Combination therapies are now recommended to treat uncomplicated malaria. We used a longitudinal design to assess the incidence of malaria and compare the efficacies of 3 combination regimens in Kampala, Uganda.Children aged 1–10 years were enrolled from randomly selected households in 2004–05 and 2007, and were followed at least monthly through 2008. Insecticide-treated bednets (ITNs) were provided in 2006. Children were randomized upon their first episode, and then treated for all episodes of uncomplicated malaria with amodiaquine/sulfadoxine-pyrimethamine (AQ/SP), artesunate/amodiaquine (AS/AQ), or artemether/lumefantrine (AL). Risks of parasitological failure were determined for each episode of uncomplicated malaria and clinical parameters were followed. Plasmodium falciparum. The rank order of treatment efficacy was AL > AS/AQ > AQ/SP. Failure rates increased over time for AQ/SP, but not the artemisinin-based regimens. Over the 4-year course of the study the prevalence of asymptomatic parasitemia decreased from 11.8% to 1.4%, the incidence of malaria decreased from 1.55 to 0.32 per person year, and the prevalence of anemia (hemoglobin <10 gm/dL) decreased from 5.9% to 1.0%. No episodes of severe malaria (based on WHO criteria) and no deaths were seen.A total of 690 children experienced 1464 episodes of malaria. 96% of these episodes were uncomplicated malaria and treated with study drugs; 94% were due to With ready access to combination therapies and distribution of ITNs, responses were excellent for artemisinin-containing regimens, severe malaria was not seen, and the incidence of malaria and prevalence of parasitemia and anemia decreased steadily over time.ISRCTN37517549isrctn.org  Plasmodium falciparum, the most virulent human malaria parasite, is responsible for hundreds of millions of illnesses and about one million deaths each year Malaria is one of the most important infectious diseases in the world. The problem is greatest in sub-Saharan Africa, where infection with The epidemiology of malaria varies greatly across Africa, with large variations in transmission intensity and both seasonal and year-round transmission in different areas. The greater the transmission intensity, the more rapidly humans acquire the partial immunity to malaria that allows older children and adults to be relatively spared from malarial incidence and risks of severe malaria. Thus, in high transmission areas children acquire effective immunity over the first few years of life, and malarial illness is uncommon in older children and adults. In areas with lower levels of transmission, including many cities, the acquisition of immunity occurs more slowly. The level of immunity in a population directly impacts upon responses to antimalarial therapies, especially in the context of sub-optimal treatments, as the immune response plays an important role in control of infection, and there is a clear correlation between transmission intensity, level of antimalarial immunity, and response to therapy with partially effective agents We studied the comparative efficacy of 3 antimalarial combination therapies in an urban cohort of children in Kampala, a city with a level of malaria transmission that is relatively low for sub-Saharan Africa. An interim analysis identified a clear rank-order of treatment efficacy (AL > AS/AQ > AQ/SP) The protocol for this trial and supporting CONSORT checklist are available as supporting information; see A detailed description of the cohort has been published previously Parents and guardians were asked to bring their children to the study clinic for all medical care. The clinic was open daily from 8 AM to 5 PM, and after-hours care was available. Participants who presented with new medical problems underwent a standardized evaluation. Malaria was diagnosed if a child had complicated malaria ; AS/AQ ; and artemether 20 mg/lumefantrine 120 mg twice daily for 3 days . Administration of first daily doses were directly observed by study nurses; second daily doses of medication or placebo (for AQ/SP and AS/AQ arms) were given to the parents or guardians to administer in the evening. All study personnel involved in outcome assessment were blinded to treatment allocation. Study participants and their caregivers were not informed of their assigned treatment regimens. Placebos were administered to match the twice-daily schedule of AL. However, because the study drugs were not identical in appearance and taste, the study was considered single-blind. After treatment in the clinic, patients were observed for 30 minutes and the dose was readministered if vomiting occurred. Patients who vomited persistently were referred for treatment with parenteral quinine. Participants with severe malaria or danger signs were also treated with quinine. The AQ/SP treatment arm was discontinued in March 2007 after our interim analysis demonstrated inferior efficacy Participants diagnosed with malaria were asked to return on days 1, 2, 3, 7, 14, and 28 or any other day they felt ill. Follow-up evaluation consisted of a standardized medical history and physical examination. Blood was obtained by finger prick for thick blood smears and storage on filter paper on all follow-up days, except day 1. Treatment outcomes were classified according to 2005 WHO guidelines; for this analysis all failures  were combined. Patients with failure within 14 days of initiation of therapy were treated with quinine , beginning a new 28-day follow-up schedule. Symptomatic malaria diagnosed more than 14 days after a previous episode was managed according to the study protocol. Safety outcomes have been reported previously P. falciparum DNA was not identified by genotyping in a sample with a positive blood smear, parasite species was determined by amplification of 18S small subunit ribosomal DNA, as previously described Blood smears and molecular genotyping methods to distinguish recrudescences from new infections were performed as previously described The sample size for this study was calculated and outcomes were analyzed as previously described The cohort included 690 children aged 1 to 10 years at enrollment; 601 were enrolled from November 2004-April 2005, and an additional 89 from January–May 2007 . All parP. falciparum. When P. falciparum was identified, we did not search for other species, so mixed infections were not identified. However, the three other human malaria parasite species were all seen, with disease caused solely by P. malariae in 66 episodes, P. ovale in 13 episodes, and P. vivax in 7 episodes.As expected most episodes of malaria (94%) were caused by Asymptomatic parasitemia was common at study enrollment in 2004–05 (17%) The incidence of malaria was year-round, with large variation, consistent with variable year-round rainfall and malaria transmission in Kampala . IncidenFinal data from our study offer an update over a prior report Measures of anemia improved through the course of the study. For the 4 full years of observation, the annual overall prevalence of anemia, considering all measurements over the course of the year and anemia defined as hemoglobin <10 gm/dl, decreased from 6.7% in 2005 to 1.0% in 2008 . Both thRemarkably, no cases of severe malaria We followed 690 children in Kampala, Uganda for up to 4 years and identified 1464 episodes of malaria. Our study design included ready access to care for all medical problems. Thus, treatment outside of our study protocol was very uncommon, and we obtained accurate measures of malarial incidence and antimalarial drug efficacy. Children were randomized to one of 3 combination regimens with the first episode of uncomplicated malaria, and we subsequently followed them through up to 30 episodes of malaria. Although further comparisons were limited due to the sequential drop-out of two treatment arms, our results add to our interim analysis Additional efficacy comparisons beyond those previously reported were limited by our need to eliminate the AQ/SP treatment arm due to inferior efficacy in 2007 and the AS/AQ arm when the study formulation of AS was no longer available in early 2008. Consistent with earlier analyses, both ACTs demonstrated excellent efficacy, but AL was superior to AS/AQ We identified remarkable decreases in the incidence of malaria and the prevalences of asymptomatic malarial parasitemia and anemia over the course of our 4 year study. Undoubtedly, these decreases were due to multiple factors. First, a decrease in malarial incidence is expected with increasing age, although in this area of relatively low malaria transmission, changes in incidence with age were modest The most ominous feature of falciparum malaria is progression to severe malaria, with subsequent high mortality. It seems likely that prompt evaluation of febrile illnesses and prompt therapy for all illnesses diagnosed as malaria will decrease the incidence of severe malaria. In our study 1464 episodes of malaria were seen, 21 were complicated, mostly due to single seizures soon after presentation, and no episodes of severe malaria and no deaths were seen. These results further support emphasis on availability of prompt effective therapy for those with malaria as a key control strategy.It is useful to consider our results from a cohort of children in Kampala in the context of current malaria control efforts across Africa. Resources for malaria control have increased greatly in recent years, and initial gains in control have been seen, with marked decreases in malarial parasitemia and incidence in some areas. At the same time, many areas of Africa continue to have very high levels of malaria transmission and disease incidence. Our results suggest that, in an area of intermediate transmission, prompt therapy with effective agents greatly limits progression to severe disease or death. Thus, distribution of new effective therapies across Africa is a critical priority. Nonetheless, with prompt and effective therapy the incidence of malaria, although decreased, remained quite high in our cohort, and so additional control measures, including ITNs, rational use of insecticides, and, when available, an effective vaccine, are also needed for control of malaria in Africa.Protocol S1Trial Protocol.(1.12 MB PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(0.19 MB DOC)Click here for additional data file.


