Plasmodium spp neither reliably confirms malaria as the cause of the fever, nor excludes the possibility of other diseases. This is because the patient may be an asymptomatic carrier of malaria parasites and suffer from another disease.Recent WHO guidelines recommend a universal "test and treat" strategy for malaria, mainly by use of rapid diagnostic test (RDT) in all areas. The evidence for this approach is questioned here as there is a risk of over-reliance on parasitological diagnosis in high transmission situations, which still exist. In such areas, when a patient has fever or other malaria symptoms, the presence of To allow clinicians to perform their work adequately, local epidemiologic data are necessary. One size does not fit all. If parasite prevalence in the population is low, a diagnostic test is relevant; if the prevalence is high, the test does not provide information of any clinical usefulness, as happens with any test in medicine when the prevalence of the tested characteristic is high in the healthy population. It should also be remembered that, if in some cases anti-malarials are prescribed to parasite-negative patients, this will not increase selection pressure for drug resistance, because the parasite is not there.In high transmission situations at least, other diagnoses should be sought in all patients, irrespective of the presence of malaria parasites. For this, clinical skills (but not necessarily physicians) are irreplaceable, in order to differentiate malaria from other causes of acute fever, such as benign viral infection or potentially dangerous conditions, which can all be present with the parasite co-existing only as a "commensal" or silent undesirable guest. This agnosis" , which ragnosis" . The queagnosis" ,5.WHO's assumptions are discussed one by one, considering their applicability to high-transmission settings, which still exist. Basically, the « level of transmission » in malarial areas is an entomological concept. In most cases, the so-called transmission level is derived from the proportion of infected people in the general population. For practical purposes, it is considered here that « low transmission » is characteristic of areas where this proportion in the general population is below 20%. Between 20 and 50%, one can speak of « moderate transmission » and « high » above 50%. Since there is no universal definition, or cut-off figures, for "high-transmission", the discussion below will detail at what levels of parasite prevalence in the population a parasitological test supports, or does not support, clinical decisions.The specificity of a diagnostic algorithm or test almost always varies according to the prevalence of the target condition. Clinical diagnosis and parasitological tests for malaria are no exception. The specificity of clinical diagnosis of malaria varies according to age group, time, place and the epidemiology of the disease. In low-transmission settings, specificity of clinical diagnosis is low and specificity of parasitological tests (e.g. rapid diagnostic tests - RDTs) is high, whereas in high-transmission areas the reverse is the case. In chilThis assumption is incorrect, particularly in high transmission areas. In a randomized controlled trial (in Burkina Faso in 2006), comparing outcomes in patients treated either presumptively or after use of an RDT, there was no difference in outcomes between groups, i.e. thA useful tool for evaluating the clinical utility of a test is to calculate the likelihood ratio of having a disease, based on a positive or a negative result. The likelihood ratio multiplied by the pre-test odds of the disease gives the post-test odds. Figures In summary, where the prevalence of malaria infection is low, a parasitological test is useful: if it is negative, the post-test probability of malaria is almost zero. If the test is positive, the probability of malaria is greatly increased. However at higher levels of prevalence, a negative test does not rule out infection with malaria parasites, and a positive test does not greatly increase the probability of malaria infection . There is no clear threshold for applying a cut-off, but one commonly used rule is that a test is clinically useful to "rule in" or "rule out" a disease if the positive likelihood ratio is > = 5, or if the negative likelihood ratio is < = 0.2 respectively. ApplyinParasite prevalence has declined in some parts of Africa, but the median in a recent review is still 22%. Of noteP. falciparum can produce symptoms, but may also be asymptomatic . Detecting the presence of parasites can be misleading. It may divert the attention of clinicians from other diagnoses if they do not conduct an appropriate clinical examination. For example, the case of an infant is reported with a positive malaria test who was only treated with anti-malarials but subsequently died of presumed pneumonia[In a patient with fever  in a high-transmission area, the presence of parasites neither reliably confirms malaria as the cause, nor excludes the possibility of other diseases . Like mapneumonia. Paradoxpneumonia.in all patients, irrespective of the presence of malaria parasites. Some analyses assume that there is no co-infection between malarial and bacterial infections[In high transmission areas, other diagnoses should be sought nfections, but thinfections. Therefonfections.not at high risk of other life-threatening bacterial diseases, and the most common other diagnosis is viral upper-respiratory tract infection [However it is inappropriate to extrapolate findings from severely ill children in secondary care settings, to children with uncomplicated febrile illness in primary care settings, which anfection ,19. UnleAntibiotics should not be overused in primary care settings, otherwise there is a higher risk of evolution and spread of resistant bacteria . Some studies show that use of RDTs increases antibiotic prescribing in malaria-negative patients ,21  rather than clinical malaria. A more useful measure is the population attributable fraction of fever due to malaria. FurtherRapid diagnostic tests based on detection of the HRP-2 antigen often remain positive for over five weeks after the disappearance of live parasites, because they detect the HRP-2 antigen which is still present in debris from dead parasites for some time after total parasite clearance . This alMicroscopy does not help to confirm treatment failure unless it is of excellent quality, which is rare outside of research settings. In such settings, when high-quality microscopy is available, a negative film is very good at detecting the absence of malaria parasites in the blood Figure  and it iFrom the review of the evidence above it seems that a universal "test and treat" strategy is neither evidence-based nor cost-effective. The new WHO guidelines can be understood in the context of the long term goal of elimination of malaria. However, this is not achievable in the foreseeable future in many areas. WHO's assumptions justifying parasitological diagnosis may well be correct in low-transmission settings, but not in high-transmission settings. The prevalence of malaria parasitaemia has declined in some parts of Africa, so these areas are transitioning from high-transmission to low-transmission. In these areas, diagnostic tests will become increasingly important. However, many areas of sub-Saharan Africa remain high-transmission areas, and the appropriateness and cost-effectiveness of universal testing in these areas is questionable.Malaria is a complex disease and it is impossible to write clinically useful global guidelines on its management, which do not take local epidemiology into account. In practice countries contain areas with differing levels of transmission, which also depends on seasonality. It would make more sense to adapt the strategy according to local factors. This is the way health professionals deal with any infectious disease . It is perfectly possible to apply two different policies simultaneously (for example presumptive treatment for patients aged < 5 years and test-and-treat for older patients). Even whIf the WHO recommendations have little influence on clinical practice, this may not be due solely to problems of policy implementation, but also to the fact that the debate has been closed too early. Those cCost-effectiveness must also be an important consideration especially in settings where health care resources are limited. In settings of high transmission the cost saving from avoiding ACT is outweighed by the greater cost of deploying RDTs universally ,31. RDTsThere is a need for policies and clinical strategies adapted to different settings. Each area should develop its own guidelines based on sound evidence, taking into consideration both the local epidemiology of malaria and other febrile diseases and a realistic assessment of available healthcare resources. It would be a dangerous mistake to rely too much on diagnostic tests in areas where parasite prevalence is high. Clinical examination and reasoning remain necessary in order to differentiate malaria from other causes of acute disease such as benign viral infection or potentially dangerous conditions . To allow clinicians to perform their work adequately, local epidemiological data are necessary: in areas of low parasite prevalence, a diagnostic test is relevant; if the prevalence is high, the test does not provide information of any clinical usefulness. In this latter case, clinical skills are irreplaceable.Written informed consent was obtained from all individuals for publication of accompanying images.The authors declare that they have no competing interests.BG and AR wrote a first draft of the article which was substantially revised by MW and TS. All authors read and approved the final manuscript.


