Prescribing antimalarial medicines based on parasite confirmed diagnosis of malaria is critical to rational drug use and optimal outcome of febrile illness. The impact of microscopy-based versus clinical-based diagnosis of childhood malaria was assessed at primary health care (PHC) facilities using a cluster randomized controlled training intervention trial.Sixteen PHC facilities in rural Tanzania were randomly allocated to training of health staff in clinical algorithm plus microscopy  or clinical algorithm only  or no training . Febrile under-five children presenting at these facilities were assessed, treated and scheduled for follow up visit after 7 days. Blood smears on day 0 were only done in Arm-I but on Day 7 in all arms. Primary outcome was antimalarial drug prescription. Other outcomes included antibiotic prescription and health outcome. Multilevel regression models were applied with PHC as level of clustering to compare outcomes in the three study arms.A total of 973, 1,058 and 1,100 children were enrolled in arms I, II and III, respectively, during the study period. Antimalarial prescriptions were significantly reduced in Arm-I (61.3%) compared to Arms-II (95.3%) and III (99.5%) (both P < 0.001), whereas antibiotic prescriptions did not vary significantly between the arms . In Arm-I, 99.1% of children with positive blood smear readings received antimalarial prescriptions and so did 11.3% of children with negative readings. Those with positive readings were less likely to be prescribed antibiotics than those with negative . On day 7 follow-up, more children reported symptoms in Arm-I compared to Arm-III, but fewer children had malaria parasitaemia (p = 0.049). The overall sensitivity of microscopy reading at PHC compared to reference level was 74.5% and the specificity was 59.0% but both varied widely between PHCs.Microscopy based diagnosis of malaria at PHC facilities reduces prescription of antimalarial drugs, and appears to improve appropriate management of non-malaria fevers, but major variation in accuracy of the microscopy readings was found. Lack of qualified laboratory technicians at PHC facilities and the relatively short training period may have contributed to the shortcomings.This study is registered at Clinicaltrials.gov with the identifier NCT00687895. Plasmodium falciparum in sub-Saharan Africa is estimated to be 365 million clinical episodes resulting in over one million fatal cases, mostly in children underfive years of age [The annual burden of s of age . In Tanzs of age .Prompt and effective case management is critical for the successful implementation of malaria control strategy . AlthougMost of malaria endemic African countries have adopted the use of highly effective but expensive artemisinin-based combination therapy (ACT). Restricted and better directed treatment is increasingly advocated to prevent drug resistance and reduce the costs of these drug combinations ,10. EffoThe establishment and maintenance of reliable and efficient malaria microscopy at PHC level requires organized health system infrastructure, with functioning microscopes, well trained health workers, regular provision of reagents, supervision and quality control ,16. ThisPlasmodium falciparum is the dominant parasite species. In Kibaha the government health facilities consist of one hospital and 19 Primary Health Care (PHC) facilities  and Bagamoyo has one hospital and 41 PHC facilities . Most PHC facilities are in the rural areas where the majority of the population live. The health staff includes assistant medical officers (AMO), clinical officers (CO), assistant clinical officers (ACO), mother and child health aides (MCHA), trained nurses, nurse auxiliaries and laboratory assistants. They are supervised by the district medical officer (DMO) and other senior health officers from the district hospital.The study was conducted in two neighbouring districts, Kibaha and Bagamoyo, situated in the Coast region, north of Dar es Salaam Tanzania, and with estimated populations of about 103,000 and 240,000, respectively. They have similar geographical characteristics. Malaria transmission is holoendemic, with some accentuation during the rainy seasons in May-July and December to January. PHC facilities receive medicines including antimalarial drugs monthly through the essential drug program-Kit system from the Medical Store Department (MSD), a semi-autonomous institution located in part under the Ministry of Health. Microscopes are available in health centers and some dispensaries, but their use is limited because of lack of qualified/skilled laboratory technicians. At the time of study, sulphadoxine/pyrimethamine (SP) and amodiaquine (AQ) were the first- and second-line treatments for uncomplicated malaria and quinine (QN) the drug of choice for severe malaria.The study was designed as a cluster-randomized controlled intervention evaluating two intervention packages Figure . PHC facChildren attending the study PHC facilities during daytime were enrolled if they fulfilled the following criteria: a) under five years of age, b) fever (axillary temperature ≥ 37.5°C) and/or history of fever in last two days, c) able to return to the facility on day 7 after treatment or any other day if symptoms were to worsen or recur and d) the mother/guardian consented to participate. Patients with severe illness requiring inpatient care according to the IMCI guidelines were admitted or referred to the health centres or the district hospitals. Both consultation and treatments are provided free for under-five children.Based on national malaria treatment and the IMCI guidelines, a clinical training package was developed aiming at improving the knowledge and the skills of the health workers to clinically diagnose and treat malaria. The content of the package included (a) description of signs and symptoms of malaria disease; (b) history taking relevant to malaria and physical examination; (c) identification of danger signs and severe illness for referral; (d) appropriate treatment; (e) counselling patients on the use of drugs.Based on the World Health Organization (WHO) basic malaria microscopy training manual , a trainThe training for both packages included presentations of theoretical information, brain storming and group discussion. Clinical demonstrations were provided by the research team illustrating how to take history, physical examination, diagnosis, prescribing and counselling. There were also clinical practice sessions with the health care workers assessing patients under supervision with individual feedback and practical sessions on malaria microscopy which included blood smear preparation and reading, parasite counting and artefact identification. A pilot training programme conducted in rural Dar es Salaam PHC facilities demonstrated its feasibility and feedback from the participants was used to improve the final training programme.Health workers in Arm-I and II facilities received five-day group training on management of malaria on clinical basis and the health workers in Arm-I received an additional five-day group training in malaria microscopy. No training was provided to those in Arm-III. In all three arms, health workers were trained on how to fill the case record forms and how to prepare malaria blood smears. All health facility staff involved in the care of under-fives were included in the training i.e. AMOs, COs, ACOs, MCHAs, nurses, nurse assistants and laboratory assistants/attendants. In Arm-I, microscopy was performed by assistant laboratory technicians with one year training in two PHC facilities  and by the COs in three facilities .The training was held at each health facility during working hours; practical training was conducted in morning sessions and the theoretical part in afternoons. The training was facilitated by two PhD research students  assisted by a senior laboratory technician from MUCHS and supervised once a week by senior members of the team. The project provided all study health facilities with all supplies needed for making blood smears. In addition each health facility in Arm-I was provided with one light microscopy and malaria microscopy guidelines.Children presenting at the study facilities with fever were enrolled in the study and information on age, sex, duration of fever, other presenting symptoms and body temperature was recorded. Assessment of body temperatures (axillary) were performed by using mercury thermometers. In Arm-I, a thick blood smear for malaria was collected from each patient and examined by the health worker (first reading) and haemoglobin levels were estimated using the Tallquist test. The collected blood smears were forwarded weekly to MUCHS for second reading by an experienced laboratory technician for malaria microscopy. In Arm-II and Arm-III, blood smears for malaria and Tallquist test were not done on day 0. Diagnosis and prescribed drugs  were recorded at the end of the consultations.In all three arms, patients were scheduled for day 7 follow-up visits and encouraged to report at any time prior to day 7 if necessary. The health workers assessed them for new or persistent symptoms, any concomitant medication taken and any adverse event perceived. In addition, thick blood smears were taken during the follow-up visits and examined by the second reader at MUCHS. Patients who failed to attend the health facility for day 7 follow up were visited at home to assess their health status and to identify possible reasons for not attending scheduled follow up.At the end of the study special interviews were conducted with health workers in Arm-I to assess whether and under what circumstances they prescribed antimalarial treatment to patients with negative blood smear results.Thick blood smears were stained with 5% Giemsa for 20 minutes. Microscopy reading at PHC level included species identification and parasite count. The parasite density was estimated by counting the number of asexual parasites per 200 white blood cells and calculating parasites per μL, assuming a white cell count of 8,000 cells per μL. A smear was declared negative if no asexual parasites were seen after examining 100 high power fields. Every week an experienced microscopist at MUCHS re-examined all blood smears without knowledge of the first reading. The result of the second reading was compared with the first reading and if discordant results (positive vs. negative) or even two fold difference in parasite density, a third decisive microscopy reading, again blinded was to be done.Using a Tallquist paper scale method, a drop of blood absorbed on a test