or debilitating illness, such as recent significant weight loss, chronic diarrhoea, or history of chronic illness were excluded. All infants born from mothers diagnosed with placental parasitaemia were seen at birth and at two-week intervals for two years for full clinical examination and blood sampling, either at the Muheza Designated District Hospital or at the Projects sponsored mobile clinics that rotated through study villages on a weekly basis. Written informed consent was obtained from the mother prior to recruitment into the study.The study population comprised of mothers aged between 18 and 45 years who had placental Demographic information on maternal age, village of residence, gravidity, history of intermittent presumptive therapy (IPT) against malaria during pregnancy, abortion or miscarriage;as well as baby information on birthweight and gestation age were collected by trained nurses at enrollment. Bed net usage was assessed by village health workers during clinical visits and onsite home visits. The study protocol was approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania.P. falciparum by microscopy were collected by the project staff nurses from placental tissues, umbilical cord and through either finger or heel prick of new born infants. Placental blood samples were obtained after delivery by mechanically grinding full thickness placental tissue [P. falciparum positive in their thick smears and all infants born from these mothers who presented with parasitaemia within three months from birth were selected for further analysis by PCR. In order to analyse the phenomenon of malaria infections of neonates via transmission across the placental, matched umbilical cord blood samples were examined in thick blood smear and further analysed whether negative by Giemsa stain or not. About 10 μl of blood samples were bloated on 0.5 × 2 cm strips of 3 MM Whatman filter papers and dried in a laminar flow hood. After drying, the filter papers were packed in small individual plastic bags to avoid contamination and stored at room temperature until needed for analysis.Blood samples of study mothers diagnosed positive for l tissue . Cord bl® capture card kit . The DNA was amplified in two nested PCR using oligonucleotide primers of the 5' and 3' conserved regions as described by [2, 200 μM mixture of dNTPs , and 0.25 U/μl of Taq polymerase . To each PCR tubes, 5 μl of DNA was added in primary reaction and 2 μl was re- amplified in the nested PCR reaction and carried out in a PTC®-100 Peltier. The expected lengths of the bands were 509 bp and 750 bp and the previously established DNA positive control sample used was 3D7. In all PCR reactions a negative control sample with no template DNA was used.Genomic DNA was extracted from blood collected on 3 MM Whatmann filter papers using Generationribed by . The detGel electrophoresis was performed on nested PCR products to confirm the presence of PCR amplicons size of interest. Placental-cord pair, placental-infants first parasitaemia and placental-cord and infants first parasitaemia samples were run in parallel in the 2% agarose gel in 100 ml of 1 × TBE  solution. The gels were stained with 0.5 μg/ml ethidium bromide final concentration and the similarities and differences of the bands size was visualized by ultraviolet light. Fragment sizes were estimated using a DNA size marker . Nest two PCR products with single bands were purified by QIAquick PCR product purification kit using microcentrifuge before sequencing. For samples which presented more than one band (more than one genotype), the gel were cut along those bands that share the size, purified by QIAquick Gel extraction kit and then sequenced.msp2 and the second template were sequenced if differences were found between the two derived sequences.DNA sequencing was done on nest two amplicons . In prepP value < 0.05 in the analysis was considered to be significant. Chi-square test was used to determine the proportional of the placental parasitaemia among gravity . A DNASTAR Program was used to analyse the sequencing results and all results were compared with msp2 sequences in Plasmodium genome resources or Plasmodium database (PlasmoDB) by Basic Logical Alignment Search Tool (BLAST) so as to determine if each individual sequence matched with concordant msp2 gene of P. falciparum sequences previously deposited.Data were analysed using STARTVIEW statistical package. Descriptive analysis used to evaluate the means of the different variables. Paired t- tests were used to analyse and determine significance relationship between difference observed variables. Regression plots that showed the variability around means of different variables and By using PCR, placenta malaria was identified in 95 out of 100 microscopy-positive mothers and out of these 40 (42.1%) were primigravidae, 31 (34.7%) secundigravidae and 24 (25.2%) multigravidae. The mean weight of 95 babies born from placental malaria was 2.9kg with the range of 2–4.3 kg and 14.9% babies were below average birth weight while the mean gestation age was 36.3 weeks . Seventy-six out of 95 (79.8%) infants were born before the normal gestation age. Infants born from mothers diagnosed with placental malaria during delivery, presented with the first clinical malaria at the mean age of 11 weeks  weeks.P. falciparum infection in placental, cord blood and infant's peripheral blood at first parasitaemia as diagnosed by microscopy and PCR are presented in Table msp2 genes).The prevalence of Fourteen pairs of placental and cord blood samples that shared the same band size following PCR were sequenced and analysed using DNASTAR program. Sequenced samples were paired based on similarities using Megalign program. Eight (60%) out of 14 pairs were genetically related while six pairs (40%) were genetically unrelated by the difference of few nucleotides estimated at 10%. In addition, seven pairs of infant's samples at first