Plasmodium vivax.Humoral immune responses play a key role in the development of immunity to malaria, but the host genetic factors that contribute to the naturally occurring immune responses to malarial antigens are not completely understood. The aim of the present investigation was to determine whether, in subjects exposed to malaria, GM and KM allotypes--genetic markers of immunoglobulin γ and κ-type light chains, respectively--contribute to the magnitude of natural antibody responses to target antigens that are leading vaccine candidates for protection against P. vivax apical membrane antigen 1 (PvAMA-1) and merozoite surface protein 1 (PvMSP1-19) were determined by an enzyme-linked immunosorbent assay. Multiple linear regression models and the non-parametric Mann-Whitney test were used for data analyses.Sera from 210 adults, who had been exposed to malaria transmission in the Brazilian Amazon endemic area, were allotyped for several GM and KM determinants by a standard hemagglutination-inhibition method. IgG subclass antibodies to P = 0.004, P = 0.002, respectively) in subjects with the GM 3 23 5,13,14 phenotype than in those who lacked this phenotype.IgG1 antibody levels to both PvMSP1-19 and PvAMA-1 antigens were significantly higher (P. vivax malaria antigens. These findings have important implications for the effectiveness of vaccines containing PvAMA-1 or PvMSP1-19 antigens. They also shed light on the possible role of malaria as one of the evolutionary selective forces that may have contributed to the maintenance of the extensive polymorphism at the GM loci.Results presented here show that immunoglobulin GM allotypes contribute to the natural antibody responses to  Plasmodium falciparum and Plasmodium vivax  versus 0.67 RI [0.20-2.13], P = 0.002). No significant interactions between GM and KM phenotypes were found for antibody responsiveness to either PvAMA-1 or PvMSP1-19 antigens. As presented in the footnotes of Tables P = 0.025 and regression coefficient 4.56 [95% CI 2.78-6.34], P = 0.000 for PvMSP1-19 and PvAMA-1, respectively).IgG1 antibody responses to both PvMSP1-19 Table  were higA number of studies have shown that anti-malarial antibodies of IgG1 subclass contribute significantly to protective immunity against malaria ,29. A maAdditionally, GM allotypes could contribute to antibody responsiveness through their direct or indirect involvement in antibody specificity. There is growing body of evidence for the involvement of these constant-region determinants in antibody specificity usually associated with the variable region of the Ig molecule . PossiblP. falciparum malaria [P. falciparum malaria in Beninese children [P. falciparum malaria and with antibody responsiveness to malarial antigens [P. falciparum vs P. vivax), there are highly significant differences in the frequency of GM/KM allotypes between the two populations (26). For instance, none of the Sudanese subjects possessed the GM 3 23 5,13,14 Phenotype, which is associated with antibody responsiveness in the present study, underscoring the necessity of studying different populations.The possibility of GM allotype involvement in malarial immunity was suggested by the results of a previous investigation, which showed significant differences in the frequencies of these determinants between two African sympatric tribes--Fulani and Masaleit--with marked differences in susceptibility to  malaria . A recenchildren . In a poantigens . The SudHaemophilus influenzae type b polysaccharide vaccine--based on their GM 23 and KM 1 allotype status--became responders when the vaccine was conjugated to a suitable adjuvant [Because malaria is a major global health problem, there is clear urgency to develop a vaccine for protection against infection. Development of an effective vaccine, however, would require a thorough understanding of the mechanisms associated with immunity to this parasite. Furthermore, given the complexity of the malarial parasite and tremendous genetic diversity of human populations, it may be difficult to devise one vaccine that is effective against all variants of the parasite and provides protective immunity for all people. Studies of the kind reported here might be able to identify people who are more likely to respond to a given vaccine. For the subjects with non-responders genotypes, the vaccine could be fused with appropriate adjuvants to circumvent the allotypic restriction in immune responsiveness. There is precedence for converting genetic non-responders into responders to a given vaccine. For instance, subjects classified as non-responders to adjuvant -42. The adjuvant . Therefoadjuvant . Such stP. vivax malarial antigens.Although plausible mechanisms of direct involvement of GM allotypes in humoral immunity to malaria are discussed here, the associations observed in this study could also result from linkage disequilibrium between as-yet-unidentified putative immune response gene(s) for PvMSP1-19 and PvAMA-1 antigens and particular GM alleles. Therefore, it is important to expand these studies to include other ethnic groups, characterized by a different array of GM haplotypes, resulting from ethnic differences in linkage disequilibrium among GM alleles . This isP. vivax PvMSP1-19 and PvAMA-1 antigens were significantly higher in subjects with the GM 3 23 5,13,14 phenotype than in those who lacked this phenotype. If these findings were confirmed in an independent study, they could aid in identifying subjects  who are more likely to benefit from PvMSP1-19 and PvAMA-1 containing vaccines. For the non-carriers of GM 3 23 5,13,14, these antigens could be fused with appropriate adjuvants to circumvent the allotypic restriction in immune responsiveness.Anti-malarial antibodies of IgG1 subclass have been previously shown to contribute significantly to protective immunity against malaria. Results presented here show that IgG1 antibody levels to The nature of the evolutionary selective mechanism(s) that maintains polymorphisms at the GM loci is not known. Strong associations between GM allotypes and antibody levels to malarial antigens suggest that malaria may have been one of the selective forces that have contributed to the maintenance of the balanced polymorphism at these loci.The authors declare that they have no competing interests.JP conceived the study. All authors participated in the acquisition of data. EB participated in the design of the study and performed the statistical analysis. JP and EB were responsible for data interpretation and drafting the manuscript. All authors read and approved the final manuscript.


Plasmodium knowlesi is typically found in nature in macaques and has recently been recognized as the fifth species of Plasmodium causing malaria in human populations in south-east Asia. A case of knowlesi malaria is described in a Swedish man, who became ill after returning from a short visit to Malaysian Borneo in October 2006. His P. knowlesi infection was not detected using a rapid diagnostic test for malaria, but was confirmed by PCR and molecular characterization. He responded rapidly to treatment with mefloquine. Evaluation of rapid diagnostic kits with further samples from knowlesi malaria patients are necessary, since early identification and appropriate anti-malarial treatment of suspected cases are essential due to the rapid growth and potentially life-threatening nature of P. knowlesi. Physicians should be aware that knowlesi infection is an important differential diagnosis in febrile travellers, with a recent travel history to forested areas in south-east Asia, including short-term travellers who tested negative with rapid diagnostic tests. Plasmodium knowlesi occurs in nature in long-tailed and pig-tailed macaques , that are commonly found in forested areas of south-east Asia [P. knowlesi was regarded as a rare disease, occurring only sporadically in humans [P. knowlesi. This parasite is clearly the fifth species of Plasmodium, causing malaria in humans in south-east Asia [P. knowlesi and P. malariae due to their similar morphological characteristics [ast Asia . Until an humans . The recn humans -5, especn humans , Myanmarn humans , the Phin humans  and Singn humans , have chast Asia . It has ast Asia , and higast Asia . Under teristics ,3,10.9/L), moderate thrombocytopenia (58 × 109/L) and an elevated C-reactive protein concentration of 109 mg/L . Plasma creatinine was normal, while plasma bilirubin (31 μmol/L) was just above the upper limit of the normal range and alanine transaminase (1.68 μcat/L) was slightly increased .In October 2006, a 35-year-old Swedish man presented to hospital in Stockholm with a two-day history of fever, sweats, headache and fatigue. He had a temperature of 40°C, but was otherwise in good general condition with a normal respiratory rate and blood pressure. Laboratory investigations revealed normal haemoglobin concentration (154 g/L), leucopaenia  was negative, oral mefloquine therapy was started while waiting for the results of the blood film examination. Malaria parasites were detected by microscopy in 0.1% of erythrocytes. The Plasmodium species could not be identified conclusively, but Plasmodium malariae was suspected, since late trophozoites were observed and the infected red blood cells were not enlarged (Figure 9/L, respectively. Two blood cultures were negative and a pulmonary x-ray was normal.The patient had recently returned from a two-week holiday in Sarawak, Malaysian Borneo. He had trekked in the jungle of the Bario Highlands during the last seven days of his holiday, at altitudes between 800 and 1,400 m. He had not taken any anti-malarial chemoprophylaxis. His symptoms started 11 days after leaving the Bario Highlands. There was a strong suspicion of malaria, and although a rapid diagnostic test (NowP. falciparum, Plasmodium vivax, Plasmodium ovale and P. malariae were performed in Sweden. These assays were negative, but microscopy results that were suggestive of P. malariae and the patient's travel history led to the suspicion of a P. knowlesi infection. A frozen blood sample, collected when the patient was admitted to hospital, was sent to the Malaria Research Centre at University Malaysia Sarawak for further analysis. The infection was identified as a single P. knowlesi infection following DNA extraction and examination by PCR assays for P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi as described previously [P. knowlesi isolates (Figure Due to the uncertain identification by microscopy, PCR assays for eviously . PCR ampeviously . The SSUs Figure .P. knowlesi. Since the incubation period of P. knowlesi in the liver is 9 to 12 days [The nested PCR assay and molecular characterization confirmed that the patient was infected with Plasmodium knowlesi parasites morphologically resemble those of P. malariae [Plasmodium lactate dehydrogenase that have been developed for use in a rapid diagnostic test, it was shown that certain antibodies that were thought to be specific for P. falciparum and P. vivax, also bind to P. knowlesi [P. knowlesi infections by examining a larger number of patient samples.malariae ,2,10 andmalariae . Howevermalariae , particumalariae . In a reknowlesi . HoweverP. knowlesi has a 24-hr erythrocytic cycle, and high parasitaemias with fatal outcome in humans can occur [P. knowlesi infections resolve rapidly following treatment with chloroquine [P. knowlesi can multiply [Early identification and adequate anti-malarial treatment of suspected cases are essential since oroquine  and the P. knowlesi infection in a Swedish traveller returning from Malaysian Borneo, this indicates that knowlesi malaria infections in travellers are probably not rare events, particularly since south-east Asia has become a popular tourist destination.Previous reports of human knowlesi malaria infections were of people that have either lived or worked in the forest and forest fringe areas of south-east Asia . There hP. knowlesi can infect short-term visitors to areas in south-east Asia, where knowlesi transmission occurs, and mefloquine was an effective anti-malarial. The P. knowlesi infection in the Swedish traveller was not detected by a rapid diagnostic test and evaluation of rapid diagnostic tests with further samples are necessary since early identification and appropriate anti-malarial treatment of suspected cases are essential, due to the rapid growth and potentially pathogenic nature of knowlesi malaria. Physicians should, therefore, be aware that knowlesi infection is an important differential diagnosis in febrile travellers with a recent travel history to forested areas in Southeast Asia, including short-term travellers, who tested negative with rapid diagnostic tests.In conclusion, this report shows that The authors declare that they have no competing interests.UB, BS and AF wrote the paper, AF facilitated the study, UB was the physician responsible for the patient, BS supervised molecular characterization of parasites and analysis of sequence data, and PCSD was responsible for molecular detection, characterization and phylogenetic analysis of sequence data. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report.