strip of filter paper was matched against the scale standards by moving it up and down, to determine the equivalent Hb value. The Tallquist paper scale has colours which range from yellow to brown in a scale of 10%–100% which corresponds to Hb range from 1.58 to 15.8 g/dL.The primary outcome was the proportion of study children receiving prescriptions of antimalarial drugs in the respective arms. Secondary outcomes included prescriptions of antibiotics, cost of drugs and health outcome of the patients, i.e. the proportion of patients re-visiting the health facility due to clinical complaints on days 1 to 6 and the proportion reporting symptoms on day 7 follow-up. Full health improvement was determined by the proportion of the study patients in each arm alive and without complaints of fever or any other symptoms and without parasitaemia on day 7. The cost of drugs was calculated using price list from Medical Store Department. Drug costs for each patient were calculated based on the specific weight or age related drug regimen costs.The prescription rate of antimalarial drugs was assumed to be 90% in the control arm and the intervention package in Arm-I was assumed to reduce this to 70%. The minimum sample size for independent observations to give 90% power at a significance level of 5% was found to be 140 patients per comparative arm, when adjusted for multiple testing (Bonferroni). To account for secondary outcomes, potential clustering at PHC level and a loss to follow up, we decided to recruit 200 patients per facility giving approximately 1,000 patients per arm. Comparisons of the differences in outcomes for the study arms multilevel regression models were applied with PHC as level of clustering (XTMIXED command in STATA version 10). In the result section 95% confidence intervals (CI) are given and stated significances are on the 5% level. Descriptive results within treatment arms were not adjusted for clustering due to limited number of PHCs per treatment arm.Approval of the study was granted by the National Institute for Medical Research Tanzania and the Karolinska Institutet (D-nr 03-712) ethical committees. Permission to carry out the study was obtained from the district health team, wards authorities and village leaders and PHC facility workers. Informed written consent was sought from parents/guardians of the under-five children.A total of 3,131 children with fever were enrolled between July 2003 and March 2004 . SP, officially first-line antimalarial drug, was prescribed to most malaria patients (range 73.8%–100%) in 14 study health facilities and AQ, officially second-line antimalarial drug, was prescribed to most patients (48% and 79%) in the two remaining health facilities (one in Arm-I and another in Arm-III). Overall, in accordance with Tanzania national treatment guidelines 76.7% of antimalarial drugs were correctly (dosage and duration of treatment) prescribed; 9.8% were under-dosed; 10.2% were over-dosed; and in 3.3%, the treatment was correct but the prescription indicated insufficient duration. There was no significant difference in the proportion of antimalarial drugs correctly prescribed in the three arms.Table In all antibiotics were prescribed to 44.8% of all patients but there was no significant difference in the proportion of antibiotic prescriptions in the three arms Table . The antThe average number of drugs per prescription was higher in Arm-II compared to Arm-III (p = 0.003); other differences were not statistically significant. The average cost of all drugs in Arm-III was 0.170 US$. This increased by 47% in Arm-II  and by 18% in Arm-I (with microscopy) Table .A total of 934 blood smears in Arm-I were re-examined at the reference laboratory at MUCHS by the experienced microscopist  with positive blood smears but also to 50/442 (11.3%) with negative blood smears (Table Antibiotics were prescribed to 193/531 (36.3%) patients in Arm-I with positive blood smears compared to 241/442 (54.5%) patients with negative smears, indicating that malaria patients were less likely to receive antibiotics compared to patients without parasitaemia . Of the 442 patients with negative blood smears, 31% received neither antibiotic nor antimalarial drug i.e. only antipyretics and 4% had no record of any drug prescription. The influence of clinical malaria diagnosis on the antibiotic prescriptions could not be compared meaningfully in Arms II and III because almost all patients were considered as malaria and prescribed antimalarial drugs. Based on the readings of day 0 blood smears from Arm-I by the experienced microscopist at MUCHS, antimalarial drugs were prescribed to about 80% of the patients with parasitaemia and 46% without parasitaemia . In addition, 406 patients who did not turn up on day 7 follow up were visited in their homes on day 9 to 10. According to the post-interventions follow up at home; over 97% were reported to have recovered by day 7. The reasons provided by parents/guardians for not attending regular day 7 follow up included long distance to the facilities, travel or moved away and a few had no reasons.About 10% of patients in Arm-I and 5% of patients in Arms-II and III had complaints of mainly fever, vomiting, diarrhoea or cough, but there was no statistically significant difference between the arms Table . The freWhen defining full health improvement as absence of fever or any symptoms or parasitaemia, the general recovery rates on day 7 were 695/934 (74.4%) patients in Arm-I, 649/826 (78.6%) in Arm-II and 650/961 (67.6%) in Arm-III, but there was no statistically significant difference between the arms.This study documents that the use of microscope for malaria diagnosis significantly reduces prescriptions of antimalarial drugs when compared to use of clinical algorithm alone. The majority of health workers in Arm-I adhered to the blood smear results provided to them. In contrast, the practice of giving antimalarial drugs to patients with negative blood smears was significantly more frequent in other studies -22. One Prescription of antibiotics was also influenced by microscopy as patients in Arm-I with negative blood smears were more likely to be prescribed antibiotics compared to patients with positive blood smears. This is consistent with findings from northeast Tanzania . RestricThe findings from this study, further confirm the limited usefulness of clinical algorithms alone in malaria diagnosis as has been shown earlier ,12,23,24The current malaria treatment guidelines for Tanzania recommend any under-five child with fever or history of fever within the last 24 hours without evidence of other diseases should be treated for malaria even with a negative blood smear/RDT for malaria parasites . The ratThere was sub-optimal quality of antimalarial drug prescriptions among PHC health workers characterized by incorrect doses, incorrect duration and high rate of amodiaquine use in some health facilities. These prescribing patterns are similar to the results of previous cross sectional studies in Tanzania ,6. TheseClustering on PHC facilities was accounted for in the analysis and the relatively small number of PHC facilities randomized to each arm might have reduced the power of the study to show some important differences between study arms. However, this was partly compensated for by including a large number of patients. Selecting PHC facilities that were geographically accessible from Dar es Salaam might have introduced a selection bias. However the selected facilities were not different from those not included in the study in terms of number and quality of staff and availability of drugs.The overall accuracy of blood smear reading in this study was relatively low with a large variation in the estimated positive predictive values and negative predictive values in different PHC facilities. False positive smears were frequently reported, indicating a tendency among laboratory workers to report a blood smear as positive when they are in doubt. Also false negative smears were reported in some patients with high parasite counts, maybe due to insufficient reading time and poor smear quality. One such child developed severe malaria. These reading inaccuracies will result in delayed malaria diagnosis, severe illness and also undermine the management of non-malaria associated illness. A national laboratory training programme in Ghana demonstrated improvement in diagnostic accuracy of microscopy in PHC facilities . The parThe findings of the present study indicate that microscopy supported malaria diagnosis reduces over-prescription of antimalarial drugs. However, the accuracy of the microscopy readings in this study was suboptimal despite the extra training and supervision provided by the study personnel. Lack of qualified laboratory personnel at PHC facilities in Tanzania and the relatively short training period of the staff may have contributed to the shortcomings. This calls for a simple malaria diagnostic tool for PHC facilities and whether the role of RDTs may be more efficient than microscopy in such peripheral health care settings needs to be investigated. Clearly laboratory tests confirming the presence of malaria parasites will be increasingly required, especially in the context of possible future reduction in the incidence of malaria related disease ,30. ThisThe authors declare that they have no competing interests.BN planned the study, conducted the field trial, data analyses and interpretation, and wrote the report. MM conducted the field trial and data analysis. MW contributed to the study planning and design, interpretation of results and write up of the manuscript. MGP assisted in study planning, designed data analyses and interpretation. AM participated in study planning and coordinated the field trial. ZP participated in study planning and coordinated the field trial. LLG involved in study planning and review of manuscript. GT involved in study planning and review of manuscript. AB contributed to study planning, design, and coordination, interpretation of results and preparation of the manuscript. All authors read and approved the final manuscript.The study received financial support from the SIDA/Sarec bilateral project between KI and MUCHS(Bil-Tz 16/98 75007059)