parasitaemia and corresponding placental samples shared same band size by PCR and were sequenced. Upon megalignment, one pair (14.3%) was genetically related by 100% while six pairs (85.7%) were genetically unrelated.P. falciparum genotypes between and within pairs were also made. The comparison was between PCR results by looking at the bands which shared the same sizes, and sequencing results through alignment of the sequences in the phylogenetic tree. Some of the msp2 genes observed to be sharing the band size by PCR, had sequences not closely aligned in the tree. Nevertheless, pairs that were genetically related or closely genetically related by PCR were closely arranged in the tree leaves. Analysis from Plasmodium database (PlasmoDB) by Basic Logical Alignment Search Tool (BLAST) indicated that all samples originated from the msp2 gene of P. falciparum.Comparisons of t = 4.8, P < 0.001)  as compared to infants born from multigravidae. It was observed that 8 out of 18 (44.4%) infants that presented with clinical malaria within three months after birth were from multigravidae mothers. Infants diagnosed with cord malaria by PCR were observed to be more susceptible to P. falciparum infection in their early life as compared to the infants that were cord negative, with the relative risk (RR) of 2.6  may be explained by the structural diversity in the msp2 gene, which is due to the central domain containing repeats that vary in number, length and sequence as previously proposed [The proposed -19.P. falciparum parasite strain (multiple infections) may sequester in the placenta while only one strain crosses the placental and can be detected in the cord blood [P. falciparum population from the placenta to the cord, which causes clinical malaria in infants.More than one rd blood ,20,21. TThe findings from this study confirm that the parasite were congenitally acquired from the placenta suggesting that although neonates are thought to be immune to malaria, a proportional of them do get the disease as a result of transplacental transmission. Some explanation to the absence of parasites in the new-born includes the failure of parasites to grow in cord blood and fast elimination of parasites from the foetal circulation ,23.Findings from this study and others  suggest Placental malaria has been observed to have a negative outcome to the newborn. We observed similar negative outcome in this study where the mean gestation age were below the normal range (37 – 40) weeks in most infants born from mothers with placental malaria . These fP. falciparum that may lead to congenital malaria does occur as previously reported. The occurrence has important consequences for foetal and newborn development. Direct infection to the foetus could contribute to prematurity, low birth weight or increase likelihood of early and subsequent infant infections. However, the genetic unrelatedness of P. falciparum, which was detected in the placenta and infants below three months of age, signifies a rarity of congenital malaria and could be associated with the occurrence of maternal malaria. Further investigation of these relationships with assessment of other potential confounders, as well as their impact on infant's mortality and morbidity need to be done. Malaria prevention measures in pregnant women such as insecticide-treated nets and intermittent presumptive treatment should be a priority for malaria control programmes to prevent low birth weight, anaemia, intrauterine growth retardation, premature delivery and neonatal malaria.In conclusion, transplacental transmission of GM involved in developing the research proposal, conducting the genotyping study, analysing data, alignment of the sequenced results and writing the initial and the final manuscript. SK participated in designing of the study and analysis of the research results. LB assisted in writing the initial and final manuscript. All authors have seen and approved the final manuscript and are in agreement with my being the corresponding author.


Site preparation is a pre-requesite in conducting malaria vaccines trials. This study was conducted in 12 villages to determine malariometric indices and associated risk factors, during long and short rainy seasons, in an area with varying malaria transmission intensities in Korogwe district, Tanzania. Four villages had passive case detection (PCD) of fever system using village health workers.Four malariometric cross-sectional surveys were conducted between November 2005 and May 2007 among individuals aged 0–19 years, living in lowland urban, lowland rural and highland strata. A total of 10,766 blood samples were collected for malaria parasite diagnosis and anaemia estimation. Blood smears were stained with Giemsa while haemoglobin level was measured by HaemoCue. Socio-economic data were collected between Jan-Apr 2006.Plasmodium falciparum parasitaemia was significantly lower in both lowland urban,  and highlands,  compared to lowland rural. Individuals aged 6–9 years in the lowland rural and 4–19 years in both lowland urban and highlands had the highest parasite prevalence, whilst children below five years in all strata had the highest parasite density. Prevalence of splenomegaly and gametocyte were also lower in both lowland urban and highlands than in lowland rural. Anaemia (Hb <11 g/dl) prevalence was lowest in the lowland urban. Availability of PCD and higher socio-economic status (SES) were associated with reduced malaria and anaemia prevalence.Adjusting for the effect of age, the risk of Higher SES and use of bed nets in the lowland urban could be the important factors for low malaria infections in this stratum. Results obtained here were used together with those from PCD and DSS in selecting a village for Phase 1b MSP3 vaccine trial, which was conducted in the study area in year 2008. Malaria remains a major cause of morbidity and mortality in sub-Saharan Africa . Worldwieg