Uganda embraced the World Health Organization guidelines that recommend a universal 'test and treat' strategy for malaria, mainly by use of rapid diagnostic test (RDT) and microscopy. However, little is known how increased parasitological diagnosis for malaria influences antibiotic treatment among patients with febrile illness.Data collection was carried out within a feasibility trial of presumptive diagnosis of malaria (control) and two diagnostic interventions (microscopy or RDT) in a district of low transmission intensity. Five primary level health centres (HCs) were randomized to each diagnostic arm (diagnostic method in a defined group of patients). All 52,116 outpatients  aged 5 months to ninety five years presenting with fever (by statement or measured) were included. Information from outpatients and laboratory registers was extracted weekly from March 2010 to July 2011. The proportion of patients who were prescribed antibiotics was calculated among those not tested for malaria, those who tested positive and in those who tested negative.Seven thousand and forty (41.5%) patients in the presumptive arm were prescribed antibiotics. Of the patients not tested for malaria, 1,537 (23.9%) in microscopy arm and 810 (56.2%) in RDT arm were prescribed antibiotics. Among patients who tested positive for malaria, 845 (25.8%) were prescribed antibiotics in the RDT and 273(17.6%) in the microscopy arm. Among patients who tested negative for malaria, 7809 (61.4%) were prescribed antibiotics in the RDT and 3749 (39.3%) in the microscopy arm. Overall the prescription of antibiotics was more common for children less than five years of age 5,388 (63%) compared to those five years and above 16798 (38.6%).Prescription of antibiotics in patients with febrile illness is high. Testing positive for malaria reduces antibiotic treatment but testing negative for malaria increases use of antibiotics.NCT00565071ClinicalTrials.gov:  Recent World Health Organization (WHO) guidelines recommend a universal 'test and treat' strategy for malaria, mainly by use of rapid diagnostic test (RDT) and microscopy in all transmission areas . In ruraAntibiotics are routinely prescribed for colds, non-specific upper respiratory tract infections and acute bronchitis ,4, as coEven for conditions in which antibiotic use might be justified, experts have expressed concern about substantial overuse  that impAlthough the Uganda national malaria guidelines now recommend confirmation of parasitaemia before initiation of treatment, data on how results of use of malaria diagnostics influence antibiotic treatment among febrile outpatients is lacking. The current study assessed antibiotic prescribing rates among febrile outpatients attending rural health centres (HCs) where feasibility of rolling out parasitological diagnosis for malaria was being tested. The primary outcome measures were the proportions of patients with febrile illness that were prescribed antibiotics when: not tested for malaria, they test positive and when they test negative.Data collection on antibiotic treatment was carried out within a cluster randomized feasibility trial of presumptive malaria diagnosis (control) and two diagnostic interventions (microscopy and RDT). Fifteen out of twenty sub-county level government HCs in a district of low malaria endemicity in Uganda were randomly selected for the trial. HCs were the primary sampling units, and were allocated to the three diagnostic arms using simple randomization. Finally, there were five HCs per arm (diagnostic method in a defined group of patients).The trial was carried out in Bushenyi district in south-western Uganda. The district headquarter is located at about 320 km from the capital city Kampala. The district is mainly rural with a total land area of 3,949 sq. km. It is endowed with diverse natural resources that include arable land, forests, large lake water bodies , Queen Elizabeth National Park and minerals. The main economic activities are semi-intensive agriculture , fishing and trade. The district is multi-ethnic with varying customs and norms. The main inhabitants are Banyankore and Bakiga. The total population is estimated at 731,392, and with 20 public HCs at sub-county level. The population distribution and density varies with physical geography. It is concentrated in the low-lying plateau zones of Sheema, Igara and Ruhinda; and sparse in the hilly-rough and rugged terrain of Buhweju and Bunyaruguru. The climate is relatively wet. The mean annual temperature range is 12.5°-30°C. Most of the district receives 1500-2000 mm of rainfall annually. Although Bushenyi experiences low and unstable malaria transmission, people of all ages are at risk. It is epidemic-prone, with occasional malaria outbreaks occurring shortly after the rains. The annual entomological inoculation rate is not known, but it was reported to be < 10 infective bites per person per year in the neighbouring district of Kanungu .®Paracheck. The staff in HCs with microscopy or RDT were instructed to treat patients for malaria according to test results. Treatment with antibiotics followed national guidelines. HC outpatient registers were modified to record additional variables such as the presenting complaints, drugs dispensed and to indicate those prescribed but out-of stock. The trained staff were charged with training those that were off-duty on the day of training. However, additional clarification was provided during supervision. Supervision by the study team was carried out weekly during the first two months and monthly thereafter. The district laboratory focal persons provided the routine quality control procedures in both microscopy and RDT arms.A total of 74 clinical and laboratory staff received a one-day refresher training on-site. The training and subsequent study procedures were a scale-up of activities performed during the assessment of the accuracy of these malaria diagnostic methods . All staPatients presenting with fever (by statement or measured) were enrolled to receive service without parasitological confirmation of malaria. Patients were treated on the basis of signs and symptoms only.All patients presenting with fever (by statement or measured) were enrolled. The laboratory assistants prepared thick and thin blood smears by finger-prick using sterile blood lancets on separate frosted slides. Standard staining was performed using the Field's stain method. Laboratory assistants were only familiar with this staining technique. Blood films were read at magnification X1,000. Each film was graded as positive  or negative  based on inspection of 200 fields. Microscopy test results were recorded in the laboratory registers.®Paracheck" device . ® Paracheck Pfis based on the detection of histidine rich protein-2 (Pf HRP-2) produced by Plasmodium falciparum trophozoites and young gametocytes. The specimens were drawn by trained nurses or laboratory assistants using a simple finger-prick. The test preparation and interpretation were done following manufacturer's instructions and standard operating procedures. The test was considered positive when the antigen line was visible in the test window and negative when only the control band was visible. RDT results were recorded in the outpatient registers.Patients presenting with fever underwent rapid testing with the "All outpatients presenting at the study HCs with fever (by statement or measured) from March 2010 to July 2011 were enrolled. Data collection was carried out weekly by the research assistants by extracting information from the laboratory and outpatient registers.p-values) were set at 0.05 and confidence intervals (CI) were calculated at the 95% level. Socio-demographic and symptom data were presented using descriptive statistics: distribution by age, number and percent of patients with positive and negative results, and those receiving drugs.The collected data were manually checked and cleaned. Data were double entered by two trained database assistants in a customized entry template with in-built consistency checks in EpiData 3.1 software . The two data sets were validated to check for entry errors. Before analysis in Stata version 10 , the data were declared a cluster design using the "svyset command" with HCs as primary sampling units. Further, the Poisson regression model was fitted while accounting for clustering. Probability values . The study was registered with the Clinicaltrials.gov (NCT00565071).The study was carried out in 15 sub-county level government HCs located in an area of low malaria transmission intensity. Overall, 52,116 outpatients presenting with fever were enrolled: in the presumptive arm 16,971; microscopy arm 17,508; and RDT arm 17,637  and amoxicillin capsules to 5,986 (25.8%). These two antibiotics were the most commonly prescribed. Metronidazole was prescribed to 3,950 patients (17.0%), but in combination with other antibiotics. Doxycycline, erythromycin and ciprofloxacin were also prescribed, but in smaller quantities. Other drugs such as analgesics and anti-helminthics were prescribed to 49,574 patients (95.2%) and 10,330 (19.8%) respectively.In the presumptive arm 7,040 patients 41.5%) were prescribed antibiotics. In the microscopy arm 6,427 (37%) did not receive a parasitological diagnosis and of these 1,537 (23.9%) were prescribed antibiotics. In the RDT arm, 1442 (8%) did not receive a parasitological diagnosis and of these 810 (56.2%) were prescribed antibiotics  were prescribed antibiotics. In the microscopy arm of the 1,548 with positive results, 273 (17.6%) were prescribed antibiotics .Overall, 11,658 patients (52.1%) with negative results were prescribed antibiotics Table . PatientThis article reports on a large assessment of the effect of malaria diagnostics on the probability of receiving antibiotics in a Ugandan population living in an area of unstable malaria transmission. The findings indicate that the rate of antibiotic treatment was high; there was a reduction in antibiotic prescription among patients with positive malaria test results; there was an increase in antibiotic treatment in those testing negative; the rate of antibiotic prescription was higher in children under five years of age; and antibiotic prescription varied widely across HCs suggesting that prescribers' behaviour is a big factor in use of antibiotics.The level of antibiotic treatment reported here is higher than that demonstrated in Kabale , a distrPatients who were in the RDT arm were more likely than those in the microscopy arm to be prescribed antibiotics. This difference is unlikely to be attributed to use of RDT. Generally in the microscopy arm, antibiotic prescription was low among: those not tested, who tested positive and those who tested negative. The