P. falciparum. Formulated with AS02 (a proprietary Adjuvant System), it constitutes the FMP1/AS02 candidate malaria vaccine. We evaluated this vaccine's safety, immunogenicity, and efficacy in African children.The antigen, falciparum malaria protein 1 (FMP1), represents the 42-kDa C-terminal fragment of merozoite surface protein-1 (MSP-1) of the 3D7 clone of P. falciparum. We enrolled 400 children aged 12–47 months in general good health.Children were randomised in a 1∶1 fashion to receive either FMP1/AS02 (50 µg) or Rabipur® rabies vaccine. Vaccinations were administered on a 0, 1, and 2 month schedule. The primary study endpoint was time to first clinical episode of P. falciparum malaria  occurring between 14 days and six months after a third dose. Case detection was both active and passive. Safety and immunogenicity were evaluated for eight months after first immunisations; vaccine efficacy (VE) was measured over a six-month period following third vaccinations.A randomised, double-blind, Phase IIb, comparator-controlled trial.The trial was conducted in 13 field stations of one mile radii within Kombewa Division, Nyanza Province, Western Kenya, an area of holoendemic transmission of 42 antibody concentrations increased from1.3 µg/mL to 27.3 µg/mL in the FMP1/AS02 recipients, but were unchanged in controls. 97 children in the FMP1/AS02 group and 98 controls had a primary endpoint episode. Overall VE was 5.1% .374 of 400 children received all three doses and completed six months of follow-up. FMP1/AS02 had a good safety profile and was well-tolerated but more reactogenic than the comparator. Geometric mean anti-MSP-142 vaccine development should focus on other formulations and antigen constructs.FMP1/AS02 is not a promising candidate for further development as a monovalent malaria vaccine. Future MSP-1NCT00223990Clinicaltrials.gov  Plasmodium falciparum malaria kills over one million children annually in sub-Saharan Africa falciparum malaria vaccine, showed reductions of 35% for clinical disease and 49% for severe disease over an 18 months follow up period, in one to four year old Mozambican children P. falciparum vaccine target is the blood stage antigen merozoite surface protein-1 (MSP-1) Aotus monkeys vaccinated with MSP-142 have been protected from P. falciparum challenge in vitro growth inhibition activity A second promising 42 from the 3D7 clone of P. falciparum was expressed in Escherichia coli. The final product, designated falciparum malaria protein 1 (FMP1) 42 antibodies from baseline to one month after a third vaccination. These encouraging results led to the present proof-of-concept efficacy trial in the same population, which is the first to evaluate monovalent MSP-142 for efficacy in a field setting.MSP-1The protocol for this trial and the supporting CONSORT checklist are available as supporting information; P. falciparum accounts for more than 95% of the malaria infections Anopheles gambiae complex being the major vector The study was conducted at the KEMRI-“Walter Reed Project” (WRP) Kombewa Clinic (KC) in the Kombewa Division of Kisumu District of Nyanza Province in western Kenya, an area of hyperendemic malaria transmission with peaks during the long rains (March–June) and short rains (November–December). The study population, almost exclusively of the Luo tribe, predominantly Seme sub-tribe, was drawn from one mile radius catchment areas around each of 13 field stations. The field stations were staffed 24 hours/day throughout the study period to facilitate medical care. 3/mm3, for two-year olds of <3.0×103/mm3, and for three-year olds of <2.0×103/mm3.Demographic surveillance was conducted at the Kombewa Division field stations in 2003–2004. During the census, the families with children aged 12–47 months were identified and homesteads located in relation to the field stations. At the time of the study initiation, parents/guardians were invited to come to the KC for briefing and those who gave consent were later requested to bring their children to the KC for eligibility screening. Children were excluded according to the same criteria used in the previous paediatric Phase Ib study Groups that approved the study protocol were: Kenya Medical Research Institute (KEMRI) Scientific Steering Committee and Ethical Review Committee, Walter Reed Army Institute of Research (WRAIR) Scientific Review Committee, US Army Medical Research and Materiel Command (USAMRMC), Human Subjects Research Review Board (HSRRB), and the PATH Human Subject Protection Committee (HSPC). The United States Food and Drug Administration (FDA) reviewed the protocol. The study was conducted in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice. Pharmaceutical Product Development, Inc. (PPD) and the US Army Medical Materiel Development Activity (USAMMDA) monitored the study. An independent Local Medical Monitor (LMM) and a data and safety monitoring board (DSMB) closely reviewed the trial's progress.Before recruiting study participants, representatives of the study team met with local chiefs and community leaders to describe the study. Field workers then visited individual homesteads known to have potential study participants and invited the parents or guardians to visit the clinic for recruitment briefings. At each briefing session parents/guardians received a copy of the informed consent form (ICF). Groups watched a videotape detailing (in the Luo language) the study and information on the ICF, which was followed by a public question and answer session. Parents/guardians were then invited for private individual discussions with a study physician assistant or study nurse, after which they were invited to sign (or thumb print if illiterate) the ICF. For each illiterate parent/guardian, an impartial member of the community countersigned as a witness.During the screening visits, within 45 days before the first immunisation, the children had a detailed clinical history, physical examination, and blood sampling for haematology and biochemistry evaluations. The children were randomised on day 0 and received the vaccine on days 0, 29 (±9) and 57 (±9). After each vaccination the children were evaluated for solicited adverse events (AEs) on days 0, 1, 2, 3, and 6. The unsolicited AE follow-up periods ran for 30 days (vaccination days and the subsequent 29 days). Serious adverse events (SAEs) were reported throughout the duration of the study. During the efficacy follow-up period, all SAEs were reported within 24 hours of occurrence. In the event that a child was treated outside the WRP KC or the New Nyanza Provincial General Hospital copies of all available clinical records of the encounter were obtained, reviewed and maintained in the subject's record. When a study subject was admitted to NNPGH, a member of the clinical team visited the subject at least once daily and reported to the principal investigator (PI) or his designee. The PI remained in contact with the paediatrician or any other medical specialist who provided care to the subject to ensure that the locally appropriate medical care was provided.During the follow-up period, clinical malaria cases were tracked and parasite densities and immunogenicity measured. Surveillance was performed on a biweekly basis through monthly field worker home visits and monthly clinic visits (window period: ±7 days) . During the home visits to the children field workers documented axillary temperatures, solicited history of fever in the previous 24 hours and of any concomitant medications taken, and reminded parents/guardians of future appointments and the necessity of bringing sick children to the KC. Children with a fever or history of fever in the past 24 hours were taken to the KC for evaluation, including malaria blood films (MBFs) and complete blood counts (CBCs). Out-migrations, absences from Kombewa Division, and antimalarial drugs usage were documented.Clinic and home visits were staggered so that each child was evaluated (active detection) about every fortnight. During the clinic visits, an interim history and an axillary temperature were obtained, a history-directed clinical examination was performed, and blood was drawn .Clinical malaria was also identified by passive detection (when ill children presented to the KC or to field stations). Blood samples for CBC and MBF were obtained whenever an ill child presented to the clinic. At study start, parents were advised to seek medical attention for the children exclusively at the KC whenever possible Children requiring inpatient care were admitted to the NNPGH. Uncomplicated malaria was treated with a 6 dose course of artemether/lumefantrine (Coartem®) and quinine in case of severe malaria.Two microscopists independently read Giemsa-stained thin and thick malaria blood films (MBFs) and quantified them according to a standard operating procedure. Results were double data entered into a laboratory database; a third, more experienced reader resolved any discordant microscopy findings. Parasite densities were calculated against same-day CBCs.42 3D7) as the capture antigen Induction of humoral immunity was measured with an enzyme-linked immunosorbent assay (ELISA) using purified bulk FMP1  supplied in single dose vials containing lyophilised antigen with 1.0 mL of diluent (sterile water) for injection.The FMP1 antigen, described in detail elsewhere After confirming eligibility, staff drew blood for laboratory tests. The pharmacy team confirmed individual assignments to vaccine groups and then prepared the appropriate vaccine. Because the vaccines differed in volume and appearance, the pharmacy team masked the barrel of the syringe, labeled the syringe with each individual's unique randomisation code and study ID number, and passed the syringe through a small service hatch into the vaccination room.A vaccine administration team confirmed each child's identity, study number, and randomisation code before administering vaccine. A nurse administered the vaccine by slow intramuscular injection into the left anterolateral thigh. Vaccinations were performed on a 0, 1, and 2 month schedule . The nurChildren were observed for 60 minutes after each vaccination. Parents/guardians were instructed to return with children to the KC immediately for any new symptoms. The study team evaluated the children on days 1, 2, 3, and 6 after each vaccination to collect solicited  and unsolicited AEs. If a parent and child did not appear for a scheduled clinic visit, a clinician or nurse visited them on the same day to measure vital signs and collect information on any AEs that occurred during the previous 24 hours. Transportation was provided for the participants to KC, where AEs were evaluated, monitored, and clinically managed until resolution.P. falciparum malaria over a six month period commencing 14 days after a third vaccination. Clinical malaria for the primary endpoint was defined as parasite density ≥50,000 parasites/µL n the presence of fever (axillary temperature ≥37.5°C). This was based on results of an unpublished epidemiology study in children of the same group prior to the study.The primary objective was to evaluate the vaccine efficacy (VE) against first clinical episodes of P. falciparum malaria using alternate case definitions; (2) parasite density; (3) safety as evaluated by solicited adverse events (AEs) occurring in the week following each vaccination, unsolicited AEs occurring within 30 days of each vaccination, serious adverse events (SAEs) occurring throughout the study, and time to anaemia (Hb<8.0 g/dL); (4) immunogenicity as assessed by geometric means of anti-MSP-142 antibody concentration throughout the study; (5) the immunogenicity from first vaccination through the end of the follow-up period; and (6) association between anti-MSP-142 antibody concentration and the subsequent risk of clinical malaria during the follow-up period.Secondary objectives were to evaluate: (1) clinical episodes of P. falciparum malaria meeting the “Primary Case Definition”  time to parasite density ≥50,000 asexual stage P. falciparum parasites/µL of blood; (4) number of clinical episodes of malaria, per subject, meeting the primary case definition; statistical methods for recurrent event data, such as Poisson regression, were used to assess the effect of FMP1/AS02 vaccination on the average number of clinical