Sabang Municipality, in Aceh Province, Indonesia, plans to initiate a malaria elimination programme in 2013. A baseline survey of the distribution of malaria in the municipality was conducted to lay the foundations for an evidence-based programme and to assess the island’s readiness to begin the elimination process.The entire population of the municipality was screened for malaria infection and G6PD deficiency. Specimens collected included blood slides, blots and tubes for selected households.Plasmodium falciparum and 2 with Plasmodium vivax. To confirm the parasite prevalence, polymerase chain reaction (PCR) diagnosis was performed on the entire positive cases by microscopy and randomized 10% of the microscopically negative blood samples. PCR revealed an additional 11 subjects with malaria; one P. falciparum infection from the village of Paya Keunekai, and nine P. vivax infections and one mixed P. falciparum/P. vivax infection from the village of Batee Shok. The overall slide positivity rate was 0.074% (CI 95%: 0.070 – 0.078) and PCR corrected prevalence 0,590% (CI 95%: 0.582 – 0.597). Analysis of 937 blood samples for G6PD deficiency revealed two subjects (0.2%) of deficient G6PD. Analysis of several genes of the parasite, such as Pfdhfr, Pfdhps, Pfmdr1, Pfcrt, Pfmsp1, Pfmsp2, Pvdhfr, Pvdhps, Pvmdr1 and host gene, such as G6PD gene revealed that both P. falciparum and P. vivax carried the mutation associated with chloroquine resistance.Samples were collected from 16,229 residents. Microscopic examination of the blood smears revealed 12 malaria infections; 10 with Malariometric and host genetic analysis indicated that there is a low prevalence of both malaria and G6PD deficiency in the population of Sabang Municipality. Nevertheless, malaria cases were clustered in three rural villages and efforts for malaria elimination in Sabang should be particularly focused on those three villages. Plasmodium species of 2.1%[Malaria remains a major public health problem in Indonesia, with 30 million cases and 120,000 deaths annually. Recently measures of annual parasite incidence (API) have varied substantially between provinces, but the highest API is consistently detected in the eastern parts of Indonesia. Aceh Province, Sumatera, is the western most point of the archipelago, and malaria has been documented throughout. However, there have been few published reports of malaria prevalence in Sumatera in general and in Aceh in particular. Malaria is endemic in 21 districts and municipalities in Aceh Province, where the annual malaria incidences (AMI) in 2003 and 2004 were 4.94% and 3.2% respectively[in vitro study conducted during 1984–1985[in vivo study in 2004[Malaria in Sabang has been documented by several previous surveys both before and after the 2004 tsunami that destroyed much of the coastal region in Aceh. Resista984–1985, finding in 2004. MalarioThe Indonesian Ministry of Health announced a country wide malaria elimination policy in 2009 that envisioned a combat against the disease lasting from 2010 to 2030. Aceh Province aims to eliminate malaria by 2015 but for Sabang Municipality, which includes the islands of Weh, Rondo and Rubiah, the target for malaria elimination is 2013[Sabang Municipality is administratively included in Aceh Province, Indonesia. It is located at latitude 5° 49' 30" north and longitude 95° 18' 28" east  tests. All slides were reviewed by an expert microscopist. Slide positive cases were confirmed with PCR. In addition, 10% of negative cases were screened with PCR, and all blood samples from villages with microscopically positive cases. Selection of the 10% negative samples was done through computer programme. All negative samples in each village were listed sequentially in microsoft excel. A Random option was used to generate sample sequence and the samples were selected in ten-fold fashion. At the end, 1,725 samples were proportionally selected. Locations of confirmed positive cases were geocoded with GPS  according to a procedure described previously. The DNAPfdhfr, Pfdhps, pfmdr1, pfcrt[Pfmsp1, Pfmsp2[Pvdhfr, Pvdhps[Pvmdr1[Molecular analyses were performed using PCR amplification, restriction fragment length polymorphisms (RFLP), and sequencing on several genes of the parasite such as r1, pfcrt-10, Pfms1, Pfmsp2,12, Pvdhr, Pvdhps,14, Pvmdps[Pvmdr1 and hostps[Pvmdr1. The PCRps[Pvmdr1-16. PCR ps[Pvmdr1.Plasmodium falciparum infection: seven from the village of Batee Shok, two from Paya Keunekai and one from Ie Meuleu. Plasmodium vivax malaria was found in one subject from Iboih and one from Rondo island. PCR testing was performed on seven malaria falciparum slide positive cases and one malaria vivax slide positive case . PCR  revealed an additional 11 malaria-positive subjects: one mixed P. falciparum/P. vivax infection from Batee Shok, one P. falciparum from Paya Keunekai village, and nine P. vivax from Batee Shok village  tested positive for G6PD deficiency. Analysis for the presence of the most commonly found mutations: Mediterranean (563C/T), Coimbra (592 C/T), Vianchang Canton (1376 G/T), Vanua Lava (383 T/C), Mahidol (487 G/T), Chatam (1003 G/A), Kaiping (1388 G/A) and Vianchang  found none of these in the two positive subjects.A total of 16,229 subjects was analysed for weight and Hb levels. The mean of Hb level was relatively high in each age group except for the below five years, which had a Hb level slightly above or below the borderline (anaemia cut-off for Hb level is 11 g/dL). Distribution of age, children, sex and mean of Hb level per village also indicated relatively high in each group. However, due to the low prevalence of malaria, the relationship between anaemia and malaria could not be determined. Malnutrition was found in one village Kreung Raya  infection alerts to the need of a more reliable detection method as the cases might be potential source of infection as that found in South America[Mass blood surveys conducted in three periods between May and October 2010 describe a low prevalence of malaria in Sabang Municipality.  America. Malaria America. It is hP. falciparum isolates examined carried chloroquine-resistant alleles. All of the isolates examined carried the 76 T mutant allele of the pfcrt gene and in addition 30% of isolates carried the 86Y allele of the pfmdr1 gene. This result is in line with previous observations that the parasite on this island is resistant to chloroquine[P. vivax isolates examined, 10 carried the 976 F allele of the pvmdr1 gene that has been associated with chloroquine resistance[P. falciparum and P. vivax in this island are resistant to chloroquine, providing a rational basis for the use of arteminisin-based combination therapy for any malaria cases found.Genotyping found that all of the sistance. Both P.Pfdhfr gene also indicated a high proportion of the P. falciparum isolates carry mutant alleles associated with resistance to pyrimethamine. However, the frequency with which the mutant allele of Pfdhfr gene was found is slightly lower than that of a previous study done in 2006, likely due to the larger sample size of our study. Pfdhps alleles associated with sulphadoxine resistance were not found in any of the isolates examined, indicating that the parasites on this island are still sensitive to treatment with sulphadoxine-pyrimethamine, quintuple mutations in dhfr and dhps being associated with sulphadoxine-pyrimethamine treatment failure[Analysis of the  failure.The prevalence of G6PD-deficiency cases was also low (0.2%) among the 937 subjects screened. These subjects represent households and the prevalence reflects a larger population, but the G6PD deficiency prevalence found in this survey is much lower than that found in a previous survey (0.8%) that was conducted 2006 (unpublished observation). The difference might be attributed to the much higher number of subjects involved in this study. In the previous G6PD survey, the Vianchang mutation  was found in one subject but during the current survey none of the most common G6PD mutations were found among the G6PD-deficient cases.This combined malariometric and host genetic analysis indicated that both malaria and G6PD deficiency exist in low prevalence among the examined population in Sabang Municipality. In the context of the malaria elimination programme, several rational actions are implied; i) more flexible use of primaquine, the only available anti-malarial for antigametocyte and radical cures; ii) to contain malaria transmission in this island, a limited mass drug administration could be performed at the three villages where the malaria cases was detected; and iii) active case monitoring and treatment on migrants should be prepared.The authors declare that they have no competing interests.PBSA, IEPR, NRP, APNH, SSM, SK and DS performed samples collection, molecular assays, data analysis, and manuscript writing. KC, SW, FAR, MES, AI, TY, TS, JN, A, B, CB, MY, CA, KJ, and CM collected field samples. AI, H, MES, SPS, FL, WH, and DS designed the study was responsible for management and fund raising for this study. All authors read and approved the final manuscript.This study was funded by UNICEF Indonesia.Detailed number of MBS subjects and coverage percentage of enrollment.Click here for fileDistribution of respondents by age group.Click here for fileDescriptive Statistic for Hb level by village.Click here for fileDistribution of anaemic status by village.Click here for fileAge distribution of participants in MBS divided per villages.Click here for file