IL13 promoter region, IL13 -1055T>C (rs1800925), was associated with susceptibility to severe malaria in Thais. In the present study, fine association mapping for a cytokine gene cluster including IL4, IL5, and IL13 on chromosome 5q31 was conducted using the same malaria subjects to refine the region containing a primary variant or a haplotype susceptible to severe malaria.It has been previously demonstrated that a single nucleotide polymorphism (SNP) in the Plasmodium falciparum malaria .A total of 82 SNPs spanning 522 kb of the 5q31 region were analysed in 368 patients with IL13, which is in linkage disequilibrium with rs1800925 (r2 = 0.73), showed a significant association with severe malaria after adjusting for multiple testing (P = 0.046 by permutation test). This SNP was in a haplotype block spanning 97 kb (from rs2069812 to rs2240032). The detected haplotype block contained the RAD50 gene and the promoter of IL13, but not the other genes.Only rs1881457 located in the promoter region of A haplotype block in which a primary polymorphism associated with severe malaria is likely to be encoded was identified in Thai malaria patients. Plasmodium falciparum and the human chromosome 5q31 region in African populations [IL3, IL4, IL5, IL9, and IL13) and other immunologically active genes such as interferon regulatory factor-1 (IRF1). These genes are strong candidates for controlling the outcome of malaria infection.Over the course of the last decade a number of studies have provided evidence for a linkage between the blood infection level of ulations . In addiulations . The 5q3IL3, IL4, and IL13 were investigated. Of which, a SNP in the IL13 promoter region, IL13 -1055T>C (rs1800925), was found to be associated with susceptibility to severe malaria in Thais [In a previous study, three single nucleotide polymorphisms (SNPs) in the promoter regions of in Thais . HoweverP. falciparum malaria  living in northwest Thailand were enrolled in this study. All patients underwent treatment at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University. Malarial infection by P. falciparum was confirmed by a positive blood smear for the asexual form of P. falciparum. Clinical manifestations of severe and mild malaria were classified according to the following definitions and criteria. A patient was classified as severe malaria when he/she has one or more of the following signs: high parasitaemia , hypoglycaemia (glucose <22 nmol/l), severe anaemia (haematocrit <20% or haemoglobin <7.0 g/dl), and a serum creatinine level of more than 3.0 mg/dl. In the present study, patients with cerebral malaria were not analyzed. Mild malaria was characterized by fever without other any underlying causes of infections and no manifestations of severe malaria as described above. All individuals were 13 years of age or older, and the mean ages of patients with mild malaria and those with severe malaria were 25.3 and 23.8, respectively. This study was approved by the institutional review board of the Faculty of Tropical Medicine, Mahidol University, and the Research Ethics Committees of the Faculty of Medicine, The University of Tokyo, and the Graduate School of Comprehensive Human Sciences, University of Tsukuba. Informed consent was obtained from all participants.A total of 368 adult patients with Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp Blood Kit . A total of 82 SNPs within a 522 kb region on human chromosome 5q31 were genotyped by using the DigiTag2 assay .2) between SNPs were calculated to evaluate the structure of LD on 5q31-33 in 368 Thai malaria patients. The frequencies of haplotypes consisting of rs2069812, rs2299015, rs2299014, rs2243677, rs2522414, rs2299013, rs2252775, rs2522394, rs2245460, rs2301713, rs3798135, rs2237060, rs2074369, rs2240032, rs1881457, and rs1800925 were estimated only for this haplotype block. All the statistical analyses were performed by using the Haploview software version 4.0 [Pan troglodytes (chimpanzee), obtained from the NCBI BLAST database (Table Macaca mulatta (rhesus macaque) was used.The allele frequency at each SNP locus was compared between mild and severe malaria patients using the chi-square test and a permutation P value was calculated from 100000 permutations. A permutation P value of less than 0.05 was considered to be statistically significant. The pairwise LD coefficients  produced by activated Th2 cells. The Th2 cytokines down-regulate macrophage activity, and inhibit the production of pro-inflammatory cytokines such as TNF and IL1. It has been reported that increased concentrations of TNF and IL1β in serum are observed in severe malaria patients [IL13 promoter polymorphisms may influence the expression of IL13. Thus, both RAD50 and IL13 seem to be plausible candidate genes associated with severe malaria.In this study rs1881457 was found to be significantly associated with severe malaria, and this SNP was included in a haplotype block encompassing the whole patients . The IL1IL5, IL13, and IL4 are subject to coordinate regulation and are expressed in a cell lineage-specific manner [RAD50 [The genes encoding the Th2 cytokines c manner ,18. The r [RAD50 . InteresIL13, the nucleotide change at this site may affect the binding ability of some transcription factor. The TFSEARCH  program based on the TRANSFAC databases [Since rs1881457 is located in the promoter region of atabases  was usedIL13 polymorphisms, rs1800925 in the IL13 promoter has been reported to be associated with various diseases [IL13 [2 value (r2 = 0.73). Therefore, the possibility that rs1800925 is a primary SNP and the significant association of rs1881457 with severe malaria resulted from LD between these SNPs is not excluded. The future functional and association studies for rs1881457, rs1800925, and other polymorphisms, including those not analyzed in the present study, may thus help us to better understand the genetic susceptibility to severe malaria.Among diseases -24. Thises [IL13 . In the RAD50 gene and IL13 promoter region that was associated with severity of malaria was identified in a Thai population.A haplotype block spanning 97 kb encompassing The authors declare that they have no competing interests.NI carried out the genotyping, helped to conduct statistical analyses, and wrote the manuscript. NN and KT helped to genotype the samples. JP, PN and HH collected blood samples, extracted DNA, and helped to genotype the samples. JP participated in the design of the study and coordination. NT was involved in the interpretation of the data and preparation of the manuscript. JO conceived of the study, and participated in its design, performed statistical analyses, and helped to write the manuscript. All authors read and approved the final manuscript.


Dengue–malaria co-infection reports are scarce. Of 1,723 consecutive febrile patients in Cayenne Hospital, 238 had dengue (174 early dengue fever cases) and 393 had malaria ; 17 had both. Diagnosis of 1 of these 2 infections should not rule out testing for the other infection. Plasmodium falciparum) and dengue were identified in July 2005 (P. vivax) . Virus isolation or reverse transcription–PCR (RT-PCR) according to Lanciotti et al. . The asexual parasite load (PL) was quantified in percent parasitized erythrocytes for values Serologic immunoglobulin (Ig) M testing was performed on all serum samples . Dengue was detected in 238 case-patients (13.8%); among these, 73% (174/238) were confirmed by positive virologic diagnosis (isolation or RT-PCR), whereas 27% were probable dengue cases (positive IgM serology only). The first group was named early dengue cases (EDC) and the second group late dengue cases (LDC).P.vivax, 133 (33.8%) were P.falciparum, 2 were P. malariae, and 7 were mixed P.vivax and P.falciparum. Most malaria-positive case-patients had a parasite count above class 2 (371/393 [94.4 %]), indicating acute malaria.Of the 1,723 patients, 393 (22.8%) had smear-positive malaria; of those, 251 (63.9%) were P. vivax malaria infection, was hospitalized for interstitial pneumonia with severe anemia. Intubation, blood transfusion, and antimicrobial drugs were required, but he was discharged from the intensive care unit after 11 days. No causative agent was identified for this pneumonia. The second patient was hospitalized for diabetes, the third because treatment with Riamet (arthemether + lumefantrine) was only available to inpatients.Concurrent dengue (EDC and LDC) and malaria were confirmed in 17 of the 1,723 patients (1%), corresponding to 7.1% (17/238) of dengue cases and 4.1% (16/393) of malaria cases, respectively . When coMalaria and dengue must be suspected in febrile patients living in or returning from areas endemic for these infections. Although the usual places of contamination by malaria and dengue viruses are quite different in French Guiana, considering that the incubation phase is longer for malaria than for dengue and that the population’s mobility is high, a simultaneous clinical expression of the 2 diseases is plausible. Moreover, in French Guiana, dengue viruses have spread to malaria-endemic rural areas  by RT-PCR, which is faster and more specific. In published case reports  because of low parasitemia . Two patients, 1 of whom was part of the EDC group, could have been asymptomatic carriers of P. vivax (EDC were diagnosed on average after 4 days of fever, never 5. Thus, delayed complications of dengue or malaria may not be detected using this definition. Such complications could be observed in patients considered LDC. One of these patients had pneumonia, which has recently been described as a complication of P. falciparum is implicated. The distinction between severe dengue and severe malaria must be made in an emergency department or hospital setting because in both situations, early diagnosis is essential for patient care.Although acute concurrent infections were benign in our study, special attention should be given to the possibility of co-infection with malaria and dengue, especially when