episodes per subject; (5) time to anaemia ; (6) proportion of children with anaemia.Measurements of efficacy included (1) time to clinical episode of initions ; (2) timAssessment of safety included (1) occurrences of solicited AEs during a seven-day follow-up period after each vaccination; (2) occurrences of unsolicited AEs during a 30-day follow-up period after each vaccination; and (3) occurrences of SAEs between the first vaccination and the end of the follow-up period. Assessment of immunogenicity included geometric means of anti-MSP-1 titers at first, second, and third vaccinations and at 30, 115, and 200 days after the third vaccination.A year-long (2003–4) longitudinal cohort study in the same area showed monthly clinical malaria attack rates of between 20% and 55% in children aged one to three years (unpublished data). The sample size calculation for this trial assumed a 50% attack rate over the six-month follow-up period with time to clinical malaria following an exponential distribution. A trial with 200 children per study arm would have 80% power to detect a 30% reduction in the six-month cumulative incidence rate.Rabipur® rabies vaccine.A randomisation list containing sequential codes linked to a study vaccine assignment was computer generated in the USA by Statistics Collaborative, Inc. These codes were assigned to participants in the order in which they presented to the clinic on the day of first vaccination. The code was then supplied to the study drug manager and the site pharmacist, who were responsible for randomising the study participants. Randomisation was performed within blocks of eight. Each randomisation assignment was sealed in a unique, tamper-evident envelope, which was opened at the time a subject presented for the first vaccination. At the end of every vaccination day, the randomisation list was secured in a locked safe accessible only to the study drug manager, the pharmacist, and the pharmacist's assistants. The only other person in Kenya who had access to the randomisation code at the study site was the LMM for use in case emergency unblinding became necessary for safety purposes. Four hundred children were randomised in the order of presentation to the clinic on the days of first vaccination; 200 received FMP1/AS02 and 200 At the study site, only the pharmacist, pharmacy assistant and the drug manager were aware of the randomisation codes of the children. Children, parents, investigators, lab personnel and all staff performing follow-up evaluations were blinded as to vaccine assignment. Because the color and volumes of the reconstituted FMP1/AS02 and comparator vaccines differed, the barrel of the syringe was covered with opaque tape to mask its contents and labeled with the subject identification number and randomisation code. Children, parents, and the staff performing follow-up evaluations were all blinded. Vaccinations were carried out simultaneously in four separate consultation rooms, which were connected to a central pharmacy (the vaccine preparation room) by small, closable service hatches. On vaccination days, the prepared syringe was handed through a service hatch to a vaccinator for vaccine administration. For each subject, an identification number, a randomisation code from a chart, and a randomisation code on the syringe were recorded on a vaccination form. Following vaccine administration, children were assessed and follow-up visits conducted by a group of clinicians who had not been involved in the vaccinations.SQL Server 2000® database. The database also underwent 100% verification by a quality assurance team before the archiving and transmission to the statisticians. Data analysis was performed on the locked database using SAS (version 9.1) and S-Plus (version 6.2) according to a prewritten statistical analysis plan.Data were recorded on case report forms that underwent 100% verification by a quality assurance team before double data entry into a The according-to-protocol cohort included all children who received the three vaccinations and had no significant protocol violation. Analyses of efficacy were based on the according-to-protocol cohort, counting each subject's first malaria case that emerged at least 14 days after a third vaccination to the end of the follow-up period. The intention-to-treat cohort included all randomised children. Supportive analyses, based on the intention-to-treat cohort, counted all cases that emerged from the day of first vaccination.The primary endpoint was analyzed with a logrank test. A Cox proportional hazards model (SAS Proc PHREG) was used to estimate the hazard ratio along with its corresponding 95% confidence interval: VE is one minus the hazard ratio. Similar analyses were performed for other case definitions. Additional Cox models explored the effects of age, sex, baseline bednet use, field station location, and haemoglobin genotype on VE. Kaplan-Meier curves displayed time to event. An alternative estimate of VE based on cumulative incidence rates, excluding time periods when the subject was not at risk for contracting a new case of malaria, was calculated using Poisson regression. Fisher's Exact Test compared binary variables. Parameters measuring immune response were calculated and compared between groups using longitudinal models (SAS Proc MIXED) with a spatial power covariance structure.A total of 625 children between 12 and 47 months of age were screened, 535 were deemed eligible, and 400 were randomised, 200 to each of the two vaccine groups . CompliaStudy enrolment/randomisation, vaccination, and follow-up were conducted from March 2005 to December 2005.At baseline, the two groups were similar  and withA total of 374 (191 FMP1/AS02 and 183 in the comparator) children who completed the study as per the protocol were included in the according-to-protocol analysis.Rabipur® group and 97 in the FMP1/AS02 group—experienced at least one case of P. falciparum malaria meeting the primary case definition. Estimated VE was 5.1% . The two groups had similar distributions of time to malaria following a third vaccination and were essentially identical throughout the period of observation . Thus, no significant difference between arms was observed with respect to the number of clinical episodes per subject meeting the primary case definition. The intention-to-treat analysis showed 122 and 131 cases in the FMP1/AS02 and comparator groups, respectively, for a VE of 15% . Active case detection identified 16% and 12% of the FMP1/AS02 and comparator groups, respectively. The comparable proportions in the according-to-protocol group were 23% and 28%.The rate of disease meeting the primary case definition per person year was 1.70 in the FMP1/AS02 group and 1.76 in the p = 0.3) versus 48% (p = 0.017) in non-bednet users . VE did not differ as a function of age (p = 0.7); sex (p = 0.1); erythrocyte abnormalities , or alpha-thalassemia; p = 0.7); presence of parasitaemia at baseline (p = 0.4); or anti-MSP-142 antibody level either at baseline (p = 0.1) or at day 85 (p = 0.3).We performed some exploratory, subgroup analyses that were not pre-specified. The following nominal p-values, not corrected for multiplicity, reflect the results of tests measuring whether the effect of vaccine differed by subgroup. At screening, parents were asked, “Does your child always sleep under a mosquito net at home?” VE in the two-thirds of the children (n = 288) reportedly always using bednets at baseline was −20%  time to first event starting at Day 0 (intention-to-treat) using various density cutoffs,(ii) time to first event starting at 14 days after Immnusation. 3 (according-to-protocol) using various density cutoffs, (iii) intention-to-treat and according-to-protocol analyses adjusted for time not at risk due to malaria treatment, and (iv) Poisson regression (intention-to-treat and according-to-protocol), where children were considered not at risk for 7 or 28 days following each new clinical case of malaria if they were treated with quinine or artemether/lumefantrine respectively.In the summer of 2007, after all statistical analyses had been completed, we reviewed the distribution of parasite densities and found an unexpected bimodality. The distribution dipped between modes straddling the density range where microscopists switched from thick to thin film reading according to their standard operating procedures; the modes also straddled the primary efficacy endpoint cutoff, 50,000 parasites/µL.We subsequently reread 643 pairs of films from all febrile episodes of malaria with parasitemia greater than 10,000 parasites/µL (618 pairs) and a random sample of episodes with densities less than 10,000 parasites/µL (25 pairs). For this rereading, each of two microscopists read both thin and thick films from the same malaria episode. If either of the pairs of thick or thin film readings yielded discordant results, a third, expert reader performed a tie-breaking read on both types of films.The goal of the reread was to reevaluate our conclusions concerning the efficacy of the vaccine. Although the original reads performed during the course of the trial and the rereads performed in 2007 showed some discordance, and the point estimates of the various measures of efficacy changed modestly, the qualitative conclusions remained unchanged.42-specific antibodies, as measured by ELISA (p<0.0001). The highest concentration was observed on study day 85, approximately four weeks after third immunisations. At this time, the geometric mean antibody concentration in vaccine recipients was 26.3 µg/mL, compared with 1.3 µg/mL in the same group before vaccination, and 1.4 µg/mL in the comparator group at day 85 (p<0.0001). The antibody levels in vaccinees declined subsequently over time, but did not reach their prior baseline values during this follow-up period. Antibody response did not differ according to age when one-, two- and three-year-olds were compared by a longitudinal repeated measures analysis (p = 0.3). However, there was no association between peak (day 85) antibody response and time to first case of malaria (p = 0.33).The concentration of MSP-1by ELISA , rose raBoth FMP1/AS02 and the comparator vaccine had a good safety profile and were well tolerated. The FMP1/ AS02 group had more local (88% versus 8%) and vaccine-related, systemic (61% versus 16%) solicited reactions. More grade 3 AEs occurred in the FMP1/AS02 group; the majority were injection site pain or fever  mostly rIn each group, 197 individuals experienced at least one unsolicited AE during the 30 days following vaccination. The most common, malaria, upper respiratory tract infection, skin disorders, and diarrhoea, were evenly distributed between the two vaccine groups.Mean screening and monthly safety laboratory values  were similar between the two groups and remained unchanged from baseline (not shown).Rabipur®). Both groups included individuals who had episodic, transient low haemoglobin values (standard range: 6.5–13.0 g/dL)  that returned to normal without transfusion. Additionally, one case of severe anaemia (Hb<5.0 g/dL) associated with severe malaria occurred in each vaccine group. In the intention-to-treat cohort, time to first episode of Hb<8.0 g/dL did not differ statistically between the groups .Mean haemoglobin values rose in both groups throughout the study .Serious and unexpected AEs were collected throughout the study. No reported SAE was judged related to vaccination. Twenty-six SAEs occurred in 23 children: 15 in the FMP1/AS02 group and 8 in the A 15 month old male in the comparator group presented with malaria 24 days after his third vaccination, developed severe anaemia four days into antimalarial treatment, and died 38 days later. The child was hospitalised and transfused several times for a recurrent Coombs negative, haemolytic anaemia that was refractory to transfusion. No haemoglobinopathy was identified; however, three of his brothers had died of anaemia in early childhood. Autopsy found no specific aetiology.P. falciparum malaria; it did not reduce the overall incidences of clinical malaria episodes or of malaria infections, and did not reduce parasite densities  and exhibited in vitro growth inhibitory activity The reasons for the lack of efficacy are unclear. The study had adequate power to detect VE of clinical importance. In