trend of antibiotic use, however, was similar in the two diagnostic arms (decreasing among patients who test positive and increasing among negative patients). A more likely explanation for this difference in rates of antibiotic use among the diagnostic arms is prescribers' behaviour [i.e. higher likelihood of service providers more likely to treat with antibiotics in the RDT arm].The rate of antibiotic prescription generally decreased among patients with positive results. This might indicate clinicians' acceptance of malaria-positive results as the only likely cause of illness at that point and therefore restrained from prescribing concurrent medications. However, the proportion of parasitaemic patients prescribed antibiotics reported here is higher than that observed in other settings . EnormouIn this study, the chance of prescribing antibiotics increased if a febrile patient tested negative for malaria. These data are similar to what has been reported elsewhere ,26-28. TIn some settings, interventions that promote rational antibiotic use have been shown to be effective. These emphasize careful diagnosis especially of upper respiratory syndromes, deferral of antibiotic use, and a watch-and-wait approach  for which antibiotics are not immediately indicated , and a tPrescription of antibiotics in patients with febrile illness is high. Testing for malaria reduces antibiotic treatment in patients with positive results but increases in those testing negative. Antibiotic use also depends on age and prescriber behaviour. It is essential that malaria diagnostics are rolled-out in all primary level health units and guidelines for antibiotic treatment especially among children developed and distributed. In addition, continuing professional education for prescribers should be enforced.The authors declare that they have no competing interests.All authors conceived and designed the study; VB and FN collected, analysed, interpreted the data and drafted the manuscript; PM critically revised the manuscript. All authors read and approved the final manuscript


Plasmodium falciparum-infected mosquitoes is an accepted tool to evaluate preliminary efficacy of malaria vaccines. To accommodate the demand of the malaria vaccine pipeline, controlled infections are carried out in an increasing number of centers worldwide. We assessed their safety and reproducibility.Exposing healthy human volunteers to We reviewed safety and parasitological data from 128 malaria-naïve subjects participating in controlled malaria infection trials conducted at the University of Oxford, UK, and the Radboud University Nijmegen Medical Center, The Netherlands. Results were compared to a report from the US Military Malaria Vaccine Program.We show that controlled human malaria infection trials are safe and demonstrate a consistent safety profile with minor differences in the frequencies of arthralgia, fatigue, chills and fever between institutions. But prepatent periods show significant variation. Detailed analysis of Q-PCR data reveals highly synchronous blood stage parasite growth and multiplication rates.Procedural differences can lead to some variation in safety profile and parasite kinetics between institutions. Further harmonization and standardization of protocols will be useful for wider adoption of these cost-effective small-scale efficacy trials. Nevertheless, parasite growth rates are highly reproducible, illustrating the robustness of controlled infections as a valid tool for malaria vaccine development. Plasmodium falciparum (Pf)-infected mosquitoes in a controlled experimental setting is an accepted tool in malaria vaccine development. Such controlled human malaria infection (CHMI) trials can be used to investigate Pf immunology Deliberate exposure of healthy human volunteers to the bites of laboratory-reared Pf candidate (sub-unit) malaria vaccines or vaccine components (www.who.int/vaccine_research/links/Rainbow/en/index.html). To meet the demands of the growing malaria vaccine development pipeline, CHMI will likely be conducted in an increasing number of sites worldwide. We have performed a comparative analysis of safety and parasitological data from trials performed at the Radboud University Nijmegen Medical Centre (RUNMC), The Netherlands, and the University of Oxford in the United Kingdom, two of a total of five different institutions and the only non-US institutions currently routinely performing CHMI. Where possible, data were compared with a previously published report from the US Military Malaria Vaccine Program (USMMVP), Naval Medical Research Center Component, Silver Spring, Maryland Decades of extensive efforts to find an efficacious malaria vaccine have lead to the development of about 38 Volunteers participating in CHMI studies performed at the RUNMC in Nijmegen, The Netherlands and the Centre for Clinical Vaccinology and Tropical Medicine at the University of Oxford, United Kingdom were included from 1999 until 2010 and from 2000 to 2010 respectively. Data were compared with a previously published report of trials performed between 1998 and 2002 at the USMMVP, Silver Spring, Maryland Nieman etal. manuscript in preparation). All other volunteers were unimmunized.In Oxford, 65 infectivity control volunteers participated in 14 studies Data from three different cohorts were assessed. A summary of the cohort characteristics is provided in All included subjects were healthy, male and female, malaria-naïve volunteers between the ages of 18 and 50 years. MalariaAnopheles stephensi mosquitoes were infected with the NF54 strain of Pf (RUNMC) or 3D7, a clone originally derived from NF54, (Oxford) following previously described procedures Fixed numbers of mosquitoes were allowed to bite volunteers during five (Oxford) or ten (RUNMC) minutes. Fully blood-engorged mosquitoes were confirmed positive for salivary gland sporozoites by dissection . If necessary, feeding sessions were repeated until exactly the predefined number of infected mosquitoes were fully engorged, i.e. five mosquitoes, except for the RUNMC I cohort, where volunteers were exposed to the bites of four to seven mosquitoes . Monitorhttp://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm074775.htm). Mild symptoms (grade 1) did not interfere with daily activities, moderate symptoms (grade 2) interfered with daily activities, severe symptoms (grade 3) prevented daily activity. Symptom severity was not consistently assessed in the other cohorts, with exception of fever, which was graded mild when 37.5 to 37.9°C, moderate when 38 to 38.9°C and severe when ≥39°C in all cohorts. All centers recorded oral temperatures, which were measured at least once daily, either by volunteers themselves or by the attending physician at the clinical site. RUNMC also recorded auricular temperatures at the clinical site up to three times daily. Serious adverse events (grade 4) were defined according to International Conference of Harmonization Good Clinical Practice Guidelines.Trial volunteers were followed on an outpatient basis and lived in the vicinity of the hospital. An active tracking policy using mobile phones and/or home visits was operational at both institutions during the monitoring period. Adverse events were recorded at every visit. Investigators evaluated the potential relation of adverse events with trial procedures. All probable or possible related events were included in the analysis, with exception of the five volunteers for whom anti-malarial treatment was delayed by 48 hours. The USMMVP report included adverse events from day seven after challenge Clinical hematological laboratory data were available on a daily basis from day five post-challenge until three days after anti-malaria treatment for RUNMC cohorts. Biochemical parameters in RUNMC cohorts were assessed once, at three days after anti-malarial treatment. Clinical laboratory parameters in Oxford were not routinely recorded during challenge in any trials. For the USMMVP cohort, clinical laboratory parameters were reported at days 10–12 after challenge.Prepatent period was defined as time from exposure to positive thick smear. The threshold for microscopic detection of parasites varied between cohorts depending on the local standard operating procedure . CentresPf was used in Oxford to support microscopy. Parasite densities were measured by Q-PCR for RUNMC and Oxford cohorts as previously described Simultaneously, a quantitative PCR (Q-PCR) for Data were assessed in SPSS 16.0 with correction for multiple analyses. Differences between frequencies and prepatent period were compared by Kruskal-Wallis tests when comparing multiple groups or Mann-Whitney U tests when comparing two groups. Dunn's multiple comparisons test was performed as post-hoc analysis when appropriate. Analysis of parasitological PCR data was performed on log-transformed data using independent-samples t-test when comparing two groups and one-way ANOVA when comparing three groups. The multiplication rate of blood stage parasites was calculated by the ratio of the geometric mean parasitemia in the second cycle with the first cycle (day 8.6–10.5 vs 6.6–8.5) or the third cycle with the second cycle (day 10.6–12.5 vs 8.6–10.5), or if possible, the mean of both ratios.Correlations were assessed by Pearson's correlation when parametric and Spearman's when non-parametric. Two-sided p-values below 0.05 were considered significant unless stated otherwise.A total of 128 volunteers were grouped into three different cohorts: RUNMC I and II and Oxford and compared to published data from 47 infected individuals at USMMVP http://whqlibdoc.who.int/publications/2010/9789241547925_eng.pdf). Five serious adverse events (grade 4) occurred; three volunteers were admitted to the hospital for intravenous rehydration and directly observed intake of anti-malarial medication because of vomiting, two from RUNMC II and one from Oxford. These volunteers were discharged within 24–48 hours without sequelae. One volunteer from RUNMC II reported retrosternal chest pain two days after treatment with arthemeter-lumefantrine One hundred percent of volunteers developed signs and symptoms of uncomplicated malaria; there was no severe malaria according to WHO criteria . Thirteen of 110 volunteers (12%) showed a severe decrease in platelet count (<100×109/l). All instances occurred after initiation of anti-malarial treatment, three in the delayed treatment group in RUNMC I, nine in RUNMC II and one in USMMVP. In all events platelet counts fully recovered without complications of bleeding. There were no differences in mean platelet count between the cohorts at similar timepoints , a trend towards a longer prepatent period (Mann-Whitney p = 0.079), but no higher peak parasite density (p = 0.29). The platelet nadir correlated with the pre-exposure platelet count (Spearman p = 0.005).No abnormalities of urea and creatinine were found, but USMMVP reported six cases of hemoglobinuria and one case of proteinuria Prepatent period between the four cohorts was significantly different  and Oxford and always preceeded parasitemia by microscopy.Group mean parasite kinetics in RUNMC and Oxford display highly synchronized, cyclical parasite growth . Peak paA comparison of clinical and parasitological data from more than ten years' experience with CHMI in RUNMC and Oxford The analysis of adverse events from 175 non-immune participants of sporozoite challenge trials shows that serious adverse events (grade 4) are rare. One serious cardiac adverse event was reported; the true nature and patho-physiological explanation of that event remains unclear We find significant differences in frequencies of fever, fatigue, arthralgia and chills between institutions. Cohorts with a longer prepatent period or a higher peak parasitemia do not consistently show a higher frequency of adverse events. These data, combined with methodological differences in the recording of adverse events , lead us to conclude that biologically relevant variation in patho-physiology between institutions seems unlikely. Nevertheless, standardized assessment of adverse events and harmonization of solicited events would advance the interpretability and comparability of CHMI in different settings worldwide.9/l) in at least 7.5% of volunteers, as has been studied by de Mast et al. Laboratory safety parameters show a severe decrease in platelet count . Variability in prepatent period among institutions, however, cannot be explained solely by microscopy methodology. A detailed analysis of parasitemia by Q-PCR revealed a ∼10 fold difference in the parasite burden during the first blood stage growth cycle. Taking into account a replication factor of approximately 10 every 48 hours, this difference accounts for a 48 hour shorter prepatent period at RUNMC. The variation in parasite load may reflect variation in liver stage development of the parasites or, alternatively, the number of inoculated parasites. The number of inoculated parasites is estimated to vary widely (ranging from 5–10 An increase in threshold for microscopic detection of parasites leads to a prolonged prepatent period but not an increase in the number of adverse events, as illustrated by comparing the RUNMC I and II cohorts. Standardized reading of blood smears is thus essential to harmonize trial endpoints worldwide; recent efforts by the WHO have resulted in a proposed harmonization document (We show that blood stage parasite growth is cyclical and highly synchronous within and between institutions. Importantly, the duration of parasite liver stage development as well as the blood stage multiplication rate are highly reproducible. Thus vaccine efficacy can be robustly evaluated in any of the CHMI centers if a non-protected, malaria-naïve control group is included.fCHMI trials do not fully mimic conditions in endemic regions where pre-existing immunity may augment or impair vaccine efficacy. A limited number of comparisons between Phase IIa preliminary efficacy trials and Phase IIb field efficacy trials shows that results are generally in line, but more comparisons are required before definite conclusions can be drawn The present data show that CHMI can be safely conducted, but will lead to grade 3 adverse events in a proportion of volunteers. The primary parasitological outcome of such experiments is highly reproducible within institutions but may vary between trial centers. With an increasing number of CHMI centers being installed, priority should be given to initiatives to standardize challenge procedures


Asymptomatic malaria parasitaemia has been reported in areas with high malaria transmission. It may serve as a reservoir for continued transmission, and furthermore complicates diagnostics, as not all individuals with a positive malaria test are necessarily ill due to malaria, although they may present with malaria-like symptoms. Asymptomatic malaria increases with age as immunity to malaria gradually develops. As mortality and morbidity of malaria is higher among younger children it is important to know the prevalence of asymptomatic malaria parasitaemia in this population in order to interpret laboratory results for malaria correctly.Plasmodium mitochondrial genome was performed on all samples.A total of 108 children that had neither been treated for malaria nor had a fever the previous four weeks were recruited consecutively at a maternal and child health clinic (MCHC) in Dar es Salaam, Tanzania. A rapid diagnostic test (RDT) for malaria and dried blood spot (DBS) on filter paper were taken from each child. Social and clinical data were recorded. DNA was extracted from the DBS of study participants by a method using InstaGene™ matrix. PCR targeting the Median age was 4.6 months (range 0.5-38). All the RDTs were negative. PCR was negative for all study subjects.The study suggests that asymptomatic malaria may not be present in apparently healthy children up to the age of three years in Dar es Salaam, Tanzania. However, because of the small sample size and low median age of the study population, the findings cannot be generalized. Larger studies, including higher age groups, need to be done to clarify whether asymptomatic malaria parasitaemia is present in the general population in the Dar es Salaam area. In 2010, 655,000 people died of malaria, 91% of these in Africa, and there were approximately 216 million cases of malaria worldwide. Tanzania had approximately 5.48 million presumed and confirmed cases of malaria in 2011 [Plasmodium parasites, asexual or gametocytes, being detectable in the blood without the individual being ill. Infants are known to have a period of malaria immunity in the first one to two months of life, likely due to maternal antibodies, foetal haemoglobin and riboflavin deficiency, followed by an increased susceptibility to infection [Plasmodium species acquire immunity resulting in fewer clinical infections of malaria and less severe presentation with increased age [Asymptomatic malaria parasitaemia is a known phenomenon in malaria-endemic areas with high year-round transmission ,3. It isnfection . Studiesnfection . Childreased age . Infectiased age . Howeverased age , as the ased age . AsymptoAsymptomatic malaria parasitaemia causes diagnostic challenges. In a clinical setting, a patient presenting with fever or other symptoms of malaria should always be treated for malaria when having a positive test result for malaria. However, a patient with asymptomatic malaria parasitaemia may present with a febrile illness with a different cause and with symptoms clinically indistinguishable from malaria, but requiring a different treatment despite a positive malaria result ,10. ClinThe prevalence of asymptomatic parasitaemia in areas with high malaria endemicity varies greatly among study locations. A study done in western Kenya identified a very high prevalence of asymptomatic asexual parasitaemia (73.8%) and gametocytaemia (33.8%) in children under five as assessed by thick blood film microscopy . In GaboPlasmodium species compared to other common anti-malarials as they cause less persisting gametocytaemia post-treatment [Plasmodium species as they are ingested by mosquitoes and develop into sporozoites, which can be transmitted to new humans where they form the asexual stages that are pathogenic [Artemisinin combination therapy, which is currently recommended in Tanzania, has been shown to reduce the transmission potential of reatment , althougreatment . Gametocthogenic . Patientthogenic . Treatinthogenic .This study was performed to investigate for the presence of asymptomatic malaria parasitaemia among young children in Dar es Salaam, Tanzania.The study was performed as a collaboration between the University of Bergen and Muhimbili University of Health and Allied Sciences (MUHAS). Ethical clearance was obtained from the Regional Ethical Committee in Norway and MUHAS, and a research permit was obtained from Tanzania Commission for Science and Technology (COSTECH). Permission was also received from Magomeni Maternal and Child Health Clinic (MCHC).At the Magomeni MCHC in Dar es Salaam Tanzania, 108 healthy children were recruited in May 2009. This is an area of high malaria transmission  with a pA simple questionnaire was completed for each child recording the child’s age, weight, gender, any recent travel (within the previous four weeks), whether the child slept under a mosquito net, the mother’s level of education and the number of siblings the child had.Plasmodium falciparum, that detects histidine-rich protein 2 (HRP2) antigen in the blood, was performed on blood from a prick in the child’s finger according to the manufacturer’s instructions. The parent or guardian received the test results if they wished. RDT cassettes from the same lot were also tested on two children in a hospital ward with positive blood microscopy for malaria, to ensure that they were functioning correctly.An immunochromatography RDT cassette, ICT malaria Pf™ , for A drop of blood from the child’s finger was placed on a segment of a Whatman® Schleicher & Schuell filter paper, grade 589/2 . The dried blood spot (DBS) was stored in a sealed, airtight plastic pocket after air-drying completely. The DBS was later stored at-20°C until DNA extraction was performed.P. falciparum-positive sample with a parasitaemia of 20%, as identified by thin film microscopy, in Plasmodium-negative control blood. The dilutions ranged from 106 parasites (p)/μl to 2 p/μl.For determining the limit of detection of the DNA extraction method, DBS were prepared from dilutions of a Harris Uni-Core™ puncher  was used to punch out six pieces  of filter paper with dried blood, 3 mm in diameter. The filter paper punches were placed in a 1.5 ml Eppendorf tube. Between samples the puncher was cleaned by punching out six punches from a blank piece of filter paper and then plunging the tip of the puncher five times in sodium hypochlorite (NaClO) and 100% ethanol, respectively, followed by punching out three circles of blank filter paper. Using this cleaning procedure, no transfer of DNA between samples was detected.DNA extraction was done by placing the punches in 200 μl InstaGene™ Matrix  and incubating at 56°C for 30 min, carefully vortexing after 15 min and after completed incubation. Another incubation for 8 min at 100°C was done, followed by centrifugation at 12,000 rpm for 2 min in an Eppendorf Centrifuge 5417C . The supernatant was carefully removed and stored at -20°C until PCR was performed .Plasmodium mitochondrial DNA, as described by Haanshuus et al.