Plasmodium falciparum antigen merozoite surface protein 2 (MSP2).Malaria is the most important cause of mortality and morbidity in children living in the Kintampo districts in the middle part of Ghana. This study has investigated the multiplicity of infection (MOI) within asymptomatic residents of the Kintampo districts, and the influence of age and seasonality on MOI, by studying the distribution of the polymorphic P. falciparum during the period November 2003 to October 2004. Polymerase chain reaction was carried out and multiplicity of infection (MOI) was determined.DNA was extracted from an asymptomatic cohort of children and adults infected with Children under 10 years of age had an average MOI of 2.3 while adults 18 years and above had an average MOI of 1.4. Children below five years had high and low average MOIs of 2.8 in the March/April survey and 0.9 in the May/June survey respectively. A similar trend in the monthly distribution of MOI was observed for the entire cohort. IC/3D7 strains outnumbered the FC27 strains throughout the year by a ratio of about 4:1 with the difference between the prevalence of the two strains being least marked in the March/April survey, at the beginning of the rainy season. MOI was not linked to the level of malaria transmission as measured by the entomological inoculation rate.The impact of interventions, introduced since this baseline study was carried out on the parasite diversity of asymptomatic residents will be the subject of further investigations. Malaria is endemic throughout Ghana and continues to be a major cause of morbidity and mortality. In most parts of Ghana, malaria accounts for over 20% of childhood deaths. According to statistics by the National Malaria Control Programme, about 3.2 million malaria cases are recorded annually resulting in about 38,000 malaria deaths .Plasmodium falciparum[Epidemiological studies carried out in northern and southern Ghana have documented levels of malaria transmission, bed-net usage, trends in parasite density and severe anaemia, acquisition of parasitaemia in relation to age and sex, and also the prevalence of molecular markers of drug resistance in alciparum-4. In thalciparum. Until ralciparum, no malaThe recent reports from Kintampo have shown decreasing parasite densities with increasing age ,6, amidsA study of the multiplicity of malaria infections among an asymptomatic cohort of children and adults resident in the Kintampo districts, based on surveys carried out at two-monthly intervals, over a one-year period, is reported here. This study also examined the distribution of MOI and parasite densities in children less than five years, during different months of the study year as well as the relationships between monthly malaria transmission and merozoite surface protein 2 (MSP2) allelic variants.2) of the total land area of the Brong-Ahafo Region of Ghana. Located between longitudes 1°25’ and 2°1’’ west of the Greenwich meridian and latitudes 7°43’’ and 8°44’’ north of the equator, the districts’ strategic location makes them the geographical centre of Ghana. The two districts have a resident population of about 140,000 and have a climate, which lies in the transitional zone between the wet semi-equatorial and tropical continental climates. Mean monthly temperatures range between 18°C and 38°C. The area experiences distinct wet and dry seasons, with the wet season beginning in late March and ending in early November each year. Relative humidity in the area is extremely high, varying from between 90% and 95% during the wet season to between 75% and 80% in the dry seasons. Rainfall ranging between 115 cm3 and 125 cm3 is experienced annually. At the peak of the rainy season, most parts of the districts, especially the northern parts, experience flooding with inundation of vast stretches of land. These conditions provide favourable breeding conditions for mosquito vectors, including the most efficient malaria transmitting vectors, Anopheles gambiae and Anopheles funestus[.Kintampo North and South districts occupy 18.1% 7,162 km,162 km2 P. falciparum infections and morbidity from this infection in the Kintampo area as the background for subsequent malaria intervention studies. Details of the main study methodology have been previously reported [Results described in this paper were obtained during a large study, which determined the epidemiological patterns and characteristics of reported . The maireported .This study was approved by the ethics committee of the Ghana Health Service and the London School of Hygiene & Tropical Medicine.P. falciparum parasites by oil immersion microscopy. The number of parasites per microlitre was calculated as 40 times the number of parasites counted per 200 leukocytes. At least 100 high power fields were examined before a thick smear was declared negative. Moving averages and proportions were statistically estimated  and used to provide descriptions of the parasite density for each participant [During the study period, individuals who had provided consent and were asymptomatic for malaria were followed-up on a two-monthly rotational basis; thus, participants were visited six times in the year. Blood samples were collected on each visit. Thick and thin blood smears were obtained and blood blot filtermats  were prepared and labelled for each participant. Thin blood smears (fixed with methanol) and thick blood smears were stained with 10% Giemsa for 10 minutes and examined for ticipant .In the main study, a minimum number of 140 volunteers were selected in each age group to represent a cluster, to allow an estimated age specific parasite prevalence rates to within 6 -10% of the true values to be calculated for the different age groups . An averP. falciparum was performed by amplifying the polymorphic deoxyribonucleic acid (DNA) region of interest using previously described nested polymerase chain reaction (PCR) protocols [Parasite genomic material was prepared using the chelex method . Genotyprotocols ,19. PrimMethods for obtaining the entomological inoculation rates (EIR) for the study area during the period of this study have been previously described .P. falciparum genotypes (parasites) carried by the host. For the MOI analysis, study subjects were divided into four age categories  based on the presumed immunity of subject hosts and potential for use of preventive methods. Results were analysed using STATA version 10. One-way analysis of variance (ANOVA) was used to compare geometric mean MOIs between groups and trend analyses was carried out.To determine the number of clones of malaria parasites (MOI) co-infecting an individual host, the number of distinct bands for all allelic variants was counted, and the total number represented an estimate of the minimum number of Four hundred and thirty-three (72.2%) of the 600 PCR amplifications were successful. The successful PCR amplifications obtained from the six bimonthly surveys (100 samples per survey round) were 71, 86, 92, 64, 82 and 38 in the respective months of Nov/Dec, Jan/Feb, Mar/Apr, May/Jun, Jul/Aug, Sep/Oct . Malaria control needs to be stepped-up and strictly adhered to during these months. Visitors need to make prophylaxis a priority.In the Kintampo area, the rainy season spans from March to November with two peaks. The major peak season between March and July and the minor peak season September and November. This study showed that MOI was not influenced by the GMPD and vice versa. While MOI was highest in March/April GMPD was highest in May/June. Significant correlation between parasite density and MOI have been demonstrated in infants and children (1–2 years of age) but not in older individuals ,21 and hThe MOI decreased significantly with increasing age. This age-effect was however most marked in subjects older than 17 years. This finding agrees with a study in Senegal where the number of genotypes decreased with age in those aged 15 and above . That chIt is reported that an increase in transmission intensity generally correlates with a progressive increase in the average number of malaria parasite clones ,9. In thThis study has defined the influence of age and season on MOI in a central area of Ghana. These findings provide knowledge upon which the impacts of interventions  introduced since this baseline study was carried out will be analysed in future investigations involving asymptomatic individuals.ANOVA: Analyses of variance; DNA: De-oxy-ribonucleic acid; EIR: Entomological inoculation rate; GMPD: Geometric mean parasite density; MOI: Multiplicity of infection; MSP2: Merozoite Surface Protein 2; PCR: Polymerase chain reaction.The authors declare that they have no competing interest.SOA conceptualised the idea, wrote the proposal, secured funding and was the principal investigator. MA, DO and DD carried out sample collections; GD performed data management; AAB, CB and DD carried out laboratory analyses; AAB and CB drafted the initial paper; KPA and SOA supervised data collection, analyses interpretation and write up and MW, BG advised on the design and implementation of the study and also reviewed and finalised this paper for publication. All authors reviewed, read and approved the final manuscript.