malaria-naïve adults in the United States, FMP1/AS02 induced high titre, functional antibodies that recognised Rabipur® recipients, respectively). The parasite reduction effect reported with MSP combination B vaccine candidate Active and passive case detection was intense and sustained, so that undetected cases of clinical malaria would be unlikely. It is also unlikely that the frequent active follow-up and aggressive treatment of malaria pursued as part of the study design prevented the detection of VE, because majority of the proportions of endpoint cases in both groups resulted from passive case detection alone  analysis for VE as a function of age; the youngest children, although mounting the highest antibody responses, showed no difference in protection from their older counterparts. Similarly, there was no association between peak (day 85) antibody response and time to first case of malaria (p = 0.33). The absence of relationship between MSP-142–specific antibody levels and VE observed here contrasts with the positive correlation of anti-MSP-142 antibodies with protection described in epidemiologic studies based on the Camp/FUP allele of MSP-142Although FMP1/AS02 elicited a greater MSP-142 phenotypes in Kenya (unpublished data), immunity conferred by the study vaccine may be directed to a minor parasite population. Additionally, the isotype The lack of observable VE has several hypothetical explanations. Owing to the high multiplicity of infection and the diversity of MSP-1The possibility that efficacy is allele-specific is being investigated by genotyping of the parasites from breakthrough cases.Protection against severe malaria is an important objective of malaria vaccine efforts; however, too few cases of severe malaria  occurred in the present study to allow assessment of this.The exploratory analysis suggesting that the vaccine might have an impact among children who do not use bednets is intriguing but requires further critical evaluation to determine its validity. In any case, it is clear that a malaria vaccine that targets only children who do not use bednets is an unsupportable strategy.FMP1/AS02 had a good safety profile and, as expected, was more reactogenic than the comparator Haemoglobin levels were similar in both groups, rising throughout the study. Although more cases of low haemoglobin occurred in the FMP1/AS02 group, no case was judged causally related to vaccination and all resolved spontaneously. This observation led to an exploratory analysis to determine if there was a significant disproportionate drop in haemoglobin between groups. Fourteen children in the comparator group and 15 in the FMP1/AS02 group had an observed decrease from baseline of ≥3 grams of Hb at least once during the study. There was no significant difference in the risk of haemoglobin level decline to less than 8 g/dL, the cut-off value at enrolment. Thus, as in our previous studies 42 vaccine was clearly immunogenic, as evidenced by the rapid, high, and prolonged antibody response. There was a 20-fold increase from day 0 to day 85 in the FMP1/AS02 recipients, whereas the comparator group experienced a slight (11%) decrease during the same period. Although the antibody response waned after day 85, it remained well above both baseline and comparator for the duration of the follow-up period. Diminishment of malaria exposure during the dry season, or early case detection and treatment associated with the execution of the trial, might have contributed to the gradual decline in antibodies observed in the comparator group.The MSP-1P. falciparum infection in the study area as shown by a previous study The study population was chosen to represent the target population for a malaria vaccine: at risk, malaria-experienced children living in an area of intense malaria transmission. The primary objective of the trial being the efficacy of the vaccine, results should be broadly generalisable to children of the representative age bracket. As for results pertaining to the secondary objectives, safety and immunogenicity can be generalised to children of this age group exposed to P. falciparum malaria in an area of holoendemic transmission, precisely where a malaria vaccine is most needed. Because of the clearly demonstrated overall lack of efficacy in this trial, FMP1/AS02 is no longer a promising candidate for further development as a monovalent malaria vaccine. No previous Phase IIb study of the FMP1 antigen has been conducted in the field and especially in an area of holoendemic malaria transmission. However, all the previous Phase I field studies evaluated the safety and immunogenicity and as with our data presented found FMP1/AS02 to be safe and highly immunogenic 42 vaccine development efforts should focus on other antigen constructs and formulations.In conclusion, this study provides no evidence that FMP1/AS02 protects against Checklist S1CONSORT Checklist(0.04 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(1.43 MB DOC)Click here for additional data file.


The gold standard light microscopy technique has high sensitivity, but is a relatively time-consuming procedure especially during epidemics and in areas of high endemicity. This study attempted to test the sensitivity and specificity of a new diagnostic tool - the Cyscope® microscope. The results of the investigations were then used to calculate the sensitivity, specificity, and positive and negative predictive values of the Cyscope® microscope in reference to gold standard light microscopy.In this study, 293 febrile patients above the age of 18 years attending the malaria treatment centre in Sinnar State (Sudan) were interviewed using a structured questionnaire. Finger-prick blood samples were also collected from the participants to be tested for malaria using the hospital's microscope, the reference laboratory microscope, as well as the CyscopeThe sensitivity was found to be 98.2% (95% CI: 90.6%-100%); specificity = 98.3% (95% CI: 95.7% - 99.5%); positive predictive value = 93.3% (95% CI: 83.8% - 98.2%); and negative predictive value = 99.6% (95% CI: 97.6% - 100%).® microscope was found to be sensitive, specific and provide rapid, reliable results in a matter of less than 10 minutes. The Cyscope® microscope should be considered as a viable, cheaper and time-saving option for malaria diagnosis, especially in areas where Plasmodium falciparum is the predominant parasite.In conclusion, the Cyscope Malaria is a leading cause of morbidity and mortality in Africa, killing a child every 30 seconds . One of In many countries, due to poverty or lack of training and facilities, malaria is still being diagnosed clinically, an unreliable method leading to over-diagnosis and wasting precious time for patients and staff . The gol® microscope produced by the German company Partec. This is a mobile, battery-operated microscope. The slides of the microscope come ready with malaria parasite DNA specific staining reagents in dried form. All that is needed is the addition of a drop of blood and viewing the slide under the microscope, saving time and preparation. An optional add-on enables viewing the slides on a computer to facilitate the diagnostic procedure and storage and retrieval of results [Many diagnostic procedures have been developed which aim at reducing the time, preparation, and training needed to diagnose malaria. The use of Plasmodium nucleic acid-specific fluorescent dyes was found to facilitate detection of the parasites even in low parasitaemia conditions due to the contrast with the background . One rec results .® microscope in reference to the gold standard of light microscopy.Given the fact that the test is relatively cheap, this technique offers the possibility of a useful test especially for malaria endemic regions. This study was conducted out to investigate the sensitivity and specificity of the CyscopeSinnar state lies 270 km south-east Khartoum, the capital. With an average annual rainfall of 700-1000 mm, the prevalence of malaria in Sinnar state is 57/1,000 population. The malaria control programme in Sinnar State Ministry of Health is one of the best state programmes building on the history of excellence as the state during eradication era was selected for piloting eradication in Sudan. Moreover, Sinnar state hosted since early 1960s, the malaria training centre with good reputation. The centre nowadays acts as a centre for training malaria microscopists all over Sudan and also used by the national malaria control programme as a venue for short courses in epidemiology, vector control and planning for malaria.This is a cross-sectional facility-based analytical studyAll patients with fever, over 18 years of age reporting to the malaria centre  were included in the study with exclusion of anyone who is receiving or has received malaria treatment within 14 days prior to the study and severely sick patients.For the calculation of the sample size, we used the following equation :Where:0) is 0.75.N = sample size, the suspected sensitivity (p) is 0.90 and the minimal acceptable sensitivity  was multiplied by 5 to get a sample size of 210. The sample size adjustment for specificity yielded a much smaller sample (approx 52). The sample size used in the study was 293.All patients attending the site were included until the sample size was reached.® microscopy.A structured data collection form was used to record the results of the light and fluorescence microscope. After verbal informed consent was obtained, the data collection form was filled out through direct interview with the patients. The form focused on basic personal data and was used later to record the results of light and fluorescence microscopy. After the form is filled out, two finger-prick blood samples were taken; one for light microscopy, and the other for Cyscope® microscope. Thin and thick films were stained using 10% Giemsa stain. Hundreds of fields were examined at 10 × 100 magnification under oil immersion for the presence of trophozoite and gametocyte stages, and for identification of parasite species. The density was estimated by counting the number of asexual parasites per 200 white blood cells and then multiplied by 40 to get the parasite number per 8,000 white blood cells.For the light microscopy, a light microscope was used to diagnose malaria using thick and thin film, by a trained person blinded to the results of the Cyscope® malaria microscope was used to diagnose malaria by another trained person, blinded to the results of light microscopy. According to Partec [A Cyscopeo Partec , a finge® was calculated as true positives/, specificity as true negatives/, positive predictive value (PPV) as true positives/, negative predictive value (NPV) as true negatives/.Epi Info (Version 3.5.1 - 13 August 2008) was used for data entry, data cleaning, and analysis. Quantitative data was summarized using proportions and means. With the results from the light microscope as the standard, sensitivity of the CyscopeVerbal consent was taken from each participant in the study.P. falciparum) (see Table ® fluorescence microscope results showed that 20.5% (n = 60) of those tested were positive for malaria.The number of patients included in the study was 293. The mean age was 32.5 years . Females constituted 60.7% (n = 176) of the population studied. Of these, 19.8% (n = 33) were pregnant) (see Table ® fluorescence microscope while one was negative (see Table ® fluorescence microscope. This translates into a specificity of 98.3% (95% CI: 95.7% - 99.5%).Of those tested positive by the reference laboratory, 56 have been found to be positive by the Cyscope® fluorescence microscope, 56 were positive by the reference laboratory, while four were negative. This translates into a positive predictive value of 93.3% (95% CI: 83.8% - 98.2%). Of those tested negative by the Cyscope® fluorescence microscope, 232 were negative according to the reference laboratory while one was positive. This translates into a negative predictive value of 99.6% (95% ci: 97.6% - 100%).Of those tested positive by the CyscopeP. falciparum. This was also expected since P. falciparum is by far the most dominant species in Sudan [The females in the sample were slightly more than the males. This might have accounted for having 33 pregnant women in the sample, two of whom were positive for