[PCR amplification of us et al., but witBasic statistics of general parameters was performed using IBM SPSSStatistics version 19 .Demographics of the studied population are shown in Table With a positive PCR result for all triplicates of the 2 p/μl dilution, the InstaGene™ Matrix method could detect parasite concentrations of 2 p/μl or less. Mitochondrial PCR was negative for all study subjects.No cases of asymptomatic malaria were identified in the population studied. This indicates that asymptomatic malaria parasitaemia is not common in children up to three years of age in the study area, and therefore that all positive malaria tests in this population should be considered true cases of clinical malaria. The results of the study may also indicate that the asymptomatic population studied does not act as a reservoir for perpetuating malaria transmission. However, age groups not studied here and populations in more rural areas may still have asymptomatic malaria gametocytaemia.et al.[Recent studies find discrepancies in the sensitivities among different tests for malaria, with PCR generally detecting significantly more cases than microscopy and RDTs -27. The et al.. As the et al.,34. The In a study conducted in the Dar es Salaam area in 2008 it was found that 83.2% of children under five had slept under a bed net the previous night and 30.9% had slept under an insecticide-treated net (ITN) . In the et al. in 2009 in Tanzania detected anti-malarials in the blood of over 80% of the recruited children that were under five years of age despite all reporting no anti-malarial use in the previous 28 days [Children, for whom the parent or guardian reported febrile illness or anti-malarial treatment within the last four weeks were excluded from the study in order to reduce the risk of not detecting asymptomatic malaria infection due to recent anti-malarial use as well as to reduce the risk of detecting malaria parasitaemia due to symptomatic malaria illness. A study done by Hodel  28 days . In the The low median age of the study population, with 62% of participants less than six months of age, makes the findings less generalizable with respect to the general population in the area. It would be expected that younger children would not have as much asymptomatic parasitaemia as older children as it develops gradually during childhood when the child grows up in a malaria-endemic area with high perennial transmission ,5. In adDespite the study’s limitations, the finding that asymptomatic malaria parasitaemia is rare suggest that positive malaria tests should always be interpreted as clinically relevant malaria in this population. The study also supports the notion that there is no asymptomatic parasite reservoir for transmission in young children, which encourages the continuation of current eradication efforts implementing early treatment with effective drugs and the use of ITNs and indoor residual spraying to eradicate malaria. The finding that PCR was negative for all the healthy children in this study lends support to the notion that false positivity is not a problem with the currently used mitochondrial PCR. A quantitative real-time PCR, targeting the same mitochondrial sequence, would be a valuable tool to further increase the understanding of low-level and asymptomatic parasitaemia and its impact on malaria transmission.This study indicates that there is likely no, or very little asymptomatic parasitaemia in very young children in the study area. All positive results for malaria in young children with clinically suspected malaria should be assumed to be illness due to malaria, and children with positive malaria test should receive adequate treatment for malaria. However, due to the small sample size and low median age of the population studied the results of the study cannot be generalized to other populations, health facilities or areas. A similar study should be performed on a larger population with a broader age range.The authors declare that they have no competing interests.GEAS and BB were involved in all stages of the study. MGT was involved in the laboratory work especially with selecting methods for DNA extraction from dried blood spots. MF coordinated the fieldwork and arranged the cooperation with the MCHC. NL participated in the study design. All co-authors contributed to the data interpretation and writing of the manuscript. All authors have read and approved the final manuscript.


Plasmodium species in southern Papua, Indonesia.Ric Price and colleagues use hospital-based surveillance data to estimate the risk of severe anemia and mortality associated with endemic Please see later in the article for the Editors' Summary Plasmodium co-endemicity is poorly documented. We compared the hematological profile of patients with and without malaria in southern Papua, Indonesia.The burden of anemia attributable to non-falciparum malarias in regions with P. malariae infection had the lowest hemoglobin concentration , followed by those with mixed species infections , P. falciparum , and P. vivax ; p-value for all comparisons <0.001. Severe anemia (hemoglobin <5 g/dl) was present in 8,151 (3.7%) patients. Compared to patients without malaria, those with mixed Plasmodium infection were at greatest risk of severe anemia (adjusted odds ratio [AOR] 3.25 [95% CI 2.99–3.54]); AORs for severe anaemia associated with P. falciparum, P. vivax, and P. malariae were 2.11 (95% CI 2.00–2.23), 1.87 (95% CI 1.74–2.01), and 2.18 (95% CI 1.76–2.67), respectively, p<0.001. Overall, 12.2% (95% CI 11.2%–13.3%) of severe anemia was attributable to non-falciparum infections compared with 15.1% (95% CI 13.9%–16.3%) for P. falciparum monoinfections. Patients with severe anemia had an increased risk of death . Not all patients had a hemoglobin measurement, thus limitations of the study include the potential for selection bias, and possible residual confounding in multivariable analyses.Clinical and laboratory data were linked for all patients presenting to a referral hospital between April 2004 and December 2012. Data were available on patient demographics, malaria diagnosis, hemoglobin concentration, and clinical outcome, but other potential causes of anemia could not be identified reliably. Of 922,120 patient episodes , a total of 219,845 (23.8%) were associated with a hemoglobin measurement, of whom 67,696 (30.8%) had malaria. Patients with P. vivax is the dominant cause of severe anemia in early infancy, mixed P. vivax/P. falciparum infections are associated with a greater hematological impairment than either species alone, and in adulthood P. malariae, although rare, is associated with the lowest hemoglobin concentration. These findings highlight the public health importance of integrated genus-wide malaria control strategies in areas of Plasmodium co-endemicity.In Papua Please see later in the article for the Editors' Summary Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Of these, P. vivax is the commonest and most widely distributed, whereas P. falciparum causes the most deaths—about a million every year. All these parasites enter their human host when an infected mosquito takes a blood meal. The parasites migrate to the liver where they replicate and mature into a parasitic form known as merozoites. After 8–9 days, the merozoites are released from the liver cells and invade red blood cells where they replicate rapidly before bursting out and infecting more red blood cells. Malaria's recurring flu-like symptoms are caused by this cyclical increase in parasites in the blood. Malaria needs to be treated promptly with antimalarial drugs to prevent the development of potentially fatal complications. Infections with P. falciparum in particular can cause anemia (a reduction in red blood cell numbers) and can damage the brain and other vital organs by blocking the capillaries that supply these organs with blood.Malaria—a mosquito-borne parasitic disease—is a global public health problem. Five parasites cause malaria—Plasmodium co-endemicity). Public health officials in such regions need to know whether non-falciparum malarias are a major cause of anemia when designing malaria control strategies. If P. vivax, for example, is a major cause of anemia in an area where P. vivax and P. falciparum co-exist, then any malaria control strategies that are implemented need to take into account the biological differences between the parasites. In this hospital-based cohort study, the researchers investigate the burden of severe anemia from the endemic Plasmodium species in southern Papua, Indonesia.It is unclear what proportion of anemia is attributable to non-falciparum malarias in regions of the world where several species of malaria parasite are always present , patients with mixed Plasmodium infection were more than three times as likely to have severe anemia as patients without malaria. Patients with P. falciparum, P. vivax, or P. malariae infections were about twice as likely to have severe anemia as patients without malaria. About 12.2% of severe anemia was attributable to non-falciparum infections, 15.1% was attributable to P. falciparum monoinfections, and P. vivax was the dominant cause of severe anemia in infancy. Finally, compared to patients without anemia, patients with severe anemia had nearly a 6-fold higher risk of death.The researchers used hospital record numbers to link clinical and laboratory data for patients presenting to a referral hospital in southern Papua over an 8-year period. The hemoglobin level (an indicator of anemia) was measured in about a quarter of hospital presentations . A third of the presentations who had their hemoglobin level determined  had clinical malaria. Patients with P. vivax makes to severe anemia in infancy, show that the hematological (blood-related) impact of P. malariae is most apparent in adulthood, and suggest, in contrast to some previous reports, that mixed P. vivax/P. falciparum infection is associated with a higher risk of severe anemia than monoinfection with either species. These findings, which need to be confirmed in other settings, highlight the public health importance of implementing integrated malaria control strategies that aim to control all Plasmodium species rather than a single species in regions of Plasmodium co-endemicity.These findings provide a comparative assessment of the pattern of anemia associated with non-falciparum malarias in Papua and an estimate of the public health importance of these malarias. Although the accuracy of these findings may be affected by residual confounding  and other limitations of the study design, non-falciparum malarias clearly make a major contribution to the burden of