Plasmodium falciparum disease was evaluated in 104 travellers with imported malaria .Most clinicians in developed, non-malaria endemic countries have limited or no experience in making clinical assessments of malaria disease severity and subsequent decisions regarding the need for parenteral therapy or high-level monitoring in febrile patients with imported malaria. In the present study, the diagnostic accuracy of plasma soluble Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), neopterin and procalcitonin levels as biomarkers for severe TREM-1, neopterin and procalcitonin were determined in serum using commercially available ELISA or EIA tests. The diagnostic performance of these biomarkers for severe disease was compared with plasma lactate, a well-validated parameter for disease severity in patients with malaria, as reference. Severe malaria was defined according to the modified WHO criteria.P. falciparum malaria had significantly higher neopterin and procalcitonin levels on admission when compared to patients with uncomplicated P. falciparum malaria or non-P. falciparum malaria. Receiver Operating Characteristic (ROC) curve analysis showed that neopterin had the highest Area-Under-the-ROC curve (AUROC 0.85) compared with plasma lactate (AUROC 0.80) and procalcitonin (AUROC 0.78). At a cut-off point of 10.0 ng/ml, neopterin had a positive and negative predictive value of 0.38 and 0.98 whereas procalcitonin, at a cut-off point of 0.9 ng/ml, had a positive and negative predictive value of 0.30 and 1.00.No significant differences in TREM-1 levels were detected between the different patient groups. Patients with severe Although the diagnostic value of neopterin and procalcitonin is limited, the high negative predictive value of both neopterin and procalcitonin may be helpful for a rapid exclusion of severe malaria disease on admission. This may be a valuable tool for physicians only occasionally dealing with ill-returned travellers from malaria-endemic regions and who need to decide on subsequent oral anti-malarial treatment or timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment. Travellers from industrialized countries and inhabitants of malaria-endemic regions clearly represent two distinct worlds of malaria . The gloIn striking contrast, in non-endemic industrialized countries malaria is only seen as an occasionally imported disease  and is uP. falciparum malaria, these rapid tests do not provide any information about the severity of the infection. Moreover, although artesunate, which is now considered the parenteral drug of choice for treatment of severe falciparum malaria, is available as an orphan drug in The Netherlands, it is currently only in stock in some specialized centres but certainly not available in every Dutch hospital. Some of these general hospitals do not even have any drug in stock for the treatment of malaria [Industrialized countries, however, have to face other -more trivial- problems. For instance, the expertise on diagnosis and treatment of malaria is usually focussed in some specialized hospitals and institutes but many ill-returning travellers may present to non-specialized hospitals or even general practitioners. Making a proper diagnosis of malaria may be troublesome under these circumstances, for instance, by lack of experience in the examination of malaria thick and thin blood smears and in the assessment of parasite load. These non-specialized centres therefore often rely on rapid diagnostic tests for the diagnosis of malaria . Althoug malaria . To prev malaria ,12.The Harbour Hospital is a 161-bed general hospital located in Rotterdam. It also harbours the Institute for Tropical Diseases, which serves as a national reference centre. In the period 1999-2008 almost 500 cases of imported malaria were diagnosed . For theP. falciparum malaria if they met one or more of the WHO criteria for severe malaria, as modified by Hien et al [Patients were classified as having severe en et al :• A score on the Glasgow Coma Scale of less then 11 .• Anaemia (haematocrit < 20%) with parasite counts exceeding 100,000/μl (roughly corresponding to 2% parasitaemia) on a peripheral blood smear.• Jaundice (serum bilirubin > 50 μmol/l) with parasite counts exceeding 100.000/μl on a peripheral blood smear.• Renal impairment (urine output < 400 ml/24 h and serum creatinine > 250 μmol/l).• Hypoglycaemia (blood glucose < 2.2 mmol/l).• Hyperparasitaemia (> 10% parasitaemia).• Systolic blood pressure < 80 mm Hg with cold extremities (indicating shock).In previous studies ,13,15 th® Malaria Test, Binax Inc., Maine, USA) and by conventional microscopy of stained thick and thin blood smears. In case of P. falciparum infections, parasite density was determined. When the parasitaemia was less than 0.5% infected erythrocytes, parasites were counted per 100 leucocytes in thick smears. When the parasitaemia was equal or higher than 0.5% infected erythrocytes, infected erythrocytes were counted in thin blood smear and expressed as a percentage of the total erythrocytes. The number of parasites per microliter was subsequently calculated from these data.On admission, blood samples were taken for analysis of the red blood cell count, haematocrit, white blood cell count, platelet count, serum electrolytes, total bilirubin, serum creatinine, liver enzymes, and blood glucose. In addition, a serum sample was taken on admission which was stored at -20°C until analysis. For the determination of plasma lactate, a separate blood sample was drawn on admission without congestion and placed on melting ice after which it was immediately analysed after isolation of plasma. Malaria was diagnosed by QBC (Quantitative Buffy Coat) analysis, by a rapid diagnostic antigen test for malaria . Procalcitonin levels in serum samples were determined using a commercially available EIA test . All tests were performed according to manufacturer's instructions. Detection limits were 3.88 pg/ml for TREM-1, 0.2 ng/ml for neopterin and 0.05 ng/ml for procalcitonin, respectively. According to the manufacturers, normal serum values are < 100 pg/ml for TREM-1, < 3 ng/ml for neopterin and < 0.1 ng/ml for procalcitonin.P. falciparum malaria and their corresponding 95% confidence intervals. Of each test a Receiver Operating Characteristic (ROC) curve, a graphical plot of sensitivity (true positive rate) versus 1-specificity , was constructed as a summary statistic and the area under the ROC curve (AUROC) and its corresponding 95% confidence intervals were calculated. Youden's index J (J = sensitivity+specificity-1) was used to choose the most appropriate cut-off point for each biomarker. All statistical analyses were performed using SPSS 15.0.For comparison between groups, the Mann-Whitney U-test was used and p-values of < 0.05 were considered statistically significant. The diagnostic performance of each biomarker was reported as sensitivity, specificity, positive and negative predictive value for severe P. falciparum infection  and 78 patients were diagnosed with P. falciparum infection. The general characteristics of all patients are shown in Table In total 104 travellers with imported malaria were included in this study, of which 26 patients were diagnosed with a non-P. falciparum malaria were further characterized by significantly lower platelet counts and haemoglobin levels and by significantly higher plasma lactate, bilirubin and C-reactive protein levels and erythrocyte sedimentation rates, respectively . Five patients had an impaired conscious level but a GSC above 11; eight patients had a parasitaemia > 5%, respectively. The first arterial blood gas analysis on ICU showed a median bicarbonate level of 22 mmol/l (range 17 to 26 mmol/l) and a median base deficit of 2 (range -3 to 8). Median GCS was 15 (range 9 to 15). One patient needed mechanical ventilation. Eleven patients received exchange transfusion as an adjunct therapy. No case fatalities were observed. The laboratory results on admission of travellers with imported severe P. falciparum malaria, uncomplicated P. falciparum malaria and non-P. falciparum malaria  and travellers with non-P. falciparum malaria (p < 0.0001)  Figure . ROC cur) Figure . As showP. falciparum malaria when compared to travellers with uncomplicated P. falciparum malaria (p = 0.0022). However, no significant differences were noted in comparison to travellers with non-P. falciparum infections (p = 0.17)  Figure . ROC cur p = 0.002. HoweveP. falciparum malaria when compared to travellers with uncomplicated P. falciparum malaria (p = 0.0012) and travellers with non-P. falciparum malaria (p = 0.0040). ROC curve analysis of plasma lactate levels showed an AUROC of 0.80 (95% CI 0.65-0.96) compatible with a good accuracy , but never as "normal" or "low" [Unfortunately, the applicability of these tests in the initial clinical assessment of patients with severe or "low" . This isPlasmodium species vivax, ovale and malariae, increased neopterin and procalcitonin serum levels were also observed in the majority of these patients, although levels were lower than compared with severe P. falciparum malaria patients. Although speculatively, these observations suggest that the mechanism whereby neopterin and procalcitonin levels increase in malaria, is not specific for severe P. falciparum malaria alone. Therefore, it may not accurately reflect the pivotal pathophysiological events in complicated P. falciparum malaria, such as the sequestration of infected red blood cells in the microcirculation of vital organs and disturbance of microcirculatory flow. Whereas an increased plasma lactate level conceivably reflects a significant reduction in microcirculatory flow in vital organs, the elevated neopterin and procalcitonin levels are probably the result of activation of a common inflammatory host response evoked by infection with the respective Plasmodium parasites. In fact, some reports even suggest that P. falciparum malaria per se is not associated with a stronger host response than P. vivax or P. ovale malaria, but that the parasite burden of the causative Plasmodium species may also modulate the extent of the host inflammatory response [Although severe or fatal malaria rarely results from infections with the non-sequestering response .P. falciparum malaria, the high negative predictive value of both neopterin and procalcitonin may be helpful for a rapid exclusion of severe P. falciparum malaria on admission. This may be a valuable tool - particularly if available as a rapid diagnostic test - for physicians only occasionally dealing with ill-returned travellers and who need to decide on subsequent oral anti-malarial treatment or a timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment.In conclusion, although neither neopterin nor procalcitonin can probably serve as a useful single diagnostic tool for severe The authors declare that they have no competing interests.RW participated in the design of the study and coordination, performed the experiments and the statistical analyses and drafted the manuscript.MW participated in the statistical analyses and drafting of the manuscript.PP participated in the design of the study.JH participated in the design of the study and revising the manuscript.RK is responsible for collection of patient materials and database management.AB participated in the design of the study and revising the manuscript.PG participated in the design and coordination of the study and in drafting and revising the manuscript.All authors have seen and approved the final version.