Severe falciparum malaria (SM) pathogenesis has been attributed, in part, to deleterious systemic host inflammatory responses to infection. High mobility group box 1 (HMGB1) protein is an important mediator of inflammation implicated in sepsis pathophysiology.Plasmodium falciparum infection, enrolled in a prospective observational case-controlled study, using a commercial enzyme-linked immunosorbent assay. The utility of HMGB1 to distinguish severe malaria  from uncomplicated malaria  patients and fatal (n = 21) versus non-fatal (n = 82) malaria, at presentation, was examined. Receiver operating characteristic curve analysis was used to assess the prognostic accuracy of HMGB1. The ability of P. falciparum-parasitized erythrocytes to induce HMGB1 from peripheral blood mononuclear cells was assessed in vitro. The effect of an anti-HMGB1 neutralizing antibody on disease outcome was assessed in the experimental Plasmodium berghei ANKA rodent parasite model of SM. Mortality and parasitaemia was assessed daily and compared to isotype antibody-treated controls.Plasma levels of HMGB1 were quantified in a cohort of febrile Ugandan children with P. falciparum infection. In vitro, parasitized erythrocytes induced HMGB1 release from human peripheral blood mononuclear cells. Antibody-mediated neutralization of HMGB1 in the experimental murine model of severe malaria failed to reduce mortality.Elevated plasma HMGB1 levels at presentation were significantly associated with SM and a subsequent fatal outcome in paediatric patients with These data suggest that elevated HMGB1 is an informative prognostic marker of disease severity in human SM, but do not support HMGB1 as a viable target for therapeutic intervention in experimental murine SM. Plasmodium falciparum malaria remains a leading cause of global morbidity and mortality[ortality. Recent ortality and incrortality have theortality.High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein with recently described properties as a mediator of inflammation. ReleaseStudies in experimental models of sepsis, a life threatening syndrome of systemic inflammation with pathophysiological features resembling SM , suggestP. falciparum infection and the association of HMGB1 release with disease severity and mortality. The potential therapeutic efficacy of HMGB1 neutralization was investigated in a murine model of SM using an anti-HMGB1 monoclonal antibody (mAb) with previously validated therapeutic benefit in experimental sepsis models.The potential role of HMGB1 in the pathogenesis of infectious syndromes associated with pronounced systemic inflammation suggests that HMGB1 may contribute to disease severity and outcome in malaria. This study examined HMGB1 release during P. falciparum infection were eligible for enrollment in a prospective observational nested case-controlled study conducted at Mulago Hospital’s Acute Care Unit in Kampala, Uganda between October 15, 2007 and October 30, 2009, as described[Febrile pediatric patients (ages 6 months to 12 years) with microscopy-confirmed escribed. ExclusiUpon enrollment, clinical and demographic data and venous blood samples were collected. Citrate plasma derived from venous blood samples was aliquoted and stored at −80°C until testing. Thin and thick blood smears obtained at presentation were reviewed at a reference parasitology laboratory by two independent experts to determine parasite density using leucocyte counts and confirm malaria diagnosis. Samples for biomarker testing were derived from the larger study cohort based on availability of sufficient volume of unthawed plasma samples. Patients who fulfilled World Health Organization (WHO) sub-categorization of malaria syndromes, including cerebral malaria (CM), severe malaria anaemia (SMA) and/or respiratory distress with either hypoxia or lactic acidosis, and werEthical approval for the study was obtained from the Mulago Hospital Research Ethics Committee, Makerere University Faculty of Medicine Research Ethics Committee, Uganda National Council for Science & Technology, and Toronto Academic Health Sciences Network Research Ethics Board . Written informed consent was obtained from the parents or guardians of all participants.Plasma HMGB1 levels were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s instruction (Shino-Test Corporation).6 cells/well in RPMI 1640 medium supplemented with 10% FBS and 2.5% gentamycin (Invitrogen) in the presence of P. falciparum (confirmed Mycoplasma-free ITG strain)-infected erythrocytes , uninfected erythrocytes , lipopolysaccharide , or RPMI 1640 alone for two, six, or 24 hours at 37°C and 5% CO2. At each of the time point, supernatant was collected and concentrated 10-fold with Vivaspin 10000 MWCO centrifugal concentrators at 15,000 g, 10 min, room temperature.Human PBMCs were isolated from malaria naïve volunteers using Ficoll-Paque . Cells were cultured at 1.5 × 10Cultured supernatants were concentrated 10-fold with Vivaspin 10000MWCO centrifugal concentrators , resolved by 10% SDS-PAGE and transferred onto a PVDF membrane for immunoblotting. Blots were probed with with a rabbit polyclonal anti-HMGB1 antibody  and secondary HRP-conjugated anti-rabbit antibody (1:3000). Protein bands were visualized using SuperSignal West Pico Chemiluminescent substrate (Pierce).6 freshly isolated Plasmodium berghei ANKA (MR4)-PEs. Parasitaemia was monitored by thin-blood smear stained with modified Giemsa (Sigma). Mice were monitored twice daily for signs of experimental severe/cerebral malaria . Mice judged as developing severe/cerebral malaria were euthanized by CO2. Experiments mice were performed in accordance with the University Health Network Animal Care Committee guidelines and regulations.Infection in eight- to 10-week old female C57BL/6 mice (Jackson Laboratories) was initiated by intraperitoneal (ip) injection of 1 × 10For antibody administration, animals were randomized to receive either anti-HMGB1 monoclonal antibody , isotype  or vehicle (PBS) control via ip injection every other day beginning one day prior to PbA infection until day 5 (d5) post PbA infection (pi).2. Blood was centrifuged and plasma was collected and stored at −80°C. Plasma cytokine  and chemokine (MCP-1) levels were measured using the mouse inflammation cytometric bead array kit , according to the manufacturer’s protocol. Plasma HMGB1 levels were quantified (i) using a commercial enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s instruction (Shino-Test Corporation) and (ii) by western blot analysis, as follows. Plasma samples were diluted in cell lysis buffer , mixed with an equivalent volume of the 2× Laemmli buffer containing 100 mM dithiothreitol, and boiled for 5 min. Plasma samples were separated by SDS-PAGE, and transferred onto PVDF membrane for immunoblotting. Blots were probed with rabbit HMGB-1 monoclonal antibody . For quantification, blots were scanned and band densities determined by using the NIH Image-J software.Peripheral blood was collected by either saphenous venipuncture on day 0  and d5 post pi in heparinized tubes (Starstedt) or, for time course analysis experiments, blood was collected at specified time points by cardiac puncture into heparinized syringe following euthanasia by COt-test. Multiple groups were compared using Kruskal-Wallis test with Dunn’s multiple comparison test. Receiver operating characteristic (ROC) curves and area under the curve (AUC) were generated to assess the predictive accuracy. To correct for multiple comparisons, p values were adjusted using Bonferroni-Holm’s correction. ECM survival studies were compared by Log-rank test and visualized by generation of Kaplan-Meier plots. Statistical significance was defined as a p value of less than 0.05, unless otherwise specified.Statistical analysis was performed using Prism v4 (GraphPad). Differences between groups were assessed using a non-parametric Mann–Whitney or Fischer test, as appropriate, except for the comparison of HMGB1 expression in plasma collected from the same mouse at different time points, which was measured by the paired P. falciparum malaria compared to UM .In this study, extracellular HMGB1 was quantified in plasma from 103 febrile Ugandan children with either uncomplicated malaria (UM) or SM  was utilized. In this model, ECM-susceptible C57BL/6 mice develop clinical signs of severe illness approximately five to six days after PbA infection. HMGB1 levels in ECM-susceptible mice were determined by Western blot and ELISA. For Western blot analysis, quantitative comparisons of the HMGB1 band densities were made only for bands blotted on the same membrane Figure A and B. Based on the hypothesis that HMGB1 released during infection contributes to disease pathogenesis, the therapeutic efficacy of an HMGB1 neutralizing monoclonal antibody (2G7) was investigated in ECM-susceptible mice, at a dose previously shown to confer protection in experimental sepsis models. Treatmein vitro[Because the immune response to infection is recognized as an important mediator in the development of ECM pathogenesis-16 and ain vitro and reduin vitro, the abiP. falciparum malaria, confirming and extending previous reports of elevated extracellular HMGB1 in fatal paediatric falciparum cases[Prognostic and diagnostic biomarkers of underlying pathological processes may represent promising tools to supplement clinical and laboratory assessment and improve