anemia in southern Papua. In particular, these findings reveal the large contribution that http://dx.doi.org/10.1371/journal.pmed.1001575.Please access these websites via the online version of this summary at PLOS MedicinePerspective by Gosling and HsiangThis study is further discussed in a malaria ; the 2012 World Malaria Report provides details of the current global malaria situationInformation is available from the World Health Organization on malaria (in English and Spanish), including information on different Plasmodium species and a selection of personal stories about malariaThe US Centers for Disease Control and Prevention provide information on Plasmodium falciparum malaria and on Plasmodium vivax malariaThe Malaria Vaccine Initiative has fact sheets on malaria and on anemia (in English and Spanish)MedlinePlus provides links to additional information on WorldWide Antimalarial Resistance Network on antimalarial drug resistance for P. falciparum and P. vivaxInformation is available from the  Plasmodium infection and is responsible for substantial morbidity Plasmodium species Anemia is a common manifestation of P. falciparum endemicity, repeated infections from an early age induce robust immunity to clinical disease and a low risk of severe anemia beyond childhood P. falciparum invariably co-exists with other Plasmodium species—the most important of which is P. vivax. In these regions, less intense parasite exposure during early life delays the development of immunity and gives rise to the potential for symptomatic and complicated infections at all ages.In regions of high P. falciparum and the other Plasmodium species complicate analyses of the pattern and public health impact of malarial anemia in co-endemic areas. Plasmodium vivax infection has been shown to reduce the risk of anemia secondary to P. falciparum malaria, possibly by providing a degree of cross-species immunity P. vivax infection and mixed-species infections suggesting that the hematological impact of the non-falciparum malarias may have been underestimated Biological differences and interactions between Plasmodium species in a co-endemic setting.In the present study we used clinical and laboratory data from Mitra Masyarakat Hospital in southern Papua, Indonesia, to establish the comparative hematological profiles of patients infected by the different Ethical approval for this study was obtained from the Health Research Ethics Committees of the University of Gadjah Mada, Indonesia, Menzies School of Health Research, Darwin, Australia, and the Oxford Tropical Centre, Oxford, UK.Anopheles koliensis, An. farauti, and An. punctulatus. The estimated average annual incidence of parasitemia is 876 episodes per 1,000 people, 58% due to P. falciparum, 37% due to P. vivax, 3% due to mixed infection, and 1.8% due to P. malariaeP. falciparum, 6.4% for P. vivax, 1.9% for mixed infection, and 0.6% for P. malariaeMimika District lies in south-central Papua, the easternmost province of Indonesia. Its geography, climate, and demographics have been described elsewhere Until November 2008, Rumah Sakit Mitra Masyarakat (RSMM) was the only referral hospital in the district; since 2008 RSMM treated approximately 80% of patients with malaria attending an inpatient facility in the district. RSMM has 110 beds, a high dependency unit, a 24-hour emergency department, and a busy outpatients department that reviews approximately 300 patients per day, 6 days per week. Treatment is provided free of charge to Papuans and at a cost for non-Papuans.P. falciparum (Paracheck) are also performed in some cases. In 2004, a random selection of 1,083 positive slides was re-read by an independent expert microscopist with more than 10 years of experience. Concordance was 90% with 1.7% of cases reported as negative on the second reading and 4% of monoinfections reclassified as mixed infections. Complete blood counts are ordered according to clinical indication and are performed using a Coulter Counter (JT Coulter).Protocols dictate that all patients presenting to the outpatients department with a fever or symptoms consistent with malaria and all inpatients, regardless of diagnosis, should have a malaria blood film. As a result, reliance on clinical symptoms alone to make the diagnosis of malaria is discouraged and rare. Microbiological diagnosis of malaria is usually based on a thick film examination, though confirmatory thin films and histidine rich protein (HRP2)-based rapid diagnostic tests for Every patient presenting to RSMM for the first time (regardless of department) is assigned a unique hospital record number (HRN). All clinical, laboratory, and pharmacy data from the first and subsequent presentations are linked to this unique HRN. Hospital clerks record basic demographic and administrative information, mortality data, and the diagnoses given by the attending doctor  for each patient presentation to hospital. Ethnicity is recorded as the patient's “Suku” (clan), which is self-reported to the admission clerk on presentation to the hospital. For the purposes of analyses, ethnicity was categorized as Highland Papuan, Lowland Papuan, or non-Papuan on the basis of the location of the clans' village(s). Hematological results are generated by coulter counter (JT Coulter) and collated in a separate laboratory database. Pharmacy data are entered manually into an electronic database by the pharmacist fulfilling the prescription. Since data were gathered from routine hospital surveillance informed consent was not requested from participants, however all records were anonymized to ensure patient confidentiality.Plasmodium species.Clinical data were merged with hematology data by creating all possible pairwise combinations for each HRN. Pairs in which the laboratory record fell between the date of presentation and discharge (the same day for outpatient visitations) were kept and if more than one hemoglobin measurement was available for a single event, the lowest was taken . For thePlasmodium species with comparison to patients without malaria, presence of severe anemia (hemoglobin less than 5 g/dl), the population attributable fraction (PAF) of severe anemia associated with infection by the different Plasmodium species at all ages from infancy through to adulthood, and all-cause mortality. Continuous hemoglobin data were analysed using linear regression and binary anemia data (such as severe anemia and death) were analysed using logistic regression. Since some patients appeared in the database multiple times, robust standard errors were calculated using the Huber-White sandwich estimator.The primary outcomes in this study were the mean hemoglobin concentration (g/dl) associated with infection by the different Plasmodium species , sex, self-reported ethnicity , age group , and year of presentation (2004 through to 2012). All of these factors, as well as the interaction between age and Plasmodium species, were associated with clinically important differences in hematological status and included in multivariable models. Department (outpatient versus inpatient) and the number of presentations with malaria in the preceding 2 months  were also included in univariable analyses; however, both factors were omitted from multivariable analyses since department was deemed to be a consequence of severe disease rather than a confounder, and the number of preceding malaria episodes would potentially obscure the net hematological effect of Plasmodium infection. Fractional polynomials were used to allow for the non-linear relationship between age (as a continuous exposure) and the mean hemoglobin and risk of severe anemia, because they make no assumption regarding the shape of this association n = 1114, 0.51%) were excluded since they would not have had ex utero exposure to malaria. Adults over 63 years (the 99th percentile) of age  were also excluded.Univariable analyses were performed for each of the following variables: Plasmodium infection were calculated for multiple age groups from infancy through to adulthood from multivariable logistic regression models using the punaf module for STATA, which derives PAFs using the formulae provided in Greenland and Drescher P. ovale infections were excluded  from the multivariable models. All statistical analyses were done in STATA version 12.1 (StataCorp).Adjusted PAFs of severe anemia associated with P. falciparum accounting for 53.3% of monoinfections, P. vivax for 32.3%, P. malariae 2.7%, and P. ovale 0.06%. Mixed species infections were detected in 19,569 (11.6%) presentations, which in 18,489 (94.5%) cases were mixed P. falciparum and P. vivax infections.Between April 2004 and December 2012, there were 922,120 patient presentations to RSMM Hospital constituting 837,989 (90.9%) outpatient visitations and 84,131 (9.1%) inpatient admissions . The agePlasmodium vivax infection was the dominant cause of malaria in patients under 3 years of age in both the outpatient and inpatient setting. Thereafter, P. falciparum was the most common malaria parasite.The absolute number of patient presentations with malaria over the study period peaked during the second year of life but as a proportion of all patient presentations was highest during the early teens . PlasmodP. ovale (which was rare), 30.6% of malaria outpatient presentations were matched with a hemoglobin measurement compared to 15.5% of non-malaria presentations. The corresponding figures for patients admitted to the wards were 93.9% and 76.4%, respectively. Patients who had a hemoglobin measurement were slightly younger than those who did not have a measurement, the median age being 23.0 years versus 24.5 years in those without malaria and 15.9 years versus 18.2 years for those with malaria; p<0.001 for both comparisons. Measurement of hemoglobin was also more common in males compared to females (odds ratio = 1.02 [95% CI 1.01–1.03]) and Papuans compared to non-Papuans (odds ratio = 1.13 [95% CI 1.12–1.15]); p<0.001 for both comparisons.Overall, 219,845 (23.8%) patient presentations  were matched with at least one hemoglobin measurement. Of these 85,000 individuals, 40,427 (47.6%) had more than one presentation to hospital; the number of presentations ranging from 1 to 56 (median = 2). Excluding P. falciparum  = 0.82] or mixed 0.24 g/dl [SD = 0.88]) compared to 0.17 g/dl (SD = 0.80) in patients with P. vivax and 0.14 g/dl (SD = 0.72) in those with P. malariae; p<0.001.The mean of the minimum hemoglobin concentration recorded for each patient presentation was 10.18 g/dl and the maximum 10.34 g/dl. The mean difference between minimum and maximum hemoglobin was greatest for patients with P. vivax either alone or mixed  compared to those with P. falciparum alone , P. malariae , or those without