malaria. As expected, the prevalence of malaria within the sample was around 20%. All positive samples were ® fluorescence microscope. The calculated sensitivity of the Cyscope® fluorescence microscope in diagnosing malaria was above the 95% threshold while the specificity was above the 90% threshold [® fluorescence microscope were also high (above 90%) in comparison to the gold standard of light microscopy in the reference laboratory.The accepted level of sensitivity for a rapid diagnostic test in diagnosing malaria is a sensitivity of 95% at a parasite density of 100 parasites/μl . In thishreshold . The pos® fluorescence microscope to be a reliable diagnostic tool that is very sensitive and specific in diagnosing falciparum malaria. Since this is the predominant species in Sudan, and is the species causing most mortality and complications, this is very relevant and very useful. It is expected that the microscope will show similar results for other malaria species, but this could not be ascertained by this study. Further studies are needed to determine effectiveness in diagnosing other Plasmodium species. Further studies are also needed to determine the effectiveness of the Cyscope® fluorescence microscope in diagnosing malaria in cases of low parasite densities.In conclusion, this study has found the CyscopeThe authors declare that they have no competing interests.HSH participated in the design and coordination of the study and performed the statistical analysis. OSI participated in coordinating the study. MMA participated in coordinating the study in Sinnar State. MEM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Accurate information about the burden of malaria infection at the district or provincial level is required both to plan and assess local malaria control efforts. Although many studies of malaria epidemiology, immunology, and drug resistance have been conducted at many sites in Indonesia, there is little published literature describing malaria prevalence at the district, provincial, or national level.Two stage cluster sampling malaria prevalence surveys were conducted in the wet season and dry season across West Sumba, Nusa Tenggara Province, Indonesia.Plasmodium falciparum accounted for 70% of infections; in the dry season P. falciparum and Plasmodium vivax were present in equal proportion. Malaria prevalence varied substantially across the district; prevalences in individual sub-villages ranged from 0–34%. The greatest malaria prevalence was in children and teenagers; the geometric mean parasitaemia in infected individuals decreased with age. Malaria infection was clearly associated with decreased haemoglobin concentration in children under 10 years of age, but it is not clear whether this association is causal.Eight thousand eight hundred seventy samples were collected from 45 sub-villages in the surveys. The overall prevalence of malaria infection in the West Sumba District was 6.83%  in the wet season and 4.95%  in the dry. In the wet season Malaria is hypoendemic to mesoendemic in West Sumba, Indonesia. The age distribution of parasitaemia suggests that transmission has been stable enough to induce some clinical immunity. These prevalence data will aid the design of future malaria control efforts and will serve as a baseline against which the results of current and future control efforts can be assessed. Accurate information about the burden of malaria infection at the district or provincial level is required both to plan local malaria control efforts and to measure the impact of such efforts. Although many studies of malaria epidemiology, immunology, and drug resistance have been conducted at many sites in Indonesia -7, there9°40'S, 120°00'E. The island is divided into two districts, East Sumba and West Sumba. The population of West Sumba is approximately 400,000. Most residents are subsistence farmers. The climate is tropical, with a dry season from May to November and a wet season from December to April. Since completion of the study, West Sumba District has been divided into three separate districts.Sumba is a member of the Lesser Sunda Archipelago, located in East Nusa Tenggara Province, Indonesia, at The study consisted of two point prevalence surveys with two-stage cluster sampling. The primary unit of random selection was the sub-village, the smallest administrative unit for which there were census data. Selection of clusters used probability proportional to size (PPS) sampling, based on Government of Indonesia census data collected in 2005. Within each sub-village all residents of a set of houses chosen as follows were selected. Sumban villages are built around a central cluster of megalithic tombs. A pointer was spun in the village centre and the nearest house in the direction indicated by the pointer was visited. Beginning with this house, houses were visited in an expanding counterclockwise spiral until approximately 100 subjects were enrolled. At each household the participation of every individual who had slept in that house on the previous night was requested, and the sex and age of all individuals were recorded. Informed consent was obtained from subjects or their parents or guardians. The same set of 45 clusters was sampled twice, once in March 2007 (wet season) and once in August 2007 (dry season). Although the same 45 clusters were sampled in both surveys, no attempt was made to resample the same individual households in the clusters; instead a new random selection of households in each cluster was performed for the second survey.The use of human subjects in these studies was approved by scientific and ethical review boards of the Naval Medical Research Unit #2, the Eijkman Institute, and by the Indonesian National Institute of Health Research and Development, and was conducted in accordance with regulations governing the protection of human subjects in medical research. Informed consent was obtained from all adult subjects and from the parents or legal guardians of minors.For each enrollee, the weight, height, and axillary temperature were measured, and inquiry was made if they had experienced fever in the previous 24 hours. All children between 2 and 9 years of age were examined for splenomegaly. Patients with signs and symptoms of malaria, including fever, chills, malaise, fatigue or other systemic complaints were tested with the Parascreen Rapid Test for Malaria . If positive, the patient received a three-day course of artemesinin/lumefantrine according to Indonesian Ministry of Health (MoH) guidelines. From finger or heel prick blood samples haemoglobin concentration was measured with a Hemocue device, and thick and thin blood films for malaria diagnosis were prepared. All malaria smears were read the same day, and if positive, a medical team returned to the sub-village the next day with anti-malarial treatment according to the above MoH guidelines.3 assuming a white blood cell count of 8000/mm3. A second certified microscopist reviewed all positive smears and 10% of negative smears. A third certified microscopist reviewed discrepant results; the majority reading was considered definitive. There were 195 discrepant slides, or less than 10% of the total.Thick and thin blood films were stained with Giemsa and examined by a certified microscopist using 1000× oil immersion light microscopy. At least 200 ocular fields were read before a slide was considered negative. Parasite densities were counted as parasites per 200 leukocytes and reported as parasites/mmAll data were recorded on standardized case report forms, double entered into MS Access  and exported for analysis in STATA , SPSS , and Mathematica 5.2 . All statistical tests were two-tailed and significance was defined as p < 0.05.Two two-stage, cluster sampling malaria prevalence surveys were conducted in March (wet season) and August (dry season) of 2007. In the first survey 45 sub-villages were sampled out of a total of 300 in West Sumba. Within each sub-village a mean of 19.2 households  were sampled and within each household, samples were obtained from a mean of 5.2  individuals per household, for a total of 4,480 subjects. A total of 567 subjects who resided in the sampled households could not be contacted or enrolled. In the second survey, in the same 45 sub-villages, a mean of 18.3 households  were sampled per sub-village with a mean of 5.3  individuals per household, for a total of 4,375 subjects. In this survey, 906 individuals resident in the selected households could not be enrolled. Overall, it was possible to enroll and sample 8,870 out of 10,343 residents of the selected households (86%). The age and sex breakdown of the enrolled subjects and the missing members of the selected households are shown in Table Plasmodium spp in the two survey periods. The raw prevalences of malaria infection in the sampled subjects in the wet and dry season were 6.92%  and 5.03% , respectively. The 95% confidence intervals for the two-stage cluster design were adjusted using a finite population correction for the first stage and Taylor series linearization to calculate the standard error. Confidence intervals corrected for the study design were approximately three-fold wider than the uncorrected intervals , but there was little difference in the prevalence of P. vivax and P. malariae between seasons. Presumably, the higher prevalence of P. falciparum in the wet season is due to increased abundance of the vector mosquito species in this season. If a substantial fraction of P. vivax and P. malariae infections are due to relapse from the hypnozoite stage and chronic asymptomatic infection, respectively, then the lack of seasonality in these infections may be unsurprising.The prevalence of Malaria prevalence varied greatly between clusters and across West Sumba. Figure P. falciparum and P. vivax was more common in children and teenagers than in adults in either the wet season or the dry season, and children with parasitaemia tended to have higher parasitaemia than adults. Figure P. falciparum and P. vivax parasitaemia. The greatest prevalence of infection, for both P. falciparum and P. vivax, in either wet or dry season, occurred in subjects aged 5–20 years. The parasitaemia was highest in the youngest children and declined in the older age groups. The observed age distribution of prevalence and parasite density here is similar to that seen in meso-endemic areas of sub-Saharan Africa and in long-term migrants to Papua, and suggests that malaria transmission is sufficiently intense and stable to have induced at least partial clinical immunity. In Africa, young infants are protected by maternal antibodies [The age distribution of parasite prevalence and parasitaemia density provides suggestive information about the level of naturally acquired immunity to malaria and, indirectly, about the long term intensity and stability of malaria transmission. In populations not previously exposed to malaria who move to a malaria endemic area, for example recent Javanese migrants to Indonesian Papua, malaria is equally prevalent in all age groups . On the tibodies . Among 1P. falciparum transmission in Africa, severe malarial anaemia is the most common form of severe malaria [2 = 0.2). It is possible that this association indeed reflects reductions in haemoglobin concentration resulting from prolonged or repeated malaria infection, but it may simply reflect a poorer general health status in the sub-villages with higher malaria prevalence.In areas of high  malaria ,14 and t malaria -17. In WPlasmodium falciparum was the predominant species during the wet season; in the dry season P. falciparum and P. vivax were present in roughly equal proportion (Table Two stage cluster sampling malaria prevalence surveys covering the West Sumba District of East Nusa Tenggara Province, Indonesia in the wet and dry seasons of 2007 were conducted. In the district as a whole malaria was hypoendemic Table . Plasmodon Table  suggestson Table , but it The authors declare that they have no competing interests.DS and WOR designed the study, participated in data collection, analysed the data, and participated in drafting the manuscript; K, PA, S, RMD, FC, IER, IA, IE, AS, and AR participated in data collection and analysis; MK participated in data collection and analysis and wrote the first draft of the manuscript.