Cerebral malaria carries an unacceptable case fatality rate in children despite timely and adequate chemotherapy. To improve the survival rate, adjunctive therapies previously tested mainly focused on the modulation of the inflammatory response, without definitive effect in humans. In this context, a new adjunctive strategy using a neuroprotective drug: erythropoietin  was proposed.An open-labelled study including cerebral malaria children (Blantyre coma score below 3) was conducted in Mali. The objective was to assess the short-term safety (seven days) of erythropoietin at high doses  combined to quinine.35 patients with unrousable coma were included in the study. None of expected side effects of erythropoietin were observed during the seven days follow-up. No significant increase in the case fatality rate (7/35 patients) was observed compared to other studies with mortality rates ranging from 16 to 22% in similar endemic areas.These data provide the first evidence of the short-term safety of erythropoietin at high doses combined to quinine. A multicentre study is needed to assess the potential of Epo as an adjunctive therapy to increase the survival during cerebral malaria.ClinicalTrials.gov ID: NCT00697164 Malaria is one of the most common life-threatening diseases in sub-Sahara Africa with a dramatic impact on public health: more than one million deaths each year. Cerebral malaria (CM) carries unacceptable case fatality rate in children despite timely and adequate chemotherapy. The SEAQUAMAT group provided the first evidence of artesunate superiority  compared to quinine  during severe falciparum malaria in adults -5. It waEpo has recently been considered in a new light as a tissue-protective cytokine. Its neuro-protective properties have attracted considerable attention: Epo and its receptor (EpoR) are part of a highly potent endogenous neuro-protective system in the brain . SimilarThe effect of neuro-protective therapies was not previously investigated during malaria in humans, although the manifestations of cerebral malaria partly share features with neurological stroke, neuron damage related to ischaemia-reperfusion stress or acute non-specific neurological disorders. From experimental malaria, it was speculated that the progression from cerebral malaria to death could be counteracted by Epo high doses at the onset of clinical phase of the disease .in vivo/in vitro bioactivity ratio is more than 20% higher for epoietin-beta than for epoietin-alpha, because epoietin-beta is less sialylated than epoietin-alpha [While the safety of Epo in chronic diseases is known, using high doses for a short period of time is a major issue to be addressed in severe cerebral malaria patients. Indeed, in September 2008, FDA has been made aware of preliminary safety concerns from a clinical trial conducted in Germany investigating the use of epoietin-alfa high doses  to treat acute ischemic stroke, while investigators considered this as premature alert. Early in 2009 another clinical trial using epoietin-alfa high doses  in extremely premature infants to prevent/attenuate brain injury was also FDA hold. In both cases, epoeitin-alfa was used and FDA did not communicate their final conclusions and recommendations to the public. The in-alpha ,19. DesiTo document the safety of epoietin-beta high doses (1500 U/Kg) at day 7 post-admission, combined to quinine, the local recommended drug for cerebral malaria, a clinical trial in Bamako  including children admitted with highly critical stage cerebral malaria (Blantyre coma score <3) was conducted. The safety was defined as the lack of severe adverse effects attributable to Epo.This was an open-labelled study conducted in Gabriel Toure Hospital, one of the national referral hospitals of Bamako, Mali. Ethical approval was obtained from the Institutional Review Board (IRB) of the Faculty of Medicine, Pharmacy and Dentistry of Bamako, and from the board of trustees of Roche Foundation for Anaemia Research (RoFAR). The trial was registered . The funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript. Written informed consent was obtained from parents or legal guardians of patients prior to enrolment.Plasmodium falciparum infection , were included in the study after consent obtained from parents or legal guardians. All other categories of severe malaria, including severe anaemia (defined by haemoglobin levels below 5 g/dL), respiratory distress and acute renal failure were excluded. Children with other neurological disorder , known sickle cell disease and inter-current infections were excluded.Children between six months and 14 years old, presenting cerebral malaria with a Blantyre coma score <3 due to ®) from admission to day 3 (three doses). Considering the very low risk of side effects with a short-term rHuEPO treatment, 35 patients were included, which is within the range of a generally accepted sample size for this purpose.Cerebral malaria patients received anti-malarial treatment according to the recommendation of Mali National Malaria Control Programme. They received quinine intravenously 20 mg salt/kg bw on admission then 10 mg salt/kg bw every eight hours from day 1 to day 3. Then, they received artesunate-amodiaquine combination  from day 4 to day 6. Adjunctive therapy was based on intravenous infusion of 1500 U/kg/day rHuEPO  incidence of adverse effects, ii) incidence of abnormal laboratory values, iii) rate of neurological disorders evaluated by clinical examination and Blantyre coma score.An independent local Medical Monitor and an external Data and Safety Monitoring Committee, composed by four members, were designed and evaluated adverse events and other safety data. Reports of cases were regularly sent to the data monitoring committee. The case causalities were assessed using the standardized WHO-UMC system by DSMC members. All the investigators completed IRB certificate for clinical trial and passed the evaluation from the Collaborative Institutional Training Initiative . The study adhered to the international conference on Harmonization Guidelines for Good Clinical Practices and was conducted in accordance with the Declaration of Helsinki.The following data were collected from all enrolees on a standardized case report form. Demographic details , history of present illness , physical and biological findings were gathered on admission and every day during the follow-up. Standard operating procedures were developed, applied to all patients and were strictly respected. The clinical team was trained in operating and maintaining the instruments. Biological quality controls for all the biological parameters were performed once a week. Biological abnormalities were double-checked. Regular meetings were held to ensure that data quality was maintained.Clinical and biological monitoring was in agreement with the WHO guidelines for cerebral malaria . Tempera®, Hemocue), blood saturation , blood lactate , blood gas analysis , diuresis, haemoglobinuria , were recorded daily. Blood glucose levels  were recorded every eight hours for the first two days, then daily. Creatininaemia and alanine aminotransferase assays were referred to local reference laboratory.Parasitaemia (Giemsa-stained thick smears), haemoglobin level . None of the patients presented severe hypoglycaemia except two, who were treated before admission with a three-day course of quinine (13 mg/kg/day) or high dose chloroquine  given by a primary health care officer.Table t-test) or disease characteristics (Fisher F-test) between the surviving and the deceased patients , there were no significant differences in demographic  was included and treated at admission with Epo, quinine, diazepam intramuscularly and phenobarbital for repeated seizures, but without parasite blood stage documentation. He was later classified as protocol violation. He died one hour after admission without aetiologic diagnosis.- One child  received a total dose of 700 mg of chloroquine the week before admission and was admitted with a Blantyre score at 1, repeated seizures, profound hypoglycaemia and respiratory pauses. He received at admission quinine and Epo, plus 5 mg diazepam and 40 mg phenobarbital, and died three hours later.- One child  was admitted with repeated seizures that were not terminated by initial injection of diazepam (10 mg) intramuscularly. He received successively two more diazepam injections and 120 mg phenobarbital during the first 30 hours, while he presented respiratory pauses. Mechanical ventilation and oxygen were not available and the patient died 44 hours post-admission after respiratory distress. He received two doses of Epo.- One child  was admitted with a Blantyre coma score of 2, with seizures and respiratory distress. Clinical improvement was observed after two doses of quinine and Epo. The children received enteral nutrition prepared by his family without septic care at 20, 24 and 28 hours post-admission. He suddenly presented vomiting during four hours, hypotension, mydriasis and respiratory arrest at 32 hours post-admission. No bacterial culture could be obtained during the time of the lethal shock due to the lack of these facilities during the night.- One child  was admitted with deep acidosis  not taken into account by medical team until 24 hours when the blood pH was measured at 7.0. He received Ringer 500 mL after 24 and 48 hours without biological and clinical improvement and died after 51 hours.- Two children (aged 7 & 9) died immediately and two hours post-admission, respectively, the first deceased child was nearly impossible to treat using intravenous route due to severe hypotension and the second with high blood lactate level (11.7 mmol/L). They received quinine and Epo treatment at admission, but no resuscitation was possible.The five first of these deaths were classified by the DMC as unlikely due to Epo according to the WHO-UMC system. The others two children were classified as non-evaluable.28/35 patients survived at day 7 post-admission (80%). Although the study was not designed to detect a treatment effect on the time to recovery, the Blantyre coma score increased from stages 1–2 to stages 4–5 in less than 48 hours.Epo is a safe drug used for years to treat chronic anaemia caused by renal failure or cancer. Here much higher doses of that drug were used during a very short period of time. Side effects suspected to be due to high doses were monitored.t-test > 0.05), while it became significant at day 7 (Student t-test = 0.03).None of the known side effects of EPO including increase in blood pressure , and venP. falciparum infection or to resuscitate the patients using aggressive fluid therapy [Child mortality from malaria is still unacceptable in sub-Saharan Africa, while ACT was deployed in many endemic areas. These drugs are quite effective in clearing the blood stage parasites within 48 to 72 hours . Most de therapy . More re therapy .The haematopoietic growth factor Epo has been used as a drug for the treatment of anaemia for almost two decades. Since Epo and Epo-Receptor are expressed in several organs , Epo function is not limited to the haematopoietic system . SystemiSince the pathological mechanisms involved in ischaemia share features with cerebral malaria, and none of the anti-malarial drug is effective to control the progression of the disease in the first hours of treatment, there was an opportunity for the use of Epo as an adjunctive therapy in the early stage of cerebral malaria .Children (median age: 6) referred to hospital after up to 6 days of fever were included. These children were living in urban or peri-urban settings with mesoendemic transmission level. The overall sex ratio was 1.7  compared to 0.75  for patients who died at hospital.Most of the patients received incomplete treatment  in primary health care centres and were referred to hospital in the absence of clinical improvement, leading to the late stages of unrousable coma observed at admission. Among the patients included, there was a similar number of patients showing a Blantyre coma score of 1 and 2 (17 and 18 respectively). The aim of this study was to investigate the safety of Epo during cerebral malaria. Patients suspected to present respiratory distress or renal failure before admission could lead to a bias in the interpretation of the results. While severe malaria is often a combination of severe anaemia and cerebral malaria, patients with a significant decrease in haemoglobin levels were not included to avoid any interference in the data analysis with the potential effect of Epo on erythropoiesis. However, it has to be remembered that authors  demonstrSeven patients/35 died during the trial. Five were classified as unlikely due to Epo high doses by the DSMC and none of these deaths should be attributed to Epo. Among them, two patients received only one Epo dose and were admitted with extremely severe symptoms and deep coma. In the emergency situation, patients admitted with low grade coma score were included and received Epo treatment. Some patients were included despite the presence of respiratory distress. Nevertheless, it was decided to present these information in intention-to-treat protocol to avoid any bias in data analysis.It is well recognize that good supportive care and management of complications are essential in the treatment of severe malaria . PaediatThe comparison of case fatality rates with other cerebral malaria studies in different settings are always controversial. However, considering the severity of the disease at admission and lack of standard supportive care for children included in this study, the observed mortality rate of 20% could be analysed in the light of the case fatality rate of 16.8% in Kenya ; 19,3% iTaken altogether, these data demonstrated that the short term safety (seven days) of Epo high doses administered during three days was obtained in children presenting extremely severe cerebral malaria. Before these findings can be considered as a clinical directive, the long-term safety of Epo need to be further evaluated in larger-scale, longer-term clinical studies. A randomized, double-blinded, multi-centre trial will provide definitive information on the potential of Epo as an adjunctive therapy in humans and the effect of Epo plus anti-malarial drug combination on CM case fatality rate . The conEthical approval was obtained from the Institutional Review Board (IRB) of the faculty of Medicine, Pharmacy and Dentistry of Bamako, and from the board of trustees of Roche Foundation for Anemia Research (RoFAR). The trial was registered .The authors declare that they have no competing interests.SP designed the study and acts as principal investigator. ALB was in charge of erythropoietin and biological management. SK was principal clinical investigator, SS, AB, ED, KB were clinicians and biologists involved in patients care. AD & OD supervised the study. All authors contributed to the interpretation of the analysis, read and approved the final manuscript.