size, structure), accessibility of the site, availability of health services and malaria burden in the area. Site preparation involving local communities is also likely to reduce ethical issues which could otherwise compromize implementation of the trials [In many parts of the world, adequate treatment of malaria is becoming increasingly difficult due to worsening problems of drug resistance, which have rendered cheap drugs such as chloroquine and sulphadoxine/pyrimethamine ineffective ,6. Hencee trials .In 2004, the African Malaria Network Trust (AMANET) received a grant from AIDCO, which, among other things, was to support capacity strengthening of selected African Research institutions to be able to conduct malaria vaccine trials. Site characterization for future trial sites was one of the most important components of the grant. The National Institute for Medical Research at its Centre based in Tanga, Tanzania, was one of the beneficiaries of the sub-grants given by AMANET. One of the objectives of the project was thus to conduct repeated malariometric surveys in 12 villages to describe distribution of malaria parasites as well as anemia and splenomegaly. This was done during short and long rainy seasons in urban and rural areas of Korogwe district between November 2005 and May 2007. Results from this study were used together with other data to provide guidance in the selection of villages suitable for conducting malaria vaccinesAnopheles gambiae s.s and Anopheles funestus are the main vectors of malaria [Plasmodium falciparum is the predominant malaria species accounting for more than 90% of all infections, the rest being Plasmodium malariae and Plasmodium ovale [The study was conducted in 12 villages of Korogwe district, Tanga region, north-eastern Tanzania. The population of Korogwe district was estimated to be 261,004 in 2002, with a growth rate of 1.4% per annum . The dis malaria . Plasmodum ovale .The 12 study villages are grouped into three strata namely: lowland rural, lowland urban and highlands  follow ups which are being implemented since January 2006. Population of individuals below 20 years was estimated to be 5400, lowlands rural, 6200 lowland urban and 2100 in the highlands. A socio-economic survey was conducted as part of the DSS between January and May 2006. The sample size was calculated based on population survey design in each stratum. At 95% confidence interval and 80% power, a sample size required was 985, 1130 and 385 in rural lowland, lowland urban and highlands, respectively. A list of individuals to participate in the surveys was randomly generated from the census database.Ethical clearance was granted by the Medical Research Co-ordinating Committee of the National Institute for Medical Research, Tanzania. Prior to the study, sensitization meetings were held in each village to explain the objectives and methodology of the study as well as seeking for community consent. Informed consent was obtained both orally and in writing from all individual participants or parents/guardians in case of children during the survey.Demographic information of each selected individual including name, sex, age and village of residence were recorded on a morbidity questionnaire. Thereafter, clinical and physical examination was done to assess participant's general clinical condition. Assessment of spleen enlargement was done according to Hackett's classification. Axillary temperature was measured using electronic thermometer. Individuals found to be sick were treated appropriately or referred to the nearby health facilities depending on the patients' clinical condition.A blood sample was collected for thick and thin smears preparation for malaria parasite quantification and identification, haemoglobin estimation and for other bioassays. Haemoglobin levels were estimated using HemoCue machine . Blood smears were stained with Giemsa and asexual parasites were counted against 200 white blood cells (WBCs) while sexual parasites were counted against 500 WBCs. A blood slide was declared negative after reading 100 high power fields. For quality control, 10% of the blood smears were randomly selected and read by another microscopist. Smears with discordant results were re-read by a third microscopist. Results of two microscopists with difference of less than 20% were adopted according to the standard operating procedures.Data were double entered and verified in Microsoft Access, while consistence checks and analyses were done using STATA software version 8.0 . Principal component analysis (PCA) was used to categorize households into different socio-economic scores. Household scores were grouped into three categories at ratios 40:40:20 as low, medium and high SES's . VariablTable In the four cross sectional surveys, a total of 10,766 blood samples were collected, 4,778 (44.4%) from lowland rural, 4,424 (41.1%) from lowland urban and 1,564 (14.5%) from highlands. Parasite prevalence during long rain seasons was 32.8% (95%CI: 31.0–34.7) in lowland rural, 11.8% (95%CI: 10.5–13.2) in lowland urban and 10.0% (95%CI: 8.0–12.3) in highlands; while in the short rains it was 35.1% (95% CI: 33.2–37.1) in lowland rural, 10.8% (95% CI: 9.6–12.2) in lowland urban and 13.1% (95% CI: 10.8–15.7) in highlands. The age specific prevalence is shown in Figure Plasmodium falciparum parasitaemia was significantly lower in both lowland urban,  and highlands,  compared to lowland rural. Use of bed nets was associated with low parasite prevalence, , whereas individuals from low and medium SES were at higher risk of malaria infection, OR = 2.02  and OR = 2.47 , respectively, when compared to high SES. Table Plasmodium falciparum was the predominant species accounting for 97.6% of all malaria infections while Plasmodium ovale and Plasmodium malariae were 0.3% each, and the rest were mixed infections.Adjusting for the effect of age, the risk of Overall gametocyte prevalence was