Schistosoma haematobium infection on specific antibody responses and cytokine production to recombinant merozoite surface protein-1-19 (MSP1-19) and schizont extract of Plasmodium falciparum in malaria-infected children.Malaria and schistosomiasis coinfection frequently occurs in tropical countries. This study evaluates the influence of 19, as well as IgG1 and IgG3 to schizont extract were significantly increased in coinfected children compared to P. falciparum mono-infected children. Stimulation with MSP1-19 lead to a specific production of both interleukin-10 (IL-10) and interferon-γ (IFN-γ), whereas the stimulation with schizont extract produced an IL-10 response only in the coinfected group.Specific IgG1 to MSP1-Our study suggests that schistosomiasis coinfection favours anti-malarial protective antibody responses, which could be associated with the regulation of IL-10 and IFN-γ production and seems to be antigen-dependent. This study demonstrates the importance of infectious status of the population in the evaluation of acquired immunity against malaria and highlights the consequences of a multiple infection environment during clinical trials of anti-malaria vaccine candidates. Plasmodium falciparum (Pf) malaria remains one of the major public health issues in tropical countries and the vast majority of childhood deaths due to malaria occur in sub-Saharan Africa Pf malaria is slowly acquired after several infections and is dependent on the intensity and duration of the individual exposure to the parasite P. falciparum merozoite surface protein 1 (MSP1) and especially its highly conserved C-terminal EGF-like module pair, known as MSP1-19 is one of leading candidate antigens for a vaccine against the malaria parasite blood stage P. falciparum Ab responses et al. recently demonstrated that in Ghanaian school children, there was an increase in specific IL-10 production in helminth-infected individuals, compared to non-helminth infected Protective immunity to Plasmodium infection Schistosoma coinfection during uncomplicated P. falciparum malaria unbalances the regulation of the associated inflammatory response Intrinsic features of the immune system that develop with age but also with the presence of other chronic infections may influence malaria immunity S. haematobium (Sh) on the specific isotype Ab response and its associated cytokine production to PfMSP1-19 and to schizont extracts of P. falciparum asexual blood stage antigens (Ags) in children living in a particular malaria endemic area, where schistosomiasis appeared 15 years ago The present study evaluates the impact of coinfection by P. falciparum without a confirmed schistosomiasis (Pf)  and malaria infected children presenting a coinfection with S. haematobium (Pf-Sh) . Comparable mean age and sex ratio were obtained. Absence of clinical morbidity for malaria and schistosomiasis was a criterion of selection. The detection of schistosomiasis infection was performed using the referent parasite criteria (presence of eggs in urine or/and in feces). Schistosoma infected patients presenting pathological cases of schistosomiasis were treated but not included in the study. The Taba Tache and Taba Dar Salam villages are located down-river from Diama dam on the Senegal River where schistosomiasis is absent as previously described (13–14) and as confirmed by the present study.The studied population was a cohort of 79 malaria infected children living in the same area of the Senegal River basin , as previously described Pf infection was detected by Quantitative Buffy Coat (QBC) (Becton Dickinson) and parasites were then counted and identified on blood smears. These tests are commonly used in malaria endemic areas and blood smears represent the referent criterion to detect malaria infection Pf were observed on positive slides. No other Plasmodium species infection was detected. The studied population was considered positive for malaria when Pf was detected with the QBC test and confirmed by blood smear observation, over a one-month period. In coinfected children, the mean parasitaemia of P. falciparum was not significantly different from the respective Pf mono-infected groups (Mann–Whitney U-test). Mean of parasitaemia was 1617 per mm3/blood for Pf infected children versus 340 per mm3/blood for coinfected children. During pre-selection, subjects showing high positivity in QBC (+ + +) were excluded and immediately treated for malaria. Children presenting clinical symptoms of mild or severe malaria morbidity were not selected. The studied population did not therefore present clinical symptoms of malaria morbidity. None of the selected children had received anti-malaria treatment in the month preceding the study.Sh eggs was evaluated in three samples of urine (urine filtration) using microscopy. In the studied area and population, no Schistosoma mansoni (Sm) infection was detected using Kato–Katz method.The presence of Schistosoma infection in these villages by evaluating the schistosome worm circulating anodic antigen (CAA) in sera, as previously described In the villages of Taba Tache and Taba Dar Salam, schistosomiasis infection has never been previously detected . In addition to the complete absence of schistosome eggs in faeces and urine, we have confirmed the absence of Sm and Sh eggs was evaluated in three samples of faeces (Kato–Katz method) or urine (urine filtration), respectively. The intensity mean of urinary Sh was 57 eggs/10 ml of urine.In Lampsar village, the presence of None of the selected children had received anti-schistosome treatment in the previous 6 months.The study followed ethical principles and was approved by the ethics committee of the Senegalese Ministry of Health. Written informed consent was obtained from the study population by their parents or guardians.P. falciparum antigens were recombinant PfMSP1-19 and schizont total antigens. PfMSP1-19 was produced in Pichia pastoris as a hexa-His fusion 19 was provided by J. McCormick and W. Morgan from National Institute for Medical Research . The schizont antigenic preparation of P. falciparum is a soluble extract of schizont lysate obtained from infected erythrocyte cultures The Soluble Egg Antigen (SEA) is a total soluble extract of schistosome eggs obtained from experimental infection in guinea pigs by the Antigen Production Laboratory at the Pasteur Institute of Lille, France.19, schizont extract or SEA in sera were determined by enzyme-linked immunosorbent assay (ELISA).Specific antibody levels to PfMSP1-19 protein (0.5 µg/ml), schizont extract (0.5 µg/ml) or SEA (5 µg/ml) were coated on 96-well plates  for 2 h 30 at 37°C. Plates were then blocked in PBS containing 0.5% gelatin . The sera diluted in PBS/Tween were incubated at 4°C overnight at a 1/100 dilution for IgG2, IgG3 and IgG4, 1/50 for IgA and IgE, and at 1/1,000 for IgG1 detection to schizont extract. For MSP1-19 antigen, a 1/100 dilution for IgG2, IgG3, IgG4 and IgA, 1/50 for IgE and 1/1,500 for IgG1 were used and for SEA, a 1/1,000 dilution for IgG1 and 1/100 for IgG3. Corresponding biotinylated mAbs to human Ig isotypes , were then incubated at a 1/1,000 dilution for IgG2, IgG4; 1/2,000 for IgG1, 1/750 for IgG3; 1/1,500 for IgA and 1/250 for IgE (1 h 30 mn at 37°C). Streptavidin-horseradish peroxidase-conjugated for IgG1, IgG2, IgG3, IgG4 and IgA detection  and ExtrAvidinR peroxidase conjugate for IgE detection  were then added. These ELISA conditions have been determined according to the results of previous experiences. Colorimetric development was allowed by means of ABTS  diammonium; Sigma) in 50 mM citrate buffer (pH = 4 containing 0.003% H2O2), and absorbance (OD) was measured at 405 nm for IgG1, IgG2, IgG3, IgG4 and IgA. IgE detection was revealed by OPD . OPD was added for 30 minutes, followed by the addition of 2N H2SO4 to stop the reaction and OD was measured at 492 nm. The same ELISA procedures were performed in parallel for 30 sera from uninfected European subjects used as negative controls. Individual results were expressed as ΔOD value calculated for each isotype test according to the formula: ΔOD  =  ODx-ODn, where ODx is the individual OD value of infected patient and ODn was the arithmetic mean of individual OD value for the 30 uninfected control individuals .PfMSP1-Pf/Sh coinfected subjects were diluted with PBS/Tween solution and incubated with schistosomes Soluble Egg Antigens (SEA) at a final concentration of 200 µg/ml. The mixture (sera-SEA) was incubated with gentle shaking for 5 h at room temperature to allow the binding of specific Abs to SEA. Specific IgG1 and IgG3 responses to SEA, to schizont extract of Pf or to PfMSP1-19 were evaluated by ELISA (as described above) before and after this adsorption procedure.Individual sera of 6 mononuclear cells/ml. Blood cells were then distributed at the rate of 1 ml/well in 24-well plates (Nunc). Antigens were added to each culture well to a final concentration of 1 µg/ml for MSP1-19 and 10 µg/ml for schizont extract. After 3 days of incubation at 37°C in 5% CO2, the content of each culture well was centrifuged, and supernatants were harvested and conserved at −80°C. Supernatant concentrations of IL-10, IL-12, IL-13, IFN-γ and TGF-β were measured at day 3 by means of ELISA