malaria ; p<0.0001. Recent malaria caused by any species was most common in young children aged 1 to 5 years old with an odds ratio of 2.29  compared to adults  of the patients with a hemoglobin measurement had presented to hospital with malaria within the previous 2 months, the number of malaria episodes ranging from 1 to 6. The risk of recent malaria presentation was greatest in patients presenting with ults see .Plasmodium species  whilst the hematological statuses of patients infected with P. vivax and P. falciparum monoinfection were less severely impaired and broadly similar  followed by mixed infection (−1.01 g/dl [95% CI −1.07 to −0.95 g/dl]), P. falciparum (−0.70 g/dl [95% CI −0.73 to −0.66 g/dl]), and P. vivax (−0.58 g/dl [95% CI −0.62 to −0.54 g/dl]), p for all comparisons <0.001. Patients without a presentation to hospital with malaria within the preceding 2 months had a mean hemoglobin of 10.32 g/dl (95% CI 10.30–10.33 g/dl) compared to 9.42 g/dl (95% CI 9.39–9.45 g/dl) in patients with a single recent episode of malaria and 8.90 g/dl (95% CI 8.83–8.97 g/dl) in those with two or more episodes of malaria; p<0.001  followed by those with P. malariae (AOR 2.18 [95% CI 1.76–2.67]), P. falciparum (AOR 2.11 [95% CI 2.00–2.23]), and P. vivax (AOR 1.87 [95% CI 1.74–2.01]), p for all comparisons <0.001 . The comparative reduction in hemoglobin associated with P. malariae was greatest in adulthood whereas for P. falciparum, P. vivax, and mixed infections, it was worst during infancy  of the 219,845 patient presentations were associated with a hemoglobin concentration less than 10 g/dl, 27,106 (12.3%) less than 7 g/dl, and 8,151 (3.7%) less than 5 g/dl. Compared to patients without malaria, those with mixed s <0.001 . Patient infancy .P. falciparum was 15.1% (95% CI 13.9–16.3%), compared to 5.75% (4.97%–6.53%) for P. vivax, 0.61% (0.38%–0.84%) for P. malariae, and 5.89% (5.28%–6.50%) for mixed species infections. These attributable fractions were unchanged when pregnant women were excluded. The fraction of severe anemia attributable to P. vivax, P. malariae, or mixed species infections was 12.2% (95% CI 11.2%–13.3%). The fraction of severe anemia attributable to P. vivax was highest in infancy, when it rose to 30.4% (95% CI 26.4%–34.2%) compared to 20.5% (95% CI 17.2%–23.7%) for P. falciparum. Following childhood, a second peak in the fraction of anemia attributable to malaria was apparent for all species between the ages of approximately 15 and 25 years  (p<0.001).Admission for inpatient care was required in 63.6%  of the patients presenting initially to the outpatient department with severe anemia compared to 14%  of those without severe anemia  . InpatieP. falciparum infection, 0.6%  with P. vivax infection, 0.9%  with P. malariae infection, and 0.7%  with mixed infection. The odds ratio for death in patients with a hemoglobin concentration lower than 5 g/dl was 4.63 (95% CI 4.16–5.16), compared to those with a higher hemoglobin concentration, p<0.001. After controlling for age, the AOR of death associated with severe anemia was 5.91 (95% CI 5.17–6.76) for patients without concurrent malaria, 5.93 (95% CI 4.63–7.58) for those with P. falciparum infection, 5.33 (95% CI 3.10–9.17) for mixed infections, 4.43 (95% CI 2.76–7.11) for P. vivax, and 6.58 (95% CI 2.02–21.47) for those with P. malariae. The PAF of death associated with severe anaemia was 11.9% (95% CI 10.6%–13.2%). The full multivariable model for mortality is presented in The overall risk of mortality in those admitted to hospital with a hemoglobin measurement was 1.3% , the risk increasing as the hemoglobin concentration fell below 7 g/dl . In totaP. vivax is associated with a high burden of severe anemia in infancy , that mixed P. vivax/P. falciparum infection causes significantly more hematological impairment than monoinfection with either species alone, and that in adulthood P. malariae infection is associated with the lowest mean hemoglobin concentration. Approximately 12% of severe anemia at the hospital can be attributed to non-falciparum Plasmodium infection, the consequences of which include a 6-fold greater risk of admission to hospital and an increased duration of inpatient stay. More importantly, lower hemoglobin concentrations were associated with a 5-fold increased risk of death, the worst prognosis being in patients with severe anemia associated with falciparum malaria and those without concurrent malaria.Anemia is highly prevalent throughout the tropics and has major consequences for human health and socioeconomic prosperity Plasmodium species, probably reflecting more efficient transmission and faster acquisition of immunity. For a given parasite density, P. vivax evokes a greater immune response P. vivax infection were significantly more likely to have had a prior presentation with malaria in the preceding 2 months compared with the other species. Infants are particularly vulnerable to recurrent episodes of vivax malaria, a likely consequence of lower immunity and greater inherent susceptibility to infection; P. vivax has a strong predilection for reticulocytes, which are most abundant in the first 3 months of life Infancy is a time of rapid physical and cognitive development as well as increased vulnerability to infectious diseases. Our study shows that the risk of symptomatic vivax malaria is more heavily skewed towards infancy compared with other P. vivax as the predominant malaria infection in infancy and highlights its frequent association with severe anemia in this age group. Young children with severe falciparum malarial anemia have previously been shown to have an increased risk of mortality P. falciparum infection was much higher  in patients with P. falciparum (0.21 g/dl) and mixed infections (0.24 g/dl) compared to those with P. vivax monoinfection (0.17 g/dl), and the 1.5-fold greater risk of severe anemia observed following recent falciparum malaria compared to those with recent vivax malaria. In non-malarial severe anemia the greater mortality may reflect a more acute onset compounded by a greater number or severity of comorbidities. Our study was unable to assess the long term effects of malarial anemia, which in other studies has been associated with adverse developmental and socioeconomic sequelae, although defining these is challenging due to the multifactorial aetiology of anemia in malaria-endemic areas Our study confirms h higher . It is pP. vivax infection and reduced severity of P. falciparum infection P. vivax on P. falciparum is likely to be negligible. In Thailand, where transmission is less intense, severe falciparum anemia is more likely to result from a single fulminant infection in a non-immune individual and therefore any immunomodulatory effect of P. vivax would be proportionately more important.Previous studies in Thailand have shown an association between concomitant P. malariae was found to be associated with the lowest mean hemoglobin of all locally prevalent Plasmodium species, and a high frequency of severe anemia. Its hematological effects were most apparent in adulthood whereas in childhood it was associated with minimal impairment. Plasmodium. malariae is a slow-growing parasite that usually causes a low parasitemia P. malariae infections is asymptomatic and therefore unlikely to prompt presentation to a health care service Plasmodium malariae is prevalent in much of sub-Saharan Africa Somewhat unexpectedly, P. ovale excluded) and hence, comparisons of the hematological effects of the different species are likely to be valid. Those without malaria were less likely to have a full blood count, both in the outpatient and inpatient setting. However, the low threshold for hematological investigation at the hospital suggests that patients who did not have a blood test would be unlikely to have severe anemia. Furthermore the absence of malaria at the time of the clinical encounter does not preclude the contribution to anemia of prior, recently treated, asymptomatic, or submicroscopic parasitemia. Our study was also limited to observations documented during the hospital encounter and could not address out-of-hospital deaths associated with severe anaemia. However these biases are likely to have resulted in the derived malaria-attributable fractions of severe anemia and their associated morbidity and mortality being conservative, if anything underestimating the true burden of disease.Our study has several strengths. It includes patients with uncomplicated malaria presenting to the outpatients department through to those with complicated disease who were admitted to hospital. The analysis was focused on clinical events, with each patient presenting in some cases multiple times over the 8 years of the study period. All children greater than 1 week of age were included whereas other studies have excluded those under 6 months of age Our analyses may have also been subject to residual confounding. Hemoglobin and red cell polymorphisms are known to influence the likelihood of severe malarial anemia P. falciparum malaria P. vivax malaria is unclear Plasmodium infection in our study may have been overestimated by our inability to control for geohelminth infection. This is unlikely to have been important for children under 3 years old since helminth density typically peaks in adulthood and previous work has shown minimal impact on hemoglobin concentrations prior to 30 months of age Other potential confounders include nutritional status, geohelminth infestation, bacteremia, chronic disease, and pregnancy status. Apart from pregnancy status, information on these confounders was not available. Malaria and malnutrition may cluster in the same geographic areas, but the mechanisms and direction of any biological link have not been elucidated conclusively. Chronic vivax malaria has been implicated in a state of malnourishment akin to kwashiorkor P. falciparum, and possibly P. vivax, parasitemia in malaria endemic regions Pregnancy may have also confounded the association of anaemia and malaria in adults. Pregnant women are at comparatively high risk of Plasmodium infection this approach would tend to miss patients at the severe end of the spectrum.Finally the results of this study cannot necessarily be generalised to individuals with asymptomatic disease. Although cross-sectional survey data could be used to compare the hematological impact of symptomatic and asymptomatic P. vivax, P. malariae, or mixed species infections. Plasmodium vivax was associated with the greatest attributable fraction of severe anemia in infancy and is likely to be an important cause of indirect morbidity. Conversely, the hematological impact of P. malariae was most apparent in adults. In contrast to earlier reports from regions of low mixed-species endemicity, mixed P. falciparum/P. vivax infections are associated with a significantly greater absolute reduction in hemoglobin and odds of severe anemia than monoinfection with either species alone. Severe malarial anemia, including that caused by P. vivax infection, is associated with an increased likelihood of admission, a prolonged hospital stay, and, particularly with P. falciparum infections, dramatically increased mortality. These findings highlight the public health importance of integrated genus-wide rather than species-specific malaria control strategies in areas of Plasmodium co-endemicity.In conclusion our very large hospital-based study demonstrates that the non-falciparum malarias make a major contribution to the burden of anemia in southern Papua, with 12% of all severe anemia at the hospital attributable to


Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi . For MSP-3, this difference was significant only among 4–5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies.Antibodies to selected  However, such studies have often yielded inconsistent results with some studies demonstrating a protective role for antibodies to a specific antigen, while others do not Antibodies play an important role in mediating protection against clinical malaria. Purified total IgG obtained from malaria-immune African adults was successfully used to treat children and adults hospitalized with malaria, while control sera from adults not exposed to malaria had no protective effect P. falciparum infection was explored Here, we investigated why antibodies appeared to be protective in some settings but not in others. Specifically, we tested the hypothesis that a threshold concentration of antibody was required for protection and that in some settings, although antibodies were present, their concentrations were below the thresholds required for protection. Quantitative correlates of protection have been reported for vaccine-induced antibodies against many infectious diseases Our previous studies have shown that antibodies to specific merozoite antigens were associated with protection from clinical episodes of malaria in the Chonyi cohort 22.12.1.12–10) The Chonyi cohort in Kilifi, Kenya, has been extensively studied 2.1.22–10 in Junju was 29% at the time of sampling. Participants in the Junju cohort area live approximately 25 km away from those described above in the Chonyi cohort. We analyzed 304 serum samples collected in May 2008 from children aged 1–12 years. Data on 6 months of follow up in the subsequent malaria transmission season are presented here.Children aged 1–6 years in a second independent group, the Junju cohort, were originally recruited in 2005 The Kenyan national scientific and ethics committees reviewed and approved the studies.2.2P. falciparum and include the 19 KiloDalton C-terminal fragment of merozoite surface protein (MSP)-1 of the Wellcome parasite line All antigens are based on 2.319 and GLURP were measured in the Chonyi cohort only.Serum IgG responses to individual antigens were measured using a well-established standard ELISA protocol 2.4Data analysis was performed using Stata 11 . A modified Poisson regression model was used as previously described We used data from all children in the Chonyi cohort to estimate the relative concentration of antibodies that best correlated with clinical protection for each antigen as follows: (i) different antibody concentrations were applied as cutoffs for high versus low responders over a range of increasing concentrations up to the maximum concentration recorded against each antigen, (ii) a modified Poisson regression model was used to calculate the risk ratio at each cutoff value, (iii) the best fitting model was selected using the log pseudolikelihood 2.5Analyses using the protective threshold as a cutoff point were compared to conventional analyses where the cutoff is defined as (1) seropositivity, defined as the mean plus 3 standard deviations of negative controls and (2) high versus low antibody levels, defined previously by us as the ELISA OD level above which the risk of malaria was lower than the population's average risk of acquiring a clinical episode of malaria 33.1P < 0.05).Children in the Junju cohort were sampled during a period of moderate malaria transmission intensity. In spite of this, antigen-specific antibodies were readily detectable in the cohort, and antibody prevalence was 67%, 31% and 26% for AMA1, MSP-2 and MSP-3, respectively. Antibody levels increased significantly with age for all antigens tested  to those that were seronegative for antibodies to these merozoite antigens. In the second analysis, we compared children that had high levels of antibodies  to those that had low antibody levels to each of the antigens. In both these analyses, with the exception of MSP-3, antibodies to these antigens were not associated with protection from malaria .3.219.Next, we tested the hypothesis that the apparent lack of protection observed in the Junju cohort could be explained by insufficient antibody concentrations. To do this, we used data from the Chonyi cohort where antibodies to these antigens had previously been studied and were known to correlate with protection from malaria 3.3P < 0.01) except for the youngest children (0–3 years), who had similar median antibody levels to AMA1 in both cohorts (P < 0.01). The proportion of children that had protective threshold antibody concentrations against MSP-3 was significantly lower in Junju compared to Chonyi only in the 4–5 year age group  of 1.09 (0.75–1.59), 0.94 (0.65–1.36) and 0.16 (0.02–1.12). For both AMA1 and MSP-3, antibody concentrations at the protective thresholds were considerably higher than those at seropositive or high levels . For ant4An increasing body of evidence suggests that protection from malaria is dependent on high antibody concentrations Methodological differences between immuno-epidemiological studies make it difficult to interpret apparently contradictory results where antibodies to a single antigen are associated with protection from malaria in one geographical setting, but not in another 2–10) 2–10 of ≥40% whereas those of low transmission are defined by a PfPR2–10 of <5%. Areas of intermediate/moderate transmission which would experience an immediate reduction in parasite prevalence following large scale deployment of insecticide- treated nets have a PfPR2–10 of 5–40% There was a clear difference in parasite prevalence rates in children aged 2–10 years (PfPRApart from differences in malaria transmission intensity, the two cohorts belong to the same ethnic group and share similar environmental factors such as cultivation practices, rainfall, wind direction, presence of streams and rivers. However, notable differences between both cohorts were the change in antimalarial drug policy from sulphadoxine–pyrimethamine to artemether lumefantrine in 2006 19, we were not able to define a protective threshold by the methods presented here. These findings need to be validated in much larger cohorts, and in samples collected from different geographical settings. Efforts in this regard are underway. Additionally several studies have shown that quality of antibody responses, in particular the type of IgG subclasses are also important for protection Immuno-epidemiological studies have traditionally classified study participants as being seropositive or seronegative for responses to specific antigens. More recently, we and others have found that classifying individuals as having high or low levels of antibodies is a highly informative indicator of protection among children P. falciparum needs to be achieved for protection from clinical episodes of malaria. We propose a new approach to the analysis of such data that may add value to current analytical strategies. If validated in larger studies and in unrelated immuno-epidemiological cohorts, this analytical approach based on “protective thresholds” could be usefully extended to the testing of immunogenicity and potential protective efficacy of sub-unit vaccines currently under development for malaria.In conclusion, our data suggests that a “protective threshold” concentration of antibodies against specific merozoite antigens of


Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits—including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia—are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a “natural experiment” to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the “Gordian knot” of host and parasite interactions to confer malaria protection, and offer a translational model to identify the most critical mechanisms of P. falciparum pathogenesis. In the 4th century BC, Alexander the Great conquered the known Western world Plasmodium falciparum malaria are clearly needed to identify the molecular and cellular targets of next-generation therapeutics and preventive measures. The mechanisms of falciparum malaria pathogenesis remain obscure owing to the complex tangle of parasite virulence factors, host susceptibility traits, and innate and adaptive immune responses that modulate the development of distinct malaria syndromes Malaria is an ancient disease that has persisted to our modern age, intractably killing over 500,000 children in sub-Saharan Africa each year P. falciparum infection We propose that hemoglobinopathies slice the Gordian knot of falciparum malaria pathogenesis to protect children from the severe, life-threatening manifestations of the disease. Most strikingly, heterozygous hemoglobin S  and homozygous hemoglobin C  reduce the risk of severe falciparum malaria in sub-Saharan African children by 90% and 70%, respectively Here we review the proposed mechanisms by which hemoglobinopathies  protect against falciparum malaria.P. falciparum parasites enter the erythrocytic stage of their life-cycle. It is during this time that parasites sequentially invade and egress from their host RBCs and cause the signs and symptoms of malaria. While developing within the RBC, the parasite traffics proteins to the RBC surface that mediate binding to extracellular host receptors and enable the parasite to sequester in the placenta, brain, and virtually every other organ. The attenuation of malaria by repeated, sub-lethal P. falciparum infections suggests a significant role for adaptive immunity, but the targets of this attenuating immune response remain largely obscure. Though this adaptive immunity can be protective, the development of maladaptive and dysregulated immune responses can also contribute to the pathogenesis of malaria.The red blood cell (RBC) is critical for the propagation of malaria parasites . After iVariant RBCs are produced from some of the most common