highest (4.2%) in lowland rural. In lowland urban and highlands it was 1.2% and 1.3%, respectively. Regression analysis showed lower risk of gametocytaemia in both lowland urban,  and highlands,  compared to lowland rural. The odd ratio of gametocytaemia in individuals from medium and low compared to high SES households were 2.58 (95%CI: 1.59–4.19) and 2.62 (95%CI: 1.58–4.35), respectively. In villages with PCD, the carriage rate was significantly low, . There was no seasonal variation in gametocyte rates.Prevalence of splenomegaly during the long rains was 16.5%, 5.3% and 3.5% while in short rain season was 19.1%, 3.9% and 5.0% in lowland rural, lowland urban and highlands, respectively; and there was no difference between the two seasons. Similarly, lower risk was observed in individuals who were using bed nets, OR = 0.62  and in villages with PCD, OR = 0.42  while higher risk was seen in low SES, OR = 1.82  and medium SES, OR = 2.29 .Mean Hb concentration adjusted for effect of age and gender during the long rainy season in three strata was 11.32 g/dL (95%CI 11.26–11.38), 11.51 g/dL (95%CI: 11.45–11.57) and 11.65 g/dL (95%CI: 11.55–11.75), while during the short rains was 11.10 g/dL (95%CI: 11.04–11.16), 11.44 g/dL (95%CI: 11.38–11.49) and 11.07 g/dL (95%CI: 10.96–11.18), in the lowland rural, lowland urban and highlands, respectively. Table A similar pattern as that of mean Hb was seen for variables associated with risk of anaemia Table . VariablThis epidemiological study was designed to obtain data on malariometric indices in villages with different malaria transmission intensities during short and long rainy seasons in Korogwe district, north-eastern Tanzania. It was aimed at providing baseline epidemiological information needed in selection of areas for implementing malaria clinical trials and involved both rural and urban settings.Parasite prevalence is considered as the easiest way of measuring malaria transmission compared to entomological and serology measures. However, it is more affected by seasonal variation in transmission levels, survey timing, and peak transmission seasons, and hence can be seen as reliable within a short time interval . Unlike Previous studies in Korogwe have reported a decrease in malaria with increase in altitude ,13,17. HIn the present study, the finding of peak malaria parasite prevalence in children aged 6–9 years in lowland rural, while in lowland urban and highlands it was high in all individuals aged 4–19 years, suggests better immunity in the lowland rural and thus higher exposure to mosquito bites and malaria infection. Notwithstanding this, children below 5 years had the highest parasite density in all strata indicating immature immunity and higher vulnerability to malaria. As expected, children in the low malaria risk area of lowland urban had lowest parasite density. The observation of lack of seasonal variation in prevalence of gametocytes and splenomegaly seems to suggest similar transmission dynamics during short and long rains in the 2 year study period. Furthermore, the significantly higher malaria parasite prevalence observed in lowland rural during the short rains could be due to increased rainfall in October/November 2006.Data from this study found significant variation in malaria transmission within a short distance; and this might have a bearing on malaria diagnosis and management. Findings from this study suggest that trials aimed at higher malaria transmission areas should focus in the rural areas where socio-economic status is low. And socio-economic status remains an important risk factor for malaria transmission, and so efforts to combat malaria should also focus on socio-economic status of the communities.P. falciparum as well as increasing mean Hb concentration. Data from this study and others on the immunology of malaria , DSS, and PCD were used in the selection of a study site for a Phase Ib malaria vaccine trial of MSP3 . The data will also be useful in planning and implementing other trials in Korogwe district, such as a Phase 3 of RTS,S vaccine trial.This study has documented low level of malaria infections  in the lowland urban villages, which could be due to different factors; some of which could not be fitted or explained in this paper and therefore needs more exploration. Use of sensitive PCR methods for detection of asexual and sexual parasites to rule out sub-microscopic parasitaemia is recommended. Effect of PCD in the area was found to have led to a significant reduction in prevalence of both asexual and sexual forms of PCD: passive case detection; CORPs: community resource person(s); mASL: meters above sea level; % percent; < less than; dL: decilitre; OR: odds ratio; AMANET: African Malaria Network Trust; MSP3: merozoite surface protein 3; DSS: demographic surveillance system; μL: microlitre; EDTA: ethylenediaminetetraacetic acid; SES: socio-economic status; GEE: generalized estimating equation; Hb: haemoglobin; ACT: artemisinin-based combination therapy; PCR: polymerase chain reaction; CMP: Centre for Medical Parasitology; JMP: Joint Malaria Programme; NMCP: National Malaria Control Programme; NIMR: National Institute for Medical Research; MoHSW: Ministry of Health and Social Welfare; IQR: Inter-quartile range; SE: standard error; 95%CI: ninety five percent confidence interval; g/dL: grams per decilitre; GMPD: Geometric mean parasite densityThe authors declare that they have no competing interests.BPM participated in designing the study, data collection, data analysis, interpretation of results and manuscript preparation. MDS, DI, ASR and MS assisted in designing the study, data collection and manuscript preparation. HAM and SHS assisted in clinical assessment of clients. FF assisted in data management. MLK and MML participated in designing the study, supervision of field work, interpretation of results and manuscript development. All authors read and approved the manuscript.