kits according to the manufacturer's instructions . Specific cytokine production after Ag stimulation were expressed as picograms, nanograms or International Units (IU) per millilitre after the subtraction of the amount detected in medium control cultures (non-stimulated conditions) for each individual. Identical culture conditions were assessed with mononuclear cells of 30 uninfected control individuals in which no cytokine production was detected after Ag stimulation (data not shown).Sterile conditions were maintained during the whole blood culture procedure, as originally described U-test was used to compare values between both independent groups. Correlation between specific Ab levels and the intensity of P. falciparum infection was analysed using Spearman's rank correlation coefficient. All differences were considered significant at p<0.05.All data were analysed with Graph Pad Prism . After verifying that values did not assume a Gaussian distribution, the nonparametric Mann-Whitney 19 between children having been solely infected by P. falciparum (Pf) and those coinfected with S. haematobium (Pf/Sh) was assessed   . The speh groups . No relechildren .19 Ag, specific IgG2, IgG4, IgA, and IgE responses to total schizont extract were very low or similar between Pf and Pf/Sh groups  of sera from coinfected group were therefore performed in order to verify the possible presence of IgG1 and IgG3 Abs cross-reacting with schistosome and P. falciparum antigens.We cannot exclude that antigenic cross-reactions can at least partly account for increased levels of anti-Pf/Sh patient sera with SEA, indicating the efficiency of the experimental procedure  before and after Abs adsorption. The comparison shows that the mean OD values were similar between SEA adsorbed and non adsorbed sera for both schizont extract and MSP1-19 Ags . This suggests that the observed quantitative difference in the anti- P. falciparum Abs responses between both groups were not due to differences in the intensity of blood malaria parasites but could be related to the influence of Schistosoma coinfection.The correlation between specific Ab levels and the parasitaemia due to vs. mono-infected) matched on the level of Pf parasitaemia. We have obtained 25 matched-pairs with parasitaemia values varying from 3 to 3780 infected erythrocytes per mm3 of blood. The Wilcoxon t-test was used to compare the means of the matched-pairs. Regarding Ab responses against schizont Ags, the obtained value was t = 3.75 for IgG1 (p<0.01) and t = 3.23 for IgG3 (p<0.001), indicating that both groups were significantly different regarding their IgG1 and IgG3 levels when patients were matched on their parasitaemia.In addition, we have compared individual IgG1 and IgG3 responses against schizont Ags from both groups . As for specific IFN-γ secretion, difference between both groups was only observed for the schizont extract and was significantly higher in Pf/Sh-group compared to Pf-group (p = 0.02). Nevertheless, high IFN-γ production to MSP1-19 was observed in few coinfected children (12/39) but the mean value of IFN-γ secretion in this group was not statistically higher than the one observed in Pf-group (p = 0.282). Background production of IFN-γ and IL-10  was similar between both groups  . These last results indicate that the differences of cytokine production according to infection status were observed only after the stimulation by Plasmodium antigens.The production of specific IFN-γ, IL-10, IL-12, IL-13 and TGF-β cytokines after children . These cP. falciparum malaria Plasmodium Ags extract (schizont stage) and to the specific PfMSP1-19 vaccine candidate. The greatest interest of this study is that the selected children lived in the same low malaria area (confirmed by the similar Pf parasitemia between groups), without clinical manifestations, but living in villages from the same studied area, where schistosomiasis was monitored since several years (13–14). In particular, it indicated that Pf mono-infected children have never been infected by Schistosoma. We demonstrated that S. haematobium coinfection influenced the humoral immune response against malaria antigens by specifically increasing IgG1 and IgG3 Abs levels. This increase was not dependent on the age, on the intensity of malaria infection or on a potential antigenic cross-reactivity. Indeed, the IgG1 and IgG3 responses to schizont extract were higher in Pf/Sh-infected children compared to the Pf-infected group. However, only specific IgG1 Ab response to the MSP1-19 antigen was significantly increased in coinfected children suggesting that the influence of coinfection could be antigen-dependent. During human malaria, IgG1 and IgG3 are thought to play a key role in protection Following the study of the influence of schistosomiasis coinfection on the regulation of inflammatory factors in uncomplicated Sh and Pf antigens, by assessing SEA adsorption experiments. In addition, statistical analysis showed the absence of an effect of the intensity of P. falciparum parasitaemia on these specific Ab responses to malaria antigens. Altogether, the data suggest that chronic schistosome infection in malaria-infected children could affect the specific immune response to P. falciparum. In particular, S. haematobium coinfection appeared to influence the specific isotype Ab responses involved in the development of protective immunity to P. falciparum infection. Moreover, we and others have described that P. falciparum coinfection also influenced the anti-schistosome immune response in S. haematobium-infected children 19 has been observed in 1/3 of coinfected children (12/39) whereas this isotype response was not detected in children infected only by Pf. Several immuno-epidemiological studies indicate that the presence of anti-MSP1-19 IgG3 response could be dependent to the studied site. For example, a very low IgG3 antibody response to MSP1-19 antigen was observed in some malaria endemic areas 19 antigen cannot be excluded to explain the presence or the absence of this specific IgG3 response. Nevertheless, our results indicate that the presence of specific IgG3 to MSP1-19 among some children living in an endemic malaria area was observed when schistosomiasis coinfection has been established. It could be of great interest to confirm the observations of our study in other coinfection contexts. Our study indicates that it will be crucial to systematically consider the coinfection status of patients in studies concerning the anti-malaria immunity. It strengthens the necessity to screen patients for helminth infections and their immune influence before and during malaria vaccine trials, as previously suggested In the present study, we could also rule out the possible Abs cross reactivity between total S. haematobium infection on specific immune response to Plasmodium Ags involves specific cytokine production. Helminth infections seemed to influence IL-12, IL-13 and TGF-β production under stimulation with both malaria Ags. In contrast, specific IFN-γ and IL-10 production to the schizont extract were significantly higher in the coinfected group, which showed also a predominant anti-schizont IgG1 and IgG3 Ab levels. In addition, specific IL-10 production to MSP1-19 was significantly higher in the coinfected group compared to children only infected by Pf, as observed for specific IgG1 response. In vitro human studies prove that stimulation by MSP1-19 preferentially elicits IL-10 production Borrelia burgdorferiS. haematobium chronic infections . In the acute uncomplicated malaria, it has been demonstrated that peripheral blood CD4+ T cells producing both IFN-γ and IL-10 were significantly increased during drug-induced clearance of parasitaemia 19 could produce both IFN-γ and IL-10 We have demonstrated that the influence of P. falciparum IgG1 and IgG3 Abs could be differently regulated by IL-10 and IL-10/IFN-γ, respectively. This hypothesis is now under investigation by analysing the in vitro isotype production after cytokine stimulation of T cells from infected individuals.In our study, no statistical correlation of specific IFN-γ or IL-10 productions with IgG1 and/or IgG3 production has been observed. Nevertheless, we have shown an association between the production of these cytokines and the regulation of these specific isotypes according to the group of infection. With regard to previous studies The influence of environment, genetic background or nutritional status cannot be ruled out to explain the variation of specific immunity observed in our study. Nevertheless, the concomitant infections such as schistosomiasis in children may influence the acquired anti-malaria Ab response and associated cytokine production. The presence of chronic schistosomiasis infection could specifically increase IL-10 and IFN-γ production in coinfected children, a cytokine production which would positively influence the production of cytophilic anti-malaria IgG1 and IgG3 isotypes, known to be protective during malaria infection. Altogether, this study represents an approach of the regulation of anti-malaria blood stage immunity by coinfection and indicates that the helminthic infection status has to be taken into account in the malaria epidemiological and vaccine studies.