triage and clinical management of SM. In this study, HMGB1 levels at clinical presentation were increased in a cohort of Ugandan children with severe um cases. Moreoveum cases. These rP. falciparum co-culture approach, P. falciparum-PEs were shown in this study to induce HMGB1 release from human PBMCs, which may account for elevated plasma/serum levels observed in malaria patients. It was hypothesized that malaria-induced release of HMGB1 from immune effector cells could be involved in the propagation of inflammation leading to malaria-associated immunopathology. If so, HMGB1-based strategies might represent a novel therapeutic approach for severe P. falciparum infection, as proposed for sepsis[P. berghei ANKA murine model, the development of ECM is highly dependent on host genetics and immune response to infection. Mice lacking key inflammatory mediators, such as IFN-γ and members of the TNF superfamily , are protected against the development of ECM[Extracellular HMGB1 is believed to act as a danger signal and initiates a host immune response resulting in increased pro-inflammatory production. Using a peripheral blood mononuclear cell (PBMC)-r sepsis. In the t of ECM-16. Spect of ECM.Plasmodium infection and increased in the peripheral blood of ECM-susceptible mice following infection, similar to observation in human populations, suggesting a potential role for HMGB1 in disease progression. However, administration of a monoclonal anti-HMGB1 antibody (2G7), given prophylactically at a dose previously shown to confer protection in experimental sepsis models[In this study, HMGB1 release was modulated by s models, did notP. falciparum-induced CM in humans. Further studies to elucidate the role of HMGB1, using strategies not employed and/or outside the scope of the current study, are warranted. It is also important to note that this study was carried out with a single neutralizing monoclonal antibody. It is possible that additional strategies to block HMGB1, including antibodies raised against the B box subunit domain of HMBG1, may yield more favourable outcomes.Further studies are required to explain the failure of anti-HMGB-1 antibody-based intervention in this model. In the current study, anti-HMGB1 treatment did not affect circulating levels of inflammatory cytokines induced by PbA infection, although the same antibody has been shown to attenuate levels of inflammatory cytokines induced by CLP in an experimental sepsis model. This coversus those who survived. This study supports further investigation into the potential use of HMGB1 as a biomarker to assess disease severity and prognosis in paediatric malaria. Additional prospective, multicentre studies of SM in areas of varying malaria transmission are required to validate the clinical utility of HMGB1. However, based on the results of this study in a mouse model of SM, HMGB1 neutralization using anti-HMGB1 2G7 mAb does not appear to be a viable therapeutic strategy to improve clinical outcome in this model of severe malaria. Further studies are warranted to elucidate the role of HMGB1 in the pathogenesis of human and experimental SM and CM.Accumulating evidence indicates that the host response to infection contributes to the pathogenesis of SM. Improved understanding of the pathophysiological mechanisms of SM may lead to novel prognostic tools and therapeutic strategies to improve clinical outcome. In this study, HMGB1 levels at presentation were correlated with falciparum malaria disease severity in a cohort of paediatric patients, and there was a significant difference in admission HMGB1 levels between children who subsequently died from their infection The authors declare that they have no competing interests.in vitro studies. AD, CM and CMC-G contributed to the study design and collection of patient samples. KJT provided reagents. KCK and WCL were involved in the conception and design of the study and drafting the manuscript. All authors read and approved the final manuscript.SJH performed experiments, statistical analysis and drafted the manuscript. KX performed the immunoassay. HK and DCK participated in the murine studies. FW performed the


Despite major progress made over the past 10 years, malaria remains one of the primary causes of ill health in developing countries in general, and in sub-Saharan Africa in particular. Whilst a large literature has documented the frequency and severity of malaria infections for children under-five years, relatively little evidence is available regarding the impact of early childhood malaria exposure on subsequent child development.The objective of the study was to assess the associations between early childhood exposure to malaria and pre-school development. Child assessment data for 1,410 children in 70 clusters collected through the 2010 Zambian Early Childhood Development Project was linked with malaria parasite prevalence data from the 2006 Zambia Malaria Indicator Survey. Linear and logistic models were used to estimate the effect of early childhood exposure to malaria on anthropometric outcomes as well as on a range of cognitive and behavioural development measures.No statistically significant associations were found between parasite exposure and children’s height and weight. Exposure to the malaria parasite was, however, associated with lower ability to cope with cognitive tasks administered by interviewers , as well as decreased overall socio-emotional development as assessed by parents . No associations were found between malaria exposure and receptive vocabulary or fine-motor skills.The results presented in this paper suggest potentially large developmental consequences of early childhood exposure to malaria. Continued efforts to lower the burden of malaria will not only reduce under-five mortality, but may also have positive returns in terms of the long-term well-being of exposed cohorts. The importance of early child development on all dimensions of individual wellbeing in later stages of life has been well documented in developed countries. On average, developmental delays during early childhood are associated with poorer schooling outcomes, lower incomes, and substantially higher risks of ill health during adult life-4. An eset al.[Plasmodium falciparum on cognition, and found only one high-quality study which investigates the effect of asymptotic parasitaemia on cognition: in a study of school age children in Yemen, infected children were found to perform worse on fine motor skill tasks, but not to differ with respect to their cognitive scores two weeks after the initial infection[et al.[This paper examines the associations between exposure to malaria during the first two years of life and children’s subsequent development at age five. According to the latest World Health Organization (WHO) estimates, about half of the world’s population continues to be exposed to malaria today despite massive international efforts. Acute malaria is estimated to cause 225 million cases of ill health per year, resulting in over one million deaths per year, most of which occur in sub-Saharan Africa,9. Malaret al. reviewednfection. Jukes eTo evaluate the associations between early childhood exposure to malaria and children’s physical, cognitive and socio-emotional development prior to school entry, cluster-level data on malaria parasite prevalence among children under the age of five from 2006 were combined with individual-level data from a detailed child development assessment conducted in 2010.The primary data on child development used for this project stem from the Zambia Early Childhood Development Project (ZECDP). Resulting from a joint effort between the Harvard Center on the Developing Child, the University of Zambia and UNICEF Zambia, the ZECDP was launched in 2009 with the objective to generate a comprehensive assessment of children’s development before entering school, using tools that are both internationally comparable and suitable for the sub-Saharan African context. As partet al[As described in further detail in Fink et al, the ZECet al) and genet al. Languaget al.et al.