Plasmodium falciparum P126 protein is an asexual blood-stage malaria vaccine candidate antigen. Antibodies against P126 are able to inhibit parasite growth in vitro, and a major parasite-inhibitory epitope has been recently mapped to its 47 kDa N-terminal extremity (octamer repeat domain – OR domain). The OR domain basically consists of six octamer units, but variation in the sequence and number of repeat units may appear in different alleles. The aim of the present study was to investigate the polymorphism of P126 N-terminal region OR domain in P. falciparum isolates from two Brazilian malaria endemic areas and its impact on anti-OR naturally acquired antibodies.The The study was carried out in two villages, Candeias do Jamari (Rondonia state) and Peixoto de Azevedo (Mato Grosso state), both located in the south-western part of the Amazon region. The repetitive region of the gene encoding the P126 antigen was PCR amplified and sequenced with the di-deoxy chain termination procedure. The antibody response was evaluated by ELISA with the Nt47 synthetic peptide corresponding to the P126 OR-II domain.Only two types of OR fragments were identified in the studied areas, one of 175 bp (OR-I) and other of 199 bp (OR-II). A predominance of the OR-II fragment was observed in Candeias do Jamari whereas in Peixoto de Azevedo both fragments OR-I and OR-II were frequent as well as mixed infection (both fragments simultaneously) reported here for the first time. Comparing the DNA sequencing of OR-I and OR-II fragments, there was a high conservation among predicted amino acid sequences of the P126 N-terminal extremity. Data of immune response demonstrated that the OR domain is highly immunogenic in natural conditions of exposure and that the polymorphism of the OR domain does not apparently influence the specific immune response.P. falciparum isolates and that this limited genetic polymorphism does not seem to influence the development of a specific humoral immune response to P126 and its immunogenicity in the studied population.These findings confirm a limited genetic polymorphism of the P126 OR domain in  Plasmodium falciparum P126 protein is an asexual blood-stage malaria vaccine candidate antigen. P126, also referred to as SERA [in vitro, and a major parasite-inhibitory epitope has been recently mapped to its 47 kDa N-terminal extremity (octamer repeats domain – OR domain) [Saimiri and Aotus monkeys with different fractions of P126, including the Nt47 domain [P. falciparum antigen repetitive regions is thought to contribute to immune invasion and also appear to restrict the effectiveness of subunit vaccines. In fact, although the P126 structure has been proven to be remarkably conserved among P. falciparum isolates, the P126 gene is a member of a multigene family [P. falciparum isolates (n = 52) from three areas of the Brazilian Amazon have identified the occurrence of the two P126 OR fragments, the most frequent constituting six octamer repeats [P. falciparum isolates (by PCR-SSCP and sequencing) and evaluate its impact on the natural OR-antibody responses in malaria-exposed Brazilian Amazon individuals.The  as SERA , estimat as SERA . Upon re as SERA , the com as SERA . The P12 as SERA , suggest as SERA  and rein domain) ,11. More7 domain , induced7 domain ,17 and c7 domain ,18, sugg7 domain . Therefoe family . Most poe family -24. The e family  involvedThe study was carried out in two villages, Candeias do Jamari  in Rondonia state and Peixoto de Azevedo  in Mato Grosso state, both located in the south-western part of the Amazon region. This region became the target of a large influx of people from other Brazilian regions during the 70s and 80s and malaria is hypoendemic to mesoendemic and is present throughout the year with seasonal fluctuations. The economic activities in both villages were subsistence farming, but gold-mining have become an attractive trade to Peixoto de Azevedo village. The population of Candeias do Jamari is composed of natives (30%) and migrants (70%) inhabiting this area for variable times since the 1970s. The first sample set were collected in 1993 (Candeias do Jamari-1993) and the second sample set was obtained nine years later, in 2002 (Candeias do Jamari-2002). The average annual parasite incidence (API), in Candeias do Jamari, was 549 in 1993 and 173 in 2002. For reference, the Brazilian Ministry of Health considers high risk areas those with API ≥ 50. The population of Peixoto de Azevedo is consisted mainly of gold miners and they frequently come from other gold prospecting places inside the Amazon. The samples were collected in 1995 in Peixoto the Azevedo (PA group) when the API was 292. Malaria transmission in both villages occurs throughout the year, with seasonal fluctuations with an increase of incidence at the beginning and at the end of dry season when all samples were collected.P. falciparum infected individuals: 96 from Peixoto de Azevedo in 1995, 103 from Candeias do Jamari in 1993 (Candeias do Jamari-1993 group) and 78 Candeias do Jamari in 2002 (Candeias do Jamari-2002 group). OR fragments polymorphism in samples from three cohorts  were used in order to evaluate whether the OR fragment polymorphism in Brazil was present and appeared to be temporally stable in the same area (1993 and 2002) or if it was spread throughout different areas (Candeias do Jamari and Peixoto de Azevedo). All P. falciparum malaria patients who were enrolled in this study complied with the following criteria: 1) they presented symptoms; 2) they had unique P. falciparum thick blood smears; 3) they neither used chemoprophylaxis nor took antimalarial drugs (self-treatment); 4) they were all over 15 years old; 5) no females were pregnant or breast feeding and; 6) blood collection was performed the day of diagnosis before malaria treatment. The patients