human genetic polymorphisms, and for over 60 years their widespread prevalence has been hypothesized to result from their evolutionary selection by severe, life-threatening falciparum malaria in vivo, we review the evidence for each of them separately.The current understanding of falciparum malaria pathogenesis suggests four general hypotheses for investigating the nature of malaria protection by hemoglobinopathies : 1) restP. falciparum in RBCs containing variant hemoglobins rapidly followed the development of in-vitro cultivation systems by Trager and Jensen, and Haynes et al. in 1976  profiles that are distinct from those of HbAA RBCs P. falciparum parasites, as well as the fusion of these human miRNAs with extant parasite mRNA transcripts to subsequently inhibit the translation of enzymes that are critical for parasite development. Specifically, the host miRNAs miR-451 and let-7i were significantly more abundant in HbAS and HbSS RBCs, and were associated with attenuated parasite growth in these cells. In addition, the inhibition of these two miRNAs by experimental transfection of RBCs with antisense oligonucleotides partially restored parasite growth. Downstream, miR-451 appears to fuse with transcripts of the regulatory subunit of the parasite's cAMP-dependent protein kinase (PKA-R) to reduce its translation, thereby upregulating activity of its substrate PKA and ultimately disrupting multiple parasite developmental pathways. Thus, the aberrant miRNA profile of HbS-containing RBCs may modulate the intraerythrocytic maturation of P. falciparum in a way that restricts parasite growth.A recent study proposes a novel mechanism of P. falciparum erythrocyte membrane protein 1 (PfEMP1) P. falciparum glycosylphosphatidylinositol (PfGPI) Two major pathogenic phenotypes of iRBCs have been described: those that mediate binding of iRBCs to endothelial receptors (“cytoadherence”) A series of investigations suggests that the weakening of cytoadherence interactions partially governs malaria protection by the hemoglobinopathies. Early studies by Udomsangpetch et al. in-vitro measures of parasite virulence—including PfEMP1 expression and iRBC binding to host cells—and in-vivo measures of malaria severity.A recent study supports this candidate mechanism of malaria protection. Cyrklaff et al. There is an emerging recognition of the impact of aberrant host responses in the pathogenesis of malaria, particularly severe falciparum malaria  and severe malaria highlights the importance of interactions between iRBC and endothelium in the pathogenesis of severe malaria. As noted above, severe falciparum malaria is associated with low NO bioavailability Evidence from field studies supports an association between several hemoglobinopathies, adaptive immunity, and protection from malaria P. falciparum antigens of heterologous parasites in HbAS children, and others reporting no differences. These findings were explored by measuring seroreactivity to a panel of 491 P. falciparum proteins in Malian children before and after a well-defined malaria transmission season P. falciparum antigens. It remains to be fully investigated whether they enhance IgG recognition of specific variants of PfEMP1 or other surface antigens that are known to play a role in malaria immunity.A central role for antibodies in malaria immunity is suggested by the ability of polyclonal IgG from malaria-immune adults to clear parasitemias in children with malaria Antibody-mediated phagocytosis of iRBCs is believed to be an important effector mechanism in protection from malaria. Investigations with normal RBCs have demonstrated that monocytes preferentially phagocytose iRBCs compared to uninfected RBCs, and that this preference is potentiated by the binding of IgG to iRBCs P. falciparum parasites interfere with the acquisition of immunologic memory responses that contribute to subsequent control of parasitemia  deficiency, interact with co-inherited hemoglobinopathies in mitigating pathogenesis?How do hemoglobinopathies impact the resting endogenous levels of angiopoietin-1, angiopoietin-2, and heme oxygenase-1?P. falciparum antigens?Do hemoglobinopathies augment cell-mediated immunity to Do hemoglobinopathies quantitatively or qualitatively enhance the acquisition of antibodies to specific variants or domains of PfEMP1?Do hemoglobinopathies impair the mechanisms by which the iRBC and the parasite interfere with the acquisition of immune memory?in vivo, and to translate these findings into future interventions to prevent, treat, and eliminate this ancient and intractable scourge.In the spirit of Alexander, we propose that hemoglobinopathies may be nature's “Alexandrian solution” to the problem of understanding fundamental aspects of falciparum malaria. This bold slice through the Gordian knot of malaria pathogenesis represents a unique opportunity to isolate and identify the molecular correlates of falciparum malaria pathogenesis in humans


This study aimed at determining the prevalence of malaria and anemia among children in rural community of Okada, Edo State Nigeria, as well as to assess the level of use of Insecticide treated bed nets and its impact on prevalence of malaria and anemia among study population.Thick blood films from 226 children with signs and symptoms of malaria in Okada community were stained and examined for presence of malaria parasites. Hemoglobin concentration of all children was also determined using standard method.P =0.003). A significantly higher number of positive cases of malaria and anemia was observed in rainy season as compared to dry season (P<0.05). The prevalence of anemia in children was 47.3%. Malaria was a risk factor for development of anemia in children . Use of insecticide treated bed nets was recorded in 11(4.9%) of children studied, and did not significantly reduce the prevalence of malaria and anemia. However among malaria parasite infected children, its use significantly reduced the prevalence of anemia .A total of 185 (81.9%) children were infected with malaria parasite. Malaria parasitaemia was significantly affected by age (Malaria and anemia among children was high malaria intervention progammes by relevant agencies is strongly advocated. Malaria is a leading cause of morbidity and mortality worldwide. Data from World Health Organization shows that approximately 216 million cases of clinical malaria occur worldwide, with deaths of about 655 000 occurring most among African children . MalariaAnemia is a known complication of malaria. It has a profound effect on the quality of life of people by inducing such symptoms as loss of stamina, rapid heart rate and shortness of breath . It has Okada, a rural community, is the headquarters of Ovia North East Local Government Area of Edo-state. The Local Government has an estimated population of 155 344 persons . MajoritA total of 226 children , at the out-patient department of the Igbinedion University Teaching Hospital, presenting with signs and symptom of malaria were recruited for this study. The age range of study population was 0.17 – 10 years. Verbal informed consent was obtained from parents/guardians of all participating children prior to specimen collection. Data on age and use of Insecticide treated bed nets was obtained from parents /guardian of all participating children. The study was approved by the Ethical Committee of the Igbinedion University Teaching Hospital, Okada, Nigeria.About five ml of blood was collected from each subject and dispensed into ethylene diamine tetra-acetic acid (EDTA) container and mixed. Malaria was diagnosed by examination of stained thick blood films. Thick blood films were made from each blood sample and allowed to air dry. Slides were stained in 3% Giemsa stain for 30 minutes, rinsed in tap water, and allowed to air dry. The stained films were examined for malaria parasite by microscopy using a × 100 oil immersion objective lens. A total of 200 fields per film were examined. Hemoglobin concentration was determined using an autoanalyser – Sysmex KX-21 . Anemia was defined a hemoglobin concentration <11g/dl .2) or Fischer's exact test as appropriate and odd ratio analysis using the statistical software INSTAT®. Statistical significance was set at P < 0.05The data obtained were analyzed using Chi-square (XP=0.003). A significantly higher number of malaria parasite positive cases was observed during the rainy season . Gender and use of Insecticide treated bed nets (ITN) did not significantly affect the prevalence of malaria parasite infection in children. Use of Insecticide treated bed nets was recorded in 11(4.9%) of children  out of the 226 examined were infected with malaria parasite. Age was a risk factor for infection with malaria parasite, with children within the age group of =1-2 years having the highest prevalence of 90.0%, (children .P=0.019). Gender, age, and use of Insecticide treated bed nets (ITN) did not significantly affect the prevalence of anemia in children .Among malaria parasite infected children, the use of Insecticide treated bed nets (ITN) was found to significantly reduced the prevalence of anemia   .Despite recent advances made in malaria prevention and control globally, malaria still remains a major health concern in Sub Saharan Africa with very high mortality rates being recorded among children. This study focused on determining the prevalence of malaria and anemia among children in rural community of Okada, Edo State Nigeria, as well as the level of use of Insecticide treated bed nets and its impact on prevalence of malaria and anemia.Plasmodium falciparum was the only specie of malaria parasite found in this study. The finding that malaria prevalence was significantly higher in rainy season in this study has been previously documented  of children studied. This is higher than findings from other African studies –17. DiffAge was a significant risk factor for malaria parasite infection among children. This is consistent with an earlier report . ChildreThe prevalence of anemia among children was 47.3%. This is lower than a finding in Benin City , and higMalaria was strongly associated with anemia in this study. This has been earlier reported . InteresConclusively, the prevalence of malaria and anemia among children in Okada was 81.9% and 47.3% respectively. Use of insecticide treated bed nets was poor and did not significantly reduce malaria and anemia generally among children studied. Its use however was found to significantly reduced anemia among malaria infected children. Community based malaria intervention progammes, targeted at enlightenment on modes of malaria transmission, provision of ITNs, prompt identification and treatment of all cases are needed to stem malaria and associated squeal.