[The key explanatory variable of interest was malaria exposure during early childhood. Instead of relying on parental self-reports, cluster-level parasitaemia data collected as part of the Zambia 2006 Malaria Indicator Survey (MIS) were useet al., the MISet al.,29.Field work for the MIS was conducted between May and June 2006, which means that the children in the sample were between 18 and 29 months at the time of the MIS malaria testing. Even though children in the ZECDP sample cannot be linked to the MIS at the individual (child) level, the fact that generally all children born in 2004 in a given cluster were assessed in the ZECDP implies that the majority of children tested for malaria in the MIS were assessed as part of the ZECDP. Each study cluster corresponds to an enumeration area used in the Zambia 2000 census, and comprised about 100–250 households in 2000.The MIS randomly selected about 10–15% households from each cluster for the survey, and then tested all children under-five for malaria. Since the ZECDP assessed more than 90% of children from the 2004 cohort living in each cluster, most children tested in the MIS can be presumed to be in the ZECDP sample. From the original ZECDP sample , 221 children had moved since birth, and were excluded from the analysis. An additional three clusters (55 children) were excluded because no parasitaemia assessment was conducted in the MIS in their respective clusters, resulting in a final sample size of 1,410 children across 70 clusters with both child development and parasitaemia data.Given that malaria-endemic areas are not only more rural on average, but presumably also poorer and exposed to a different set of risk factors, spatial correlations between malaria prevalence and child development cannot directly be interpreted as evidence of a causal impact of malaria on development. To at least partially address this spatial confounding concern, a large number of family as well and community characteristics were included in the empirical mode. At the time of the survey, Zambia was administratively divided into nine provinces and 72 districts – the 70 clusters in the sample are distributed across 32 of these 72 districts. All models estimated include district-fixed effects, which absorb all differences in the average prevalence levels across regions, so that the resulting empirical estimates purely explore within-district variations in malaria exposure. In order to limit the risk of confounding within districts, a large number of child-level controls, including the sex and age of the child in months, survival status of both parents, years of completed schooling of the parent or caretaker, as well as a range of household assets were included. As suggested by Filmer and Pritchett, principThe statistical models estimated assess the empirical association between malaria exposure and several domains of child development: physical development, perceived behavioural and socio-emotional development, receptive vocabulary and fine-motor skills. As discussed above, all regressions control for district-fixed effects and child characteristics (sex and age), parent and household characteristics, as well as a set of cluster-level variables to address cluster-level confounding concerns. The multi-level model estimated can be described as followsyic is the developmental outcome of interest for child i in cluster c, Malariac is the 2006 malaria parasite prevalence in cluster c as assessed in the MIS, Xic is a vector of child and household controls, Фc is a vector of cluster level controls, and ID are the district fixed effects described above. The main outcome variables – the measures of child development – are observed at the child level, while each cluster or village was assumed to be characterized by a random intercept. More specifically, the error term uic in the empirical model was assumed to contain both a cluster-specific component εc and a truly random individual noise term εic. The random-intercept model estimated will yield unbiased estimates of the co-efficients of interest under the assumption that the cluster-specific intercepts are orthogonal to the other covariates included in the model[where he model.In order to provide a better sense of the estimated magnitudes on malaria exposure, the estimated impact of reducing the exposure to malaria to zero was compared to two alternative changes in household structure: an increase in adult education, and an increase in household wealth. All estimates were implemented using the Stata© 11 software package.Table Adult educational attainment was low, with an average of 8.5% years of schooling completed by the most educated household member. The bottom section of Table The average height of children in the sample was 117 centimetres; the average weight was 21.6 kilograms. Seventeen percent of children in the full sample were stunted (height < = −2SD below reference median), and 12% of children were classified as underweight (weight < = −2SD below reference median). Overall, only minor differences were found with respect to height and weight across children living in rural, and children living in urban areas.Table As shown in columns (3) and (4) of Table Table Table β = 2.1, p-value 0.094), but did not show any statistically significant association with height. Accordingly, increases in household wealth were predicted to positively contribute to children’s weight, but not to children’s height. Wealth appeared to have a mostly positive effect on other domains of development however, with shifts in household wealth predicted to improve child development between 0.12 (receptive language) and 0.39 (fine motor skills) standard deviations. Overall, the basic comparisons reported in Table The effects of wealth were more mixed. On average, wealth was weakly associated with weight (The results presented in this paper demonstrate associations between early childhood exposure to malaria and some, but not all domains of children’s pre-school development. Generally, the estimated associations with malaria appear large compared to the associations with education and wealth, the two covariates generally considered to be the principal determinants of child development. Even though the results appear plausible, it appears important to highlight a few issues affecting the interpretation of the results. First, while the malaria exposure measure used in the empirical analysis is likely an accurate assessment of the cluster-level exposure to malaria, it does not necessarily measure the actual exposure of each child. To the extent that some parents or guardians managed to shield their children from the parasite, the analyses presented here do not estimate the effect of exposure to malaria in an uncontrolled environment, but, rather, the effect of malaria exposure conditional on the average parental efforts to protect their children. The effect of malaria on child development may also be mediated by attendance of early childhood programmes, an institution less than 30% of Zambian children currently benefit from.A second, and related point, regards the duration of malaria exposure. Given the large national efforts against malaria, the 2006 prevalence rate is likely a decent proxy for malaria exposure in the first or first two years of these children’s life, but not necessarily a good measure of malaria exposure during early childhood more generally. If continued exposure to malaria matters beyond age two for child development, the results may underestimate the true effect of malaria to the extent the national campaign succeeded in lowering the prevalence of the disease.Third, while the 2006 parasitaemia data are used as proxy for the child’s exposure to malaria, it appears likely that malaria prevalence during that period is correlated with malaria exposure in pregnancy, so that some of the effects identified might reflect malaria exposure in pregnancy, rather than the effects of the child’s exposure to the parasite.Fourth, even though a large set of controls is included in the main specification to reduce the risk of confounding, the cross-sectional nature of this study does not rule out residual correlations between unobservable household or cluster characteristics and the malaria measure. If these unobservable factors have a causal effect on child outcomes, the effects of malaria may be over- or underestimated even in the fully specified model.Last, by focusing on children surviving up to age five, the study does not only abstract from the important mortality impact malaria has in the country, but also identifies the marginal impact of malaria among the selected sample of children surviving initial exposure to the disease. To the extent that these surviving children are more resilient to the parasite than the general population of children, the study may underestimate the true effect of malaria on child development. From a broader policy perspective, the empirical strategy employed in this paper was clearly not designed to fully measure the burden of malaria. Rather, this study illustrates the burden of malaria for child development, an aspect which should be considered for the broader health and economic costs generated by the disease in order to generate a comprehensive estimate of the true societal cost of malaria.Millions of children continue to be exposed to malaria in Africa today. The results of this study suggest that exposure to the disease is not only a threat to children’s survival as extensively documented in the literature, but may also undermine their cognitive development.The authors declare no competing interests.GF conceived of the study, supervised its design and implementation, and drafted the manuscript. AO took the lead in the empirical analysis of the data and literature review. MH and JM coordinated the MIS data collection, blood testing and merging of the MIS with ZECDP data set. JM provided the sampling frame for the ZECDP as well as all spatial information used in the analytical part. BM supervised all aspects of ZECDP data collection. All authors reviewed and approved the final manuscript.