sought health care at Brazilian health services 3.6 ± 1.8 days after onset of symptoms. After blood sample collection, the patients were immediately treated according to Brazilian Ministry of Health standards for malaria therapy with quinine plus doxycycline and primaquine.Written informed consent was obtained from all donors and venous blood samples were taken from P. falciparum in the thick and thin blood smears were enrolled in our study. The P. falciparum parasite density was determined by counting parasites in a predetermined number of white blood cells in thick blood films, and the number of blood parasites per milliliter was calculated [2).Thin and thick blood smears were examined for identification of malaria parasite by two expert malaria microscopists from Brazilian Malaria Health Services and from the Laboratory of Malaria Research (Fiocruz) which is a reference in malaria diagnosis from Brazilian Ministry of Health. Thick blood smears from all subjects were stained with Giemsa, and a total of 200 microscopic fields were examined under 1000-fold magnification. Thin blood smears of positive samples were examined for species identification. Only patients infected with lculated . The freg for 10 min. The pellets were mixed with 300 μL of NET buffer  containing 0.1% (w/v) Sarcosyl and 200 μg proteinase K and were kept for 3 h at 42°C. The sample was then extracted once with phenol/clorophorm/isoamyl alcohol (25/24/1 w/v) and then once with clorophorm/isoamyl alcohol (24/1 w/v). The DNA was precipitated with cold ethanol and reconstituted in 50 μL of TE buffer .The isolation of DNA from blood was carried out using the method previously described . One μL p126A (5'-AAT GAA GTC ATA TAT TTC CTT G-3') and p126B (5'-CAA TGT TGT TCT TAA TTC GAT A-3') and the second set of primers p126C (5'-GTG TTA TAT TTA ACA AAA ATG-3') and p126D (5'-CTT ACA GGA TTG CTT GGT TCG-3') [® DNA Polymerase  and 5 μL of 10× buffer . Both single and nested PCR reactions were carried out using a GeneAmp® PCR System 9700  for thirty cycles . Ten μL of PCR reaction were loaded onto a 2.5% agarose gel  in 1× TAE buffer  in the presence of ethidium bromide (0.5 μg/mL).The repetitive region of the gene encoding the P126 antigen was amplified in a Nested PCR method using the first set of primers  TCG-3') . One μL RsaI  and then extracted with phenol-chloroform and precipitated with ethanol. The samples were denatured prior to analysis by adding one μL of 0.5 M NaOH/10 mMEDTA and then electrophoresed for 3 h at 10 V/cm using 0.5 × TBE buffer (pH8.4). SSCP patterns were visualized by silver staining as described elsewhere [In order to assess sequence microheterogeneity within fragments defined as the same genotype by PCR, each amplified fragment was analysed by SSCP. The PCR sample was previously concentrated with 0,3 M of sodium acetate pH 5.2, recovered in 20 μL of distilled water and loaded onto a 2% Nusieve GTG agarose gel  in 1 × TAE buffer . The amplified fragments were individually gel-purified using Wizard PCR preps  as recommended by suppliers and resuspended in 40 μL of distilled water. PCR fragments were digested to completion with lsewhere .Taq polymerase and fluorescently labeled  with the primers p126C 5' GTG TTA TAT TTA ACA AAA ATG 3' (forward) and p126D 5' CTT ACA GGA TTG CTT GGT TCG-3' (reverse). Nucleotide sequences were obtained by the automated method  and the sequences assembled by using SeqMan II, included in the DNASTAR software package . The OR domain sequences reported for field isolates have been submitted to EMBL/GenBank Data libraries under accession number EU360815 and EU360816.Uncloned amplified fragments were sequenced using the di-deoxy chain termination procedure with The graphical representations of the predicted antigenic determinants on the OR domains were generated using the program DNASTAR software package  and the antigenic indexes were calculated using the method described by Jameson & Wolf .P. falciparum parasite isolates from different malaria endemic regions [The Nt47 synthetic peptide corresponding to the six octamer repeats (OR-II) of the P126 amino terminal region ,33 was p regions .2O2 in citrate-phosphate buffer pH 5.0. Plates were read at 492 nm in a spectrophotometer . Each sample was tested in duplicate. Sera from 25 control individuals living in Rio de Janeiro and never exposed to malaria transmission, were used to establish the normal range for the assay. The cutoff value was determined as the mean optical density (OD) 3 standard deviations of the Rio controls . To standardize the OD data obtained in different experiments and to evaluate the specific antibody levels, an OD index was calculated for each immunoglobulin determination as the ratio between the absorbance of each tested samples and the cutoff values. Sample with OD index > 1.0 was considered positive.The ELISA was performed as previously described ,19. Brie®  data bank software. Statistica  and Epi-Info 6  statistical software programs were used for data analysis. The Student's t-test was used to analyse the differences in mean values, the chi-square test was used to analyse the difference in prevalence of positive responses and of allelic frequencies, and the Spearman rank coefficient test used to analyse the correlations between variables.The data were stored in the Fox-plusP. falciparum samples were collected from malaria patients from Candeias do Jamari-1993 (n = 103), Candeias do Jamari-2002 (n = 78) and Peixoto de Azevedo (n = 96). The average age was similar among individuals from Candeias do Jamari-1993 (30 ± 12 years old), Candeias do Jamari-2002 (33 ± 14 years old) and Peixoto de Azevedo (29 ± 10 years old). The time of residence of individuals in the endemic regions were 13 ± 10 years in Candeias do Jamari-1993, 21 ± 11 years in Candeias do Jamari-2002 and 4 ± 3 