P. berghei ANKA-GFP infection during pregnancy to evoke a prominent inflammatory response in the placenta that entails CD11b mononuclear infiltration, up-regulation of MIP-1 alpha chemokine and is associated with marked reduction of placental vascular spaces. Placenta pathology was associated with decreased fetal viability, intra-uterine growth retardation, gross post-natal growth impairment and increased disease severity in pregnant females. Moreover, we provide evidence that CSA and HA, known to mediate P. falciparum adhesion to human placenta, are also involved in mouse placental malaria infection. We propose that reduction of maternal blood flow in the placenta is a key pathogenic factor in murine pregnancy malaria and we hypothesize that exacerbated innate inflammatory responses to Plasmodium infected red blood cells trigger severe placenta pathology. This experimental model provides an opportunity to identify cell and molecular components of severe PAM pathogenesis and to investigate the inflammatory response that leads to the observed fetal and placental blood circulation abnormalities.Pregnancy-associated malaria (PAM) is expressed in a range of clinical complications that include increased disease severity in pregnant women, decreased fetal viability, intra-uterine growth retardation, low birth weight and infant mortality. The physiopathology of malaria in pregnancy is difficult to scrutinize and attempts were made in the past to use animal models for pregnancy malaria studies. Here, we describe a comprehensive mouse experimental model that recapitulates many of the pathological and clinical features typical of human severe malaria in pregnancy. We used  P. falciparum transmission is most intense. Pregnancy-associated malaria (PAM) is one of the major public health problems in Africa with a high burden of maternal and fetal morbidity leading to 100,000 infant deaths per year It is estimated that more than 50 million pregnancies occur every year in malaria endemic areas, and approximately half of these occur in sub-Saharan Africa, where Together with maternal malaria induced anemia P. falciparum infected Red Blood Cells (iRBC) to sequester in the microvasculature of vital organs. Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1), a malarial variant antigen on infected erythrocytes, is involved in the adherence to host cell receptors. Infected erythrocytes can bind to endothelial receptors such as CD36 and intercellular adhesion molecule 1 (ICAM-1) It is known that the severity of malaria is related to the capacity of Although malaria in pregnancy has recently attracted many research efforts, there are ethical and logistic issues that restrict studies of human malaria infection during pregnancy and the specific pathologic bases of different PAM outcomes remain poorly understood P. chabaudi infection but did not present pathology comparable to human placental malaria evoked by P. falciparumPrevious studies concerning PAM in rodents have focused in congenital malaria P. berghei ANKA-GFP to infect BALB/c mice during pregnancy, resulting in placenta damage and inflammation, as well as IUGR/LBW. In addition, we found that P. berghei ANKA-GFP iRBC are able to bind to receptors present in mouse placental tissues providing the basis for a pathology trigger of mouse placenta pathology, comparable to the mechanism proposed for human placental malaria.We revisited the study of severe placenta malaria pathology in the mouse through an experimental model that uses This experimental model captures many pathology features analogous to severe malaria manifestations in pregnant women and may provide opportunities to investigate the pathogenesis mechanisms of malaria in pregnancy and enable experimental evaluation of PAM interventions strategies.P. berghei ANKA constitutively expressing green fluorescent protein (P. berghei ANKA-GFP) (15cy1 clone) in vivo passage in BALB/c mice, when the percentage of iRBC reached approximately 10%. Parasitemia was measured using flow cytometry analysis as described elsewhere The BALB/c mice were bred and maintained in conventional housing at the Instituto Gulbenkian de Ciência. Infection experiments were performed in adult females, between 10–15 weeks of age. Detection of the vaginal plug and measurement of body weight were jointly used to time gestation, as described by Freyre et al. 6 iRBC, and parasitemia was recorded every other day. To evidence pathological features of malaria during the course of pregnancy and in the developing fetus, this infection period was determined to be the optimal time point as earlier infections did not allow reaching pregnancy at term (data not shown), which is consistent with previous reports Pregnant mice were intravenously (IV) infected between G11 and G13 with 10P. berghei ANKA-GFP infection is lethal in BALB/c mice, foster mothers were used in newborn post-natal follow-up studies. Hence, both newborns from infected mothers and newborns from control mothers were also transferred to foster mothers to avoid weight bias due to differential maternal nourishment. The newborns were weighted every other day.As 2 narcosis, their spleens were weighted, the uterus was examined and the number of fetuses and resorptions were recorded. Resorptions were identified as small implants with no discernible fetus and placenta, corresponding to embryos that died before complete placenta vascularization. The fetuses were extracted from their amniotic envelop and viability was immediately evaluated by prompted movement reaction to touching with pliers. The lack of reactive movement indicated that the fetus had recently died and was considered an abortion. Macerated pale white fetuses were dead and were also recorded as abortions. Fetuses and placentas were separately weighted. Non-aborted fetuses were killed combining CO2 narcosis and hypothermia.Between G17 and G19, pregnant females were killed by COPlacentas from infected and non-infected females were treated in a similar way. Placentas were separated in two halves, one half was fixed in 1.6% paraformaldehyde with 20% sucrose for further processing and the other half collected for RNA extraction. Paraffin-embedded non-consecutive placenta sections were stained with hematoxylin-eosin (HE) and examined under a light microscope . For histological and morphometric analysis, placental sections were examined in a blind fashion.Fixed placenta samples were washed in PBS with 15% sucrose overnight, soaked in Tissue-Tek® (Sakura) and frozen in dry ice. For immunohistochemistry staining, freshly made frozen sections (6 μm thick) were rinsed in PBS for 30 minutes and blocked with 1% bovine serum albumin (BSA). To enhance parasite GFP signal, we used rabbit polyclonal anti-GFP antibody (Molecular Probes) and goat anti-rabbit antibody conjugated with Alexa488 (Molecular Probes). To identify macrophages/monocytes we used anti-CD11b biotinilated antibodies , followed by incubation with Rhodamin-Avidin D (Vector Laboratories). Nuclei were stained with DAPI (Invitrogen) and coverslips were mounted with aqueous mounting media . Stained sections were examined under fluorescence microscopy .HE stained placental sections were analyzed for vascular space quantification. In each section, 5 randomly selected microscopic fields in the labyrinthine region (magnification ×40) were acquired at 1280×960 resolution, using a color video camera  connected to a light microscope . We implemented an image analysis routine using ImageJ . Briefly, after acquisition, the images underwent an automated light analysis procedure where noise removal was applied to ensure color and image quality standardization across sections and specimens. The images were given a color threshold to cover the area corresponding to blood spaces lumen. The coverage percentage was calculated as the ratio between the number of pixels covered by the area defined by the threshold and the overall number of pixels in the image. The blood vascular area in each placenta was estimated from the analysis of two non-consecutive sections. The reported results correspond to individual pregnant females and represent the average result for 2-3 placentas.Total RNA from freshly collected placentas was obtained using an RNeasy Mini Kit (Qiagen), following the manufacturer protocol for animal tissues. One microgram of total RNA was converted to cDNA  using random hexamer primers. MCP-1 (CCL2) and MIP-1 alpha (CCL3) expression was quantified using TaqMan Gene Expression Assays from ABI  with TaqMan Universal PCR master mix. The gene expression quantification reactions were performed in an ABI Prism 7900HT system. Relative quantification of MCP-1 (CCL2) and MIP-1 alpha (CCL3) in each real-time PCR reaction was obtained after normalization for GAPDH expression measured in the same PCR reaction.P. berghei ANKA-GFP infected erythrocytes were synchronized as described elsewhere 8 iRBC/ml.iRBC were collected from infected animals with 10–20% parasitemia. In order to obtain mature blood stage parasite forms (trophozoites/schizonts), Proteus vulgaris, Sigma), with 30 µg/ml hyaluronidase , with heparinase II  or with PBS for 2 periods of 2 hours at 37°C. Both enzyme-treated sections and non-treated sections were blocked with 1% BSA in PBS at room temperature for 30 minutes. Fifty microliters of synchronized iRBC suspension, at the concentration of 108/ml, were overlaid onto each tissue section for 60 minutes at 37°C in a humid chamber. After washing the unbound cells, the placental sections were incubated with DAPI. For iRBC-ligand blocking experiments, synchronized iRBC were preincubated with the indicated concentrations of chondroitin sulfate A (CSA) from bovine trachea (Sigma), hyaluronic acid (HA) potassium salt from human umbilical cord (Sigma) or colominic acid sodium salt (as negative control) from E. coli (Sigma), at 37°C for 30 minutes with moderate agitation. For iRBC-ligand cleavage assays iRBC were treated with trypsin (Gibco), proteinase K (Sigma) or neuraminidase as a negative control . iRBC were pre-incubated with each enzyme at indicated concentrations for 30 minutes at 37°C. After washing, iRBC were overlaid on placental sections as described above. The slides were mounted with Mowiol and examined under fluorescence microscopy (magnification ×40). The number of iRBC adhering placental sections in each experimental condition was determined in a blind fashion, counting 50 fields in each of 3 independent experiments.Placentas from uninfected BALB/c females, obtained at G19, were treated using a previously described protocol Statistical differences between groups of mice used in this study were evaluated by the Student's t test. Mann-Whitney test was used for morphometric