Plasmodium falciparum malaria during pregnancy is a great challenge for clinicians because of the low density of parasites in the peripheral blood and parasite sequestration in the placenta. Nevertheless, few data on the use of malaria rapid diagnostic test (RDT) during pregnancy have been published.Diagnosing P. falciparum infections were assessed in 156 febrile pregnant women by microscopic examination of their blood smears and by RDT and polymerase chain reactions (PCR). In addition, 150 women were assessed at the time of delivery by microscopy, RDT, PCR and placental histology investigations. The study was conducted at the Gadarif Hospital, Eastern Sudan. The SD Bioline P. f / P. v  RDT kit was evaluated in this study.P. falciparum were detected by microscopy, RDT, and PCR, respectively. The sensitivity and specificity of the microscopy was 94.4% and 100%, respectively. The corresponding values for RDT evaluation were 83.3% and 92.0%, as compared with PCR as the gold standard.Among the febrile pregnant women, 17 (11.0%), 26 (16.7%) and 18 (11.5%) positive cases of P. falciparum as determined by RDT, PCR, and histology, respectively. The sensitivity and specificity for RDT was 17.4% and 81.7%, respectively. The corresponding values for PCR were 6.5% and 82.7%, where histology was used as the gold standard.While there were no detected cases of malaria by microscopic examination of blood smears, 27 (18.0%), 21(14.0%) and 46 (30.7%) out of the 150 placentae investigated had P. falciparum malaria detection in this setting.The RDT kit used in this study has poor performance for peripheral and placental http://www.diagnosticpathology.diagnomx.eu/vs/1092363465928479The virtual slide(s) for this article can be found here:  Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) variant surface antigen occurs in P. falciparum malaria in pregnancy, presents an enormous diagnostic challenge, especially in sub-Saharan African countries [Malaria is a major public health problem in the tropics. Around 125 million pregnant women live in malaria-endemic areas, and 32 million of these are at risk of malaria in sub-Saharan Africa [ountries .There is a need for accurate and prompt diagnosis of malaria so as to achieve the desired level of disease control. This goal is of fundamental importance because prescription of any drug during pregnancy poses a risk to the unborn child . The WorP. falciparum infections in febrile pregnant women and placental malaria. This study aimed to provide evidence-based data on the best diagnostic methods for malaria during pregnancy, based on our recent observation of the limitations of the different diagnostic methods for malaria during pregnancy in Sudan [Pregnant Sudanese women are at risk of malaria regardless of their age or parity and malaria is associated with adverse maternal and perinatal outcomes . RDT hasin Sudan -21.P. falciparum is the sole malaria species present [This study was conducted at Gadarif Hospital in Eastern Sudan during September through December 2011. The area is characterized by unstable malaria transmission and  present . The croP. falciparum malaria in Sudan [After signing informed consent documents, those enrolled in both studies  were screened and clinically examined for malaria. Information was collected on social demographics using a pre-tested questionnaire. Malaria screening was performed using microscopy, RDT and PCR during pregnancy, and placental histology at delivery. Pregnant women with a confirmed diagnosis of malaria were treated with artesunate sulfadoxine-pyrimethamine which is the first line treatment for in Sudan . The samP. falciparum (Pf) and P. vivax (Pv). These antigens are the Histidine Rich Proteine-2 (PfHRP2) and lactate dehydrogenase (PvLD). Briefly a drop of whole blood (20 μl) was added to the card pad followed by three drops of lysis reagent. The RDT result was read within 10 minutes and the results recorded immediately. The tests were interpreted as follows: the presence of one coloured band (C control line) within the results window indicated a negative result, two coloured bands (test line 1 and C line) indicated P. falciparum, two coloured bands (test line 2 and C line) indicated P. vivax, while the presence of three coloured lines (test line 1 and 2 and C line) indicated a mixed infection with P. falciparum and P. vivax. Tests were considered valid when there was a coloured line in the control and invalid if the control (C) failed to appear in the results window.The commercially available RDT kit SD Bioline P.f / P.v  was used in this study according to the manufacturer’s instructions. It is used for the qualitative detection of antigens produced by Blood smears were stained with 10% Giemsa and examined under the X100 oil immersion objective lens of a light microscope by two independent laboratory technologists that were blinded to each other's results. The number of asexual parasites was counted against 200 leucocytes, where an average leucocyte count of 8,000/μL was assumed. Before a smear was considered negative, 200 high power fields had been examined.et al.[Full thickness placental blocks (around 3 cm) were taken from each placenta and kept in neutral buffered formalin for histology. The buffer was used to prevent formalin pigment formation, which has similar optical characteristics and polarized light activity as malaria pigment . Placentet al.; i.e., uP. falciparum DNA [Parasite DNA extraction and PCR assays were performed as described in our recent work . Brieflyarum DNA .All of the study research team were trained and had continuous supervision by the pathologist and site supervisor. RDT kits were purchased centrally and delivered to the hospital by the study team. The manufacturer's storage temperature specifications (4-30°C) were adhered to by monitoring the air temperature during transportation and storage. The slide and RDT results were read by two individuals blinded to each other's results. Placental histology smears were examined by a senior pathologist, blindly (AAM). Likewise, the PCR was performed by the research team (AH and SA) who were blind to the microscopy, RDT, and histology results.The study received ethical clearance from the Research Board at the Faculty of Medical Laboratory Sciences, University of Khartoum, Sudan.Data were analysed using SPSS  software version 19.0. Sensitivity, specificity, positive and negative predictive values were determined as described previously .P. falciparum-positive cases as detected by microscopy, RDT and PCR, respectively. The geometric mean of the parasite counts for the microscopy results was 8897.8 (1031.0) rings/μl.The basic characteristics of the women enrolled in this study are shown in Table The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the microscopic analyses were 94.4%, 100%, 100% and 99.3%, respectively. The corresponding values for RDT were 83.3%, 92.0%, 57.7% and 97.7%, where PCR was used as the gold standard, Table P. falciparum infections as judged by RDT, PCR, and histology, respectively.While there were no detected cases of malaria in the microscopy examination, 27 (18.0%), 21 (14.0%) and 46 (30.7%) out of the 150 placentae investigated had The sensitivity, specificity, PPV and NPV for the RDT kit were 17.4%, 81.7%, 29.6% and 69.6%, respectively. The corresponding values for PCR were 6.5%, 82.7%, 14.3% and 66.7%, where histology was used as the gold standard, Table Likewise, the sensitivity, specificity, PPV and NPV for the RDT kit were low , where PCR was used as the gold standard, Table P. falciparum among febrile pregnant women, but low sensitivity (17.4%) and specificity (81.7%) for diagnosing placental malaria when compared with PCR as the gold standard. The performance of the same brand of kit (SD Bioline P.f / P.v) was recently compared with PCR in eastern Sudan among febrile non-pregnant patients where it was found to have a sensitivity of 69% and specificity of 84% [et al., who showed that RDT kits had high false positive and negative rates in a region of malaria hypoendemicity in Tanzania [P. falciparum  87 (71.3%) and 74 (60.7%) were not considered positive by peripheral microscopy and the HRP2 RDT, respectively.The main findings of the current study were that the RDT kit had moderate sensitivity (83.3%) and acceptable specificity (92.0%) for the diagnosis of peripheral y of 84% . Recently of 84% . The perTanzania . HoweverTanzania  have recP. falciparum malaria among febrile pregnant women in a hyper-endemic region in Uganda, when compared with microscopy as the gold standard [P. falciparum HRP2 are more sensitive than blood smears, and appear to be reliable predictors of adverse outcomes of malaria in pregnancy [Nevertheless, it has been observed that RDT had high sensitivity (96.8%) and specificity (73.5%) for the diagnosis of standard . Howeverstandard . Recentlstandard . Previouregnancy -32.Although the manufacturer’s  instructions were strictly followed, the poor performance this RDT kit in the current study is disappointing, and perhaps somewhat difficult to explain. Furthermore, the RDT used in the current study was very sensitive and specific when evaluated by WHO/ FIND . High seThe Sudanese National Malaria Control Programme recommends the use of RDT in those settings where no expert microscopy is available, and maintains microscopic examination in those places where microscopy is of an adequate level. This RDT strategy was investigated earlier in Sudan for the home management of malaria using artemisinin-based combination therapy . TherefoP. falciparum malaria in this setting.RDT has poor performance in detecting peripheral and placental The authors declare that they have no competing interests.AHK and IA coordinated and carried out the study, and participated in the statistical analysis and procedures. GKA and AMA participated in the clinical work and statistical analyses. AAM and SEE conducted the laboratory work. All the authors have read and approved the final version of this manuscript.