years in Peixoto de Azevedo, the Candeias do Jamari-1993 and Candeias do Jamari-2002 groups residing on average longer in a malaria endemic area than that in Peixoto de Azevedo . As expected, the Candeias do Jamari-2002 group had a higher mean time of residence in the malaria endemic area than the Candeias do Jamari-1993 group . The mean parasitaemia at the time of blood collection were 9,642 ± 10,301, 2,150 ± 654 and 9,288 ± 9,058 parasites/μL in the Candeias do Jamari-1993, Candeias do Jamari-2002 and Peixoto de Azevedo groups, respectively. The Candeias do Jamari-2002 group demonstrated a lower parasitaemia than the Candeias do Jamari-1993 and Peixoto de Azevedo groups . The great majority of the individuals claimed to have experienced malaria episodes previously. Only 5% (n = 9) of the individuals from Candeias do Jamari-1993 and 2002 and 9% (n = 9) of the individuals from Peixoto de Azevedo denied prior malaria infection.Throughout the study period, 277 P. falciparum wild isolates were, respectively, 7.4% and 92.6% in Candeias do Jamari-1993 and 9.8% and 90.2% in Candeias do Jamari-2002. Predominance of the OR-II fragment was evident in the P. falciparum samples from Candeias do Jamari both 1993 and 2002. Curiously, a different pattern appeared in relation to OR fragment frequencies in Peixoto de Azevedo where OR-I fragment frequency (40.5%) was slightly lower than the OR-II fragment (59.5%). In Peixoto de Azevedo there was a higher frequency for the OR-I fragment when compared to Candeias do Jamari-1993 and Candeias do Jamari-2002  while the frequency of the OR-II fragment was lower .Only two types of OR fragments were identified in the studied areas by PCR-SSCP analysis, one of 175 bp (OR-I) and the other of 199 bp (OR-II). The frequencies of OR-I and OR-II fragments found in P. falciparum mixed infection with OR-I and OR-II fragments in 4.9% (Candeias do Jamari-1993) and in 5.1% (Candeias do Jamari -2003). It is interesting to note that in Peixoto de Azevedo, there was a higher frequency of mixed infection (20.8%) than in Candeias do Jamari P. falciparum isolates . No relationship was established between infection patterns and age, sex or time of residence in endemic areas of Candeias do Jamari-1993, Candeias do Jamari-2002 and Peixoto de Azevedo.Table P. falciparum isolates from Candeias do Jamari-1993, Candeias do Jamari-2002 and Peixoto de Azevedo, a high conservation among predicted amino acid sequences of the P126 N-terminal extremity was evident. As shown in Figure Comparing the DNA sequencing of OR-I and OR-II fragments of wild The Nt47 synthetic peptide, which is based on the six octamer repeat sequence located at the N-terminal extremity of P126 (OR-II), was used in ELISA to detect specific IgM, IgG and IgG subclasses. Most of the individuals were tested for humoral immune responses against the OR-II domain.P = 0.01 by chi-square test) or Peixoto de Azevedo  . As shown in Table P < 0.05, for both – Table t-test; P < 0.05, for all analyses – Table t-test and Spearman rank coefficient test; P > 0.05, for all).There was a predominance of OR domain antibody responders, regardless of the class (IgG or IgM), in the Candeias do Jamari-2002 group (92%) when compared with those observed in Candeias do Jamari-1993  the prevalence of IgG1 and IgG3 OR-specific antibodies were significantly lower when compared with Candeias do Jamari-1993 and Peixoto de Azevedo groups .For the majority of individuals with a detectable IgG-OR antibody, subclasses of IgG were determined in Candeias do Jamari-1993 , Candeias do Jamari-2002  and Peixoto de Azevedo  groups. As shown in Table P. falciparum infections in both Candeias do Jamari-1993 and Candeias do Jamari-2002, the statistical analysis of antibody responses to the OR domain according to the infection patterns was performed only in the Peixoto de Azevedo. In this group the prevalence and/or the levels of OR-specific antibodies regardless of class and subclass – .Due to the small number of individuals with single (only OR-I) or mixed (OR-I and OR-II simultaneously)  – Table  were broP. falciparum OR-II fragment, the prevalence of OR IgM antibody responders was higher in Candeias do Jamari-2002 (81%) than in the Candeias do Jamari-1993  or Peixoto de Azevedo  groups , whereas in Candeias do Jamari-2002 there was a predominance of IgG3 OR-specific antibodies . The prevalence of IgG1 and IgG3 OR-specific antibodies was significantly lower in Candeias do Jamari-2002 when compared with Candeias do Jamari-1993 and Peixoto de Azevedo groups . However, in Candeias do Jamari from 1993 to 2003 the antibody levels were similar except for IgG1 that was significantly lower in 2002 . No association was derived with respect to age, sex, parasite densities or time of residence in a malaria endemic area and the prevalence or levels of OR IgG antibody subclass in any of three groups studied .Comparing the profiles of OR IgG subclasses among the three groups of individuals infected with the nt Table , there wP. falciparum isolates from two important Brazilian malaria endemic areas (Candeias do Jamari in 1993 and 2002 and Peixoto de Azevedo in 1995) and assess the impact of OR fragment polymorphism on specific, naturally acquired antibodies in the studied populations.The aims of this study were to investigate the polymorphism of the P126 N-terminal region octamer repeat domain (OR = Nt47 domain) in P. falciparum malaria infected individuals while in our study, mixed infection (both OR-I and ORII) was detected for the first time in inhabitants of both villages with high frequency in Peixoto de Azevedo. Also noteworthy are the differences in the OR fragment frequencies among P. falciparum isolates from Peixoto de Azevedo when compared with the isolates from other endemic areas. In Peixoto de Azevedo the OR-I fragment frequency (40.5%) was