data and Log Rank test for survival curves.P. berghei-GFP to infect BALB/c female mice that are resistant to cerebral malaria, allowing progression of the disease to hyperparasitemia states. Other parasite species/mouse strains combinations were tested and we noted that P. chabaudi did not elicit pronounced signs of placental malaria and C57Bl/6 and DBA/2 mouse strains could not survive to P. berghei-GFP infection long enough to exhibit typical manifestations of malaria in pregnancy (data not shown).Our search for an experimental model that recapitulates typical pathology features of severe malaria in pregnancy revealed that time of infection during pregnancy is critical to evidence poor pregnancy outcome and fetal growth impairments. In fact, infection at early stages led to premature pregnancy interruption while infection at G13 allowed pregnancy to proceed to later stages when fetal and placenta pathology became apparent and resembled human PAM. Moreover, such pathological features were exuberant in the experimental model here reported, which used lethal P. berghei-GFP course of infection in pregnant and non-pregnant females confirmed earlier findings that pregnancy in mice confers an increased susceptibility to malaria p = 0.007) (Comparison of = 0.007) . In addi= 0.007) . In contWe followed-up pregnancy outcome in 22 infected females and found out that malaria had a strong negative effect in pregnancy success . ApproxiPost-natal growth of viable newborns was followed up to weaning by weight monitoring and compared to 49 newborns from non-infected mothers. In this experiment, infected mothers would not survive long enough to nourish their pups and we used foster mothers to feed all newborns as described in the p = 0.01) and had higher number of aborted fetuses (p = 0.002)  by analyzing fetuses from 28 pregnant females infected at G13 and from 9 non-infected pregnant females . Uterus = 0.002) .We searched for signs of fetal impaired development in the uterus. Fetuses from uninfected healthy mothers showed pink coloration, had translucent skin with visible blood flow in the blood vessels and the placentas were replenished with blood . In contPlacenta represents the interface between mother and fetus, playing a critical role in fetal growth and development. Placental tissue of infected pregnant females revealed a number of abnormalities in comparison to non-infected controls . We repep<0.0001) in the infected pregnant group  and hyaluronic acid (HA) in P. berghei-GFP iRBC cytoadhesion. Using a set of ex vivo adhesion assays, we verified that CSA and/or HA are involved in specific interactions of P. berghei-GFP blood stages with the placental tissue . This weight difference was steadily maintained in post-natal life until weaning, even if newborn mice from infected mothers were fostered by non-infected mothers.The effects of malaria on the newborn status are believed to be caused by placental insufficiency ghei-GFP . It is kfindings . Althougfindings . We obsein vivo and in vitro but, when properly stimulated via IFNγ, are also capable of increasing erythrophagocytosis activity Although we have found important pathological changes in both basal and labyrinthine zones of mouse placenta, parasites and hemozoin were never visualized in the fetal circulation and positive parasitemia was never recorded in newborns from infected mothers. The absence of evidence for congenital infection, despite the presence of numerous iRBC in the placental maternal blood, points to the efficacy of the placental trophoblastic layer to block parasite traversing to fetal blood. The mechanism by which the trophoblastic cells prevent fetal infection is poorly understood, but several trophoblast defense mechanisms have been proposed P. falciparum infected placentas remains controversial, but the main placental candidate receptors and their cognate parasite ligands participating in iRBC adhesion have been identified. Muthusamy et al.The pattern of iRBC adherence in P. falciparum previous studies demonstrating that the placental parasite populations do not adhere in a uniform manner to immobilized CSA or GAG receptors, implying that other factors may be confounding this interaction or other receptors are involved Our data show a significant reduction of placental adhesion when the iRBC were pre-incubated with of soluble HA  or CSA , indicating that these GAG can be important adhesion receptors in this model. Chondroitinase and hyaluronidase placental sections treatment also reduced adhesion considerably but did not completely abrogate binding. This is in line with P. berghei-GFP adhesion to CSA and HA indicates that iRBC could co-express ligands for different receptors, represented either by separate adhesive molecules or by different binding sites on a single molecule. Overall, the experimental data suggest that cytoadherence of P. berghei-GFP in the placenta may involve CSA and HA as receptors and raises the hypothesis that human and murine malaria in pregnancy have similar pathogenesis basis. Future efforts towards collecting in vivo evidence for iRBC binding will help to further establish CSA and HA as key pathogenesis mediators in placental malaria.To demonstrate that the iRBC binding is receptor dependent, we treated iRBC with two different proteases and showed that binding properties of protease-treated iRBC were substantially inhibited, which was consistent with the involvement of surface membrane protein receptors in mediating adhesion. The P.berghei, our findings raise the interesting possibility that the receptors mediating adhesion in the mouse placenta could have in P.berghei iRBC cognate ligands that are related to PfEMP1. The prominent histological alterations in mouse placenta heavily infected with P. berghei-GFP resemble those described for acute P. falciparum malaria in humans The phenotypic characterization of murine PAM here presented warrants that further research is needed to describe the pathogenesis mechanisms at cell and molecular levels. Although PfEMP1 orthologues were not yet found in


Plasmodium falciparum infections and the risk of subsequent clinical malaria.Intermittent preventive treatment (IPT) of malaria involves administration of curative doses of antimalarials at specified time points to vulnerable populations in endemic areas, regardless whether a subject is known to be infected. The effect of this new intervention on the development and maintenance of protective immunity needs further understanding. We have investigated how seasonal IPT affects the genetic diversity of merozoite surface protein 2 gene. Malaria morbidity and anaemia was monitored during 12 months follow-up.The study included 2227 Ghanaian children (3–59 months) who were given sulphadoxine-pyrimethamine (SP) bimonthly, artesunate plus amodiaquine (AS+AQ) monthly or bimonthly, or placebo monthly for six months spanning the malaria transmission season. Blood samples collected at three post-interventional surveys were analysed by genotyping of the polymorphic Monthly IPT with AS+AQ resulted in a marked reduction in number of concurrent clones and only children parasite negative just after the intervention period developed clinical malaria during follow-up. In the placebo group, children without parasites as well as those infected with ≥2 clones had a reduced risk of subsequent malaria. The bimonthly SP or AS+AQ groups had similar number of clones as placebo after intervention; however, diversity and parasite negativity did not predict the risk of malaria. An interaction effect showed that multiclonal infections were only associated with protection in children without intermittent treatment.P. falciparum infections. The reduced risk of malaria in children with multiclonal infections only seen in untreated children suggests that persistence of antigenically diverse P. falciparum infections is important for the maintenance of protective malaria immunity in high transmission settings.Molecular typing revealed effects of the intervention not detected by ordinary microscopy. Effective seasonal IPT temporarily reduced the prevalence and genetic diversity of  Plasmodium falciparum remains a major threat to health and economic development in Sub-Saharan Africa Malaria is one of the most severe infectious diseases. Despite recent reports of decreasing incidence in some areas, infection by IPTi is likely to be most effective in areas with continuous high malaria transmission. However, in many areas of sub-Saharan Africa, especially those with seasonal transmission, the main burden of malaria is in older children. An alternative approach in these areas is administration of IPT to children (IPTc) during the high transmission season. Seasonal IPT reduced the incidence of clinical malaria by 17–86% depending on antimalarial drug and dosage regime Some rebound in malaria morbidity was reported following sustained chemoprophylaxis P. falciparum infections are often composed of multiple genetically distinct clones. The ability to control diverse infections may be a key feature of protective immunity. The number of clones correlates with transmission intensity and age per se be important to maintain protection in areas of intense transmission. For these reasons we have investigated how intermittent treatment affects subsequent P. falciparum infections and risk of clinical malaria and anaemia within a seasonal IPT trial in Ghanaian children In high transmission areas, immunity to malaria develops gradually following repeated exposure and A randomized, placebo-controlled IPTc trial was conducted in Hohoe district, Ghana during 2005 to 2006 In total 2227 of the 2451 participating children (aged 3 to 59 months), provided comprehensive data required for this study. The IPTc trial has been described in detail elsewhere P. falciparum parasiteamia detected by microscopy. A second definition of malaria was fever (defined as above) and a peripheral parasiteamia of >7000 parasites/µl blood, a local threshold with high specificity for clinical malaria A clinical episode of malaria was defined as fever (axillary temperature ≥37.5° C or reported fever within the past 48 hours) together with any Initial screening for eligibility was performed one month before administration of the first IPT dose (May 2005). After the six-month intervention period (June-October 2005), three cross-sectional surveys were performed: 1) at the end of the intervention period (November 2005); 2) at the end of following dry season, (April 2006); and 3) at the end of the following rainy season (November 2006) . Axillarmsp2 genotyping. DNA was extracted from whole blood spots on filter paper using ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems). P. falciparum infections