Plasmodium falciparum by analyzing the polymorphism of the msp-1 and msp-2 genes and the multiplicity of infection in children with uncomplicated malaria in southern Benin. Blood samples of children with fever or history of fever with thick smear positive P. falciparum were collected on filter paper. After extraction of DNA by Chelex®, the samples underwent nested PCR. 93 isolates from children were genotyped. For the msp-1 gene, the K1 and R033 sequences were the most represented in the study population with 85.2% and 83% prevalence, respectively. Regarding the msp-2 gene, the FC27 family was more highly represented with 99% prevalence against 81.5% for 3D7. Mixed infections accounted for 80.4% of the samples. Twenty-five alleles were identified for msp-1 and 28 for msp-2. Fourteen and ten alleles belonged to the K1 (100–500 bp) and MAD20 (100–500 bp) families, respectively. The RO33 sequence did not show any polymorphism, with only one variant (160 bp) detected. The msp-2 gene was present as 16 FC27 family fragments (250–800 bp) and 12 of the 3D7 family (350–700 bp). The multiplicity of infection was estimated at 3.8 for msp-1 and 3.9 for msp-2 with 77 (87.5%) and 84 (91.3%) samples harboring more than one parasite genotype for msp-1 and msp-2, respectively. The multiplicity of infection (MOI) was influenced neither by age nor by parasite density. This study shows a significant diversity of P. falciparum in southern Benin with an MOI unaffected by age or by parasite density.The aim of this study was to determine the genetic diversity of  P falciparum infection is among the first diseases and is also responsible for 36% of deaths among children under 5 years . Despite the enormous efforts that have been directed toward malaria control and prevention, multiple factors including insecticide resistance in the anopheline vectors, and the emergence and rapid spread of drug-resistant parasite strains are major problems for the control and prevention of malaria. Therefore, the development of an effective malaria vaccine is urgently needed. One of the limitations to the development of this vaccine against P. falciparum is the extensive genetic diversity in parasite populations limiting the efficacy of acquired protective immunity to malaria [msp-1) and merozoite surface protein-2 (msp-2), are considered prime candidates for the development of a malaria vaccine and are also suitable markers for the identification of genetically distinct P. falciparum parasite sub-populations [et al. [P. falciparum malaria in Benin. Following scale up of malaria control interventions (massive deployment of insecticide-treated nets and free treatment with ACT) in the country, it is important to reassess genetic diversity of P. falciparum in Benin to better direct control actions. This study aimed to characterize the allelic polymorphism of msp-1 and msp-2 and determined the multiplicity of infection in P. falciparum isolates collected from children with uncomplicated malaria living in the southern Benin.In Benin,  malaria . Asexual [et al.  noted a P. falciparum mono-infection with parasite density ≥1000 asexual forms per microliter, identified by microscopy on blood smears; (iii) no evidence of a concomitant febrile illness; (iv) no sign/symptoms of severe malaria as defined by WHO [The study was conducted in two highly endemic regions of southern Benin, including the departments of Littoral and Ouémé. Southern Benin is characterized by a sub-equatorial climate and a perennial malaria transmission with two peaks corresponding to the rainy seasons (April–July and mid-September–November) . Childred by WHO ; and (v)Venous blood from symptomatic children fulfilling the above criteria was collected systematically on the filter paper. Thick and thin blood smears were prepared and were stained with 10% Giemsa for rapid diagnosis. All thick blood smears were examined against 500 leukocytes. Parasite densities were recorded as the number of parasites/μL of blood, assuming an average leukocyte count of 8000/μL of blood. All slides were read in the health center’s laboratory with external quality control performed on 10% of the negative slides and all positives in the Reference Laboratory of Parasitology of the Centre National Hospitalo Universitaire of Cotonou. Parasite DNA was extracted from filter papers using the Chelex 100 resin methods  and stormsp-1 and block 3 of msp-2 [msp-1 and the 3D7 and FC27 allelic families of msp-2 were performed sequentially in accordance with the genotyping protocol of Snounou et al. [Specific primer pairs were used to amplify block 2 of of msp-2 , 23. Theof msp-2 . Analyseu et al. . Allele-msp-1 and msp-2 genes was determined by the number of PCR products corresponding to each family within the total number of samples. The number of patients with more than one amplified PCR fragment within the total population is defined as the frequency of polyclonal infections. The multiplicity of infection (MOI) was calculated as the total number of detected Plasmodium falciparum msp-1, msp-2 genotypes/total number of infected children [p value < 0.05 was chosen as threshold significance for the various statistical tests.The data were entered in the software R version 2.12.0 . The distribution of allelic families of The Ethical Committee of the School of Medicine and Health Sciences, University of Benin, gave the ethical approval for the study. Written informed consent from head of school and parents was received.Over a period of 4 months, 93 children meeting the inclusion criteria were recruited to the study. The characteristics of the study population are detailed in P. falciparum isolates was analyzed for msp-1 and msp-2 genes. The efficiency of msp-1 and msp-2 gene amplification reactions with family-specific primers was 94.6% (88/93) and 99% (92/93), respectively.The parasite DNA from the 93 msp-1 and msp-2 genes is shown in p > 0.05. For the msp-2 gene, the FC27 family was more represented but the difference was not statistically significant (p = 0.57).The distribution of the different allelic families of msp-1 and msp-2 genotypes per isolate ranged from 1 to 9. The MOI was 3.8 ± 0.35 for msp-1 and 3.9 ± 0.30 for msp-2. Multiple infections were found in 87.5% (77/88) of samples for msp-1 and 91.3% (84/93) of samples for msp-2. Overall (msp-1 + msp-2), the multiplicity of infection was 4 PCR fragments per individual infected, with 72 (81.8%) of the samples harboring more than one parasite genotype.The number of According to age, and parasite density the MOI was similar between individuals of different age and parasite density without significant difference .Table 3.msp alleles were identified (25 for msp-1 and 28 for msp-2).A total of 53 individual msp-1 gene, 14 K1 type alleles (100–500 bp); 1 RO33-type allele (160 bp) and 10 MAD20 type alleles (100–500 bp) were identified; for the msp-2 gene 16 different FC27 type alleles (250–800 bp) and 12 3D7 type alleles (350–700 bp) were detected. The analysis of K1 alleles revealed three prevalent alleles . The RO33 allele did not show any polymorphism, with only one variant (160 bp). The most prevalent Mad20 type alleles were represented by the Mad20200 and Mad20250 PCR fragments  and 3.9 (msp-2) was high compared to those reported in Benin and Congo Brazzaville [Allele-specific genotyping of eviously  with a h studies , 24. Twe studies , 26 but  studies , 17. Forin Congo , it diffin Congo , and Mayin Congo . The difin Congo , the maj [et al.  had foun [et al. , 8. The lsewhere , 26 woulzzaville  but consP. falciparum could be a potentially useful parameter in the evaluation of interventions against malaria. Several studies have shown a correlation between the MOI and parasite density, but in our study, the MOI did not increase with higher densities similar to results of other studies conducted elsewhere [The fact that the MOI was not influenced by age as shown in other countries , 18 sugglsewhere , 18.P. falciparum isolated in southern Benin. This study has demonstrated once again the link between the high level of transmission, mixed infections and multiplicity of infection already found in other countries in the subregion and elsewhere. The MOI is not influenced either by patient age or by parasite density. However, other longitudinal studies to examine the dynamics of the genetic diversity of P. falciparum, taking into account other factors such as the degree of transmission, the study of the immune response, and studies of genes conferring resistance to antimalarial drugs, are needed to better target malaria control.These results show a high genetic diversity of populations of


Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P≤0.0090), IL-2 (P≤0.0379) and IL-8 (P≤0.0233). Children also had higher IL-12 (P = 0.0001), IL-4 (P = 0.003), IL-1β (P = 0.024) and TNF (P = 0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A TH1/pro-inflammatory response was the hallmark of non-immune subjects.Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n = 48), European adults  and Mozambican adults with long-life malaria exposure (n = 99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-1 Plasmodium falciparum in the development of immunity in African children P. falciparum going to malaria-endemic areas are also more susceptible to clinical and severe malaria than adults from endemic areas The worldwide burden of disease from malaria remains a major public health problem, with approximately 216 million cases and 655,000 deaths in 2010, with the majority of deaths occurring in sub-Saharan Africa P. falciparum malaria episode and (ii) the effect of exposure on immune responses in adults during the acute and convalescent phases of a malaria episode. We hypothesized that the immune response of children during a first malaria episode would be quantitatively and qualitatively different from the immune response of naïve adults. Further, we hypothesized that during a malaria episode the immune response in naïve adults would be quantitatively and qualitatively different from immune responses in adults with different levels of prior exposure to P. falciparum infection.In this study, we aimed to determine (i) the effect of age on cytokine, chemokine and Immunoglobulin G (IgG) responses in children and adults during the acute and convalescent phases of a first Written informed consent was obtained from participants or their respective parents or guardians before sample collection. Parasitemic individuals were treated according to standard national guidelines at the time of the studies. Approval for the protocols was obtained from the National Mozambican Ethics Review Committee and the Hospital Clínic of Barcelona Ethics Review Committee.All volunteers included in this study had an acute clinical malaria episode at the moment of recruitment and/or sample collection. There were four groups of patients: (i) naïve children from a malaria endemic area with a first episode (ii) naïve adults from a non-endemic area with a first episode (travelers) (iii) other non-immune adults temporarily resident in an endemic area (expatriates) and (iv) adults from an endemic area .P. falciparum is perennial, with some seasonality of moderate intensity. Forty eight children presenting a first malaria episode were chosen from a total of 287 followed up from birth to age 24 months in a three-arm randomized, double-blind, placebo-controlled trial with monthly chemoprophylaxis with sulfadoxine-pyrimethamine and artesunate between 2005 and 2009 Children were recruited in the context of