only slightly lower than OR-II (59.5%). The high mixed infection and single OR-I fragment prevalence in Peixoto de Azevedo in comparison with isolates from the Candeias do Jamari group as well as groups from a myriad of other geographical areas, is probably due to: i) an immunological status of this population, since studied individuals from this village, mainly migrants from malaria-free areas, have been inhabiting a malaria endemic area for an average of four years, ii) a relatively recent introduction of the variant into this population as a consequence of the gold miners' nomadic behaviour within the Amazon, facilitating new variant spread throughout the region and iii) the differences in the biological development of this variant in man and/or its invertebrate host. Although in a Brazilian endemic area [In Africa, Asia and South America, including Brazil ,26,36, oIt is well recognized that the complexity of infections may vary considerably in different epidemiological scenarios according to age and immune status of the individuals ,38. In tP. falciparum from Candeias do Jamari in 1993 and 2002, a difference of 9 years, suggesting that this portion of the P126 gene was both genetically homogeneous and temporally stable, and despite differing from the Candeias do Jamari groups in terms of frequencies and infection complexity, the Peixoto de Azevedo group shared an identical degree of polymorphism in the P126 OR domain. These results are similar to a study in the same endemic areas with the MSP2 protein [The sequences herein analysed, further confirm a limited degree of genetic polymorphism in the P126 OR domain, namely variation in the number of octamer repeats (OR-I five or OR-II six) and non-synonymous substitutions in repeat units ,26,36. I protein  that rev protein -24,36, tBased on the fact that B and T-cell epitopes are located in the OR domain , variatiP. falciparum vaccine candidates [One should be aware that a similar profile of IgG, IgG-isotypes and IgM antibody responses to the OR domain was apparent in the Candeias do Jamari -1993 and Peixoto de Azevedo groups, both exhibiting a predominance of IgG1 and IgG3 OR specific antibodies together with both cytophilic and complement fixing isotypes, in accordance with the data recently reported for the Uganda population . Howeverndidates ,42,44-47P. falciparum P126 OR domain presented a similar profile of IgG, IgG subclasses and IgM antibody response to the OR domain. Therefore, infection with P. falciparum containing OR-I and/or OR-II does not influence the response to the OR-II domain suggesting cross-reactivity of two epitopes within the repetitive sequence. Indeed, ELISA competition assay has already demonstrated that human sera from a population exposed to malaria recognize OR domains containing a minimum of four repeats [One should be aware that the prevalence of antibody responses to the OR domain in the Peixoto de Azevedo group infected with different patterns of the  repeats .P. falciparum isolates in a Brazilian population. This polymorphism does not seem to influence the development of a specific humoral immune response , which is paramount considering that genetic polymorphism, even though limited in regions encoding immunodominant epitopes, is important for malaria vaccine development regarding the potential parasite escape mechanisms involved with respect to the host immune system. Thus in addition, it was confirmed that there was the predominance of cytophilic antibodies against the P126 OR domain could participate in cellular antibody cooperation mechanisms, which might play a prime role in the development of protective immune responses against P. falciparum malaria infection [In conclusion, there is a display of a limited genetic polymorphism of the P126 OR domain in nfection ,42,47. FThe authors declare that they have no competing interests.LRLR participated in the design of the study; carried out the molecular and immunological studies; performed the statistical analysis and drafted the manuscript, SLSS participated in the design of the study and helped in molecular analysis, BTS helped in immunological analysis, MLG and MGM helped in molecular analysis, FS and JOF helped in design and implementation of field studied, MGZ, DC, MFFC and CTDR helped in the design of the study, TSS and GSS helped in predictive analysis, DMB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.


Plasmodium cynomolgi and P. vivax, we conducted phylogenetic analyses of small subunit ribosomal RNA gene sequences of Plasmodium spp. We found that these orangutans are not hosts of P. cynomolgi and P. vivax. Analysis of >1 genes is needed to identify Plasmodium spp. infecting orangutans.After orangutans in Indonesia were reported as infected with  Plasmodium cause malaria and are usually host specific. For example, humans are natural hosts for P. falciparum, P. vivax, P. malaria, and P. ovale, and orangutans are naturally infected with P. pitheci and P. silvaticum  genes that are developmentally regulated , and is not the macaque malaria parasite, P. cynomolgi, as previously reported (.The VS63 sequence is clearly not Plasmodium spp. isolated from orangutans in Kalimantan, Indonesia, are P. cynomolgi or P. vivax, as previously reported by Reid et al. (. Before any conclusion about the identity of the malaria parasites infecting orangutans and their corresponding SSU rRNA gene sequences can be derived, a second or third gene of malaria parasites from these orangutans needs to be analyzed and the morphology of the corresponding blood stages needs to be described. Our study underscores the importance of using orthologous genes and sequence data from as many species as possible when inferring species within a genus from phylogenetic trees.Phylogenetic analyses of the SSU rRNA genes indicate that none of the