were characterized by genotyping of the merozoite surface protein 2 (msp2) gene using fluorescent PCR and capillary electrophoresis (CE) msp2 (block 3) followed by separate, nested reactions with fluorescently labeled primers distinguishing the respective allelic types of msp2, FC27 and IC. CE was performed on a 3730 DNA sequencer using GeneMapper® Software (v 4.0) (both Applied Biosystems). A cut off of 300 relative fluorescent units was used to distinguish true alleles from non-specific low artefact peaks Filter paper samples from microscopy positive children collected at the three cross-sectional surveys were all analyzed by msp2 clones was categorised to avoid assumptions about linearity as follows: 0 i.e. negative by microscopy, 1 and ≥2 clones. Age was grouped into five categories  and parasite densities into quartiles. Association between number of clones and treatment group, age, parasite densities, sex and clinical malaria during intervention (yes/no) was assessed by logistic regression (with clones as outcome comparing 1 with ≥2 clones). Association between number of clones and anaemia (Hb<8.0 g/dl) (with anaemia yes/no as outcome) at the respective surveys was analyzed by logistic regression.Data analysis was performed using R (v 2.9.0) and SPSS (v 17). Comparisons of parasite prevalence between treatment groups were assessed by Chi-square tests. Associations between the number of clones and parasite densities were analysed by Spearman rank. The presence of high number of clones i.e. ≥3 were compared between the groups by Fisher's exact test. When used as explanatory variable, the number of msp2 clones , sex, age, owning a bednet, treatment group and clinical malaria (yes/no) during the intervention period. Both clinical definitions of malaria (see above) were used in the risk assessments. When the more strict definition of malaria (fever and >7000 parasites/µl) was used, 28 days were subtracted from the time at risk if children had been treated with quinine due to a clinical episode with fever and <7000 parasites/µl. The proportional hazards assumption was assessed using tests and graphical diagnostics based on scaled Schoenfeld residuals. Unadjusted Kaplan Meier survival plots with time to a first clinical episode of malaria were made for all children and for the individual treatment groups, respectively.The prospective risk of clinical malaria was assessed by Cox regression analysis based on time to first or only clinical episode during the 12-month follow-up. The analysis included children who participated in the first post-intervention survey, with one or two subsequent samples. Analysis was performed for all children and then per separate treatment group. The data on clones, age and parasite densities was categorised as described above. To identify strictly asymptomatic individuals, children with clinical episode during the period 28 days before until 7 days after survey were excluded from the survival analyses since number of clones can be affected by clinical episode or recent treatment P. falciparum infection by microscopy was lower (5.2%) in children who had received monthly AS+AQ treatment compared to children given placebo (19.8%) . No such differences were detected comparing the bimonthly groups to placebo. There was a weak correlation between number of clones and parasite densities using continuous data r = 0.20 . Age was associated with diversity only in the placebo group where children older than 11 months were more likely to be infected with ≥2 clones than younger children , a difference that persisted after adjusting for parasite density and owning a bednet. Six and 12 months after the intervention, the number of clones was equally high in all groups , were detected in 60.6–70.1% of the PCR positive samples from asymptomatic children. Children in the AS+AQ monthly group were infected with 1–2 clones , a signil groups .Children with parasites were more likely to be anaemic than children without parasites . Multiple clones did not increase the likelihood of aneamia compared to single clone infection .During the intervention period, children receiving active treatment, especially with monthly AS+AQ, experienced fewer episodes of malaria than children given placebo Univariate time to event analysis including all asymptomatic children (n = 1856), showed that multiclonal infections were, compared to single clone infections, associated with a reduced risk of subsequent malaria using both clinical definitions . The risIn a multivariate analysis including all asymptomatic children and adjusting for age and treatment group, the decreased risk of disease in children with multiclonal infections at baseline remained significant for both malaria definitions /Figure 3The non-significant results in the bimonthly treatment groups could have been due to the relatively few parasite positive children. An analysis investigating the interaction between treatment/placebo and protective effect of ≥2 clones was therefore conducted. In an analysis comparing groups with similar diversity after ended intervention i.e. placebo and the bimonthly IPT groups (SP and AS+AQ) an interaction between infection diversity and treatment showed that the protective effect of having ≥2 clones was significantly higher in children with placebo compared to children who had received bimonthly IPT  The point estimates indicated that the protective effect of having ≥2 clones was relatively strong, reducing the risk by a factor of ten.P. falciparum infections, defined by msp2 genotyping, and its relation to the subsequent risk of clinical malaria in these children in Hohoe District in Ghana. During the 6 months intervention period, the incidence of clinical malaria and anaemia was substantially reduced Intermittent treatment administered during the intense malaria transmission season affected the diversity of P. falciparum infections was clearly reduced in the first survey one month after the intervention, with detection of only 1–2 clones compared to up to 6–8 clones in the other groups. This reduction was, however, only temporary and there was no difference between the groups 6 and 12 months later. Interestingly, there was a distinct difference depending on whether AS+AQ had been administered monthly (6 courses) or bimonthly (3 courses), with the last effective course one and two months before the first post-intervention survey respectively. After just one additional month, there was no difference in the number of infecting clones between the treatment and the placebo group, suggesting that multiclonal infections are rapidly accumulated in this setting.With the most intense regime of monthly AS+AQ, the prevalence and diversity of In the monthly AS+AQ group, only children who were parasite negative at the first survey after intervention developed clinical malaria during follow-up. In the placebo group parasite negative children were at lower risk of subsequent malaria, whereas no significant risk was detected in the bimonthly groups. These parasite negative children might both represent a group with lower exposure as well as children with efficient immunity. Notably, this reduced risk was only seen in the untreated children.P. falciparum infections predicted the subsequent risk of malaria, infections with ≥2 clones were associated with a reduced risk in the placebo group, representing the natural condition in this setting. Interestingly, children in the bimonthly IPT groups were infected with similar number of clones as children receiving placebo, and although there was a tendency in the AS+AQ bimonthly group, clones did not confer protection in neither of these two groups. The protective effect of multiclonal infections in the placebo group was, indeed, relatively strong reducing the risk by a factor of ten compared to the bimonthly IPT groups. This suggests that it is not only the number of clones present at a single time point, but infections over a preceding period that influence malaria immunity.When assessing whether the number of clones in asymptomatic P. falciparum infections have previously been associated with a reduced subsequent risk of clinical malaria in high transmission areas per se are important in stimulating protective immunity. Reduced levels of anti-P. falciparum IgG antibodies have been reported after sustained chemoprophylaxis Asymptomatic multiclonal The effect on malaria risk was most pronounced at the beginning of the following rainy season when the number of clinical episodes increased . The lowA limitation of the current study is that blood samples were not collected before IPT was given. Such samples would have further elucidated the effect of IPT on infection diversity in the respective treatment groups and would have provided information on whether children attain the same diversity after a six months intervention period, i.e. reflecting tolerance to certain levels of diversity. Moreover, parasite prevalence and infection diversity is likely to have been underestimated since only filter paper samples from microscopy positive children were available for genotyping. Analyses of all children by PCR would have allowed for a more correct and in depth analyses of parasite positivity and diversity since the method generally detects a larger proportion of parasitized individuals in endemic settings compared to microscopy. This would also have given the opportunity to identify continuously parasite negative individuals who could be regarded as unexposed and excluded in a separate risk analysis.Children who received placebo experienced more clinical episodes of malaria during the intervention period The parasitological failure rate was 2.3% for AS+AQ and 6.8% for SP, and the re-infection rates at day 28 was 16% for AS+AQ and 19% for SP in the original IPTc study Seasonal IPT reduced the prevalence of anemia by 30–45%, but with no prolonged effect beyond the intervention period The risk of clinical malaria during follow up was increased in infants (3–11 months) given monthly AS+AQ, and there was a tendency in this direction in the bimonthly treatment groups. A similar rebound in morbidity in infants given IPT was suggested in another high transmission setting in Ghana P. falciparum infections. The number of clones and parasite negativity predicted a lower risk of malaria only in previously untreated children, which suggests that continuous exposure and persistent infections are important for maintenance of malaria immunity. Genotyping of P. falciparum infections was a useful tool to more in depth study the effect of different drug regimes for seasonal IPT on malaria exposure and morbidity.In conclusion, our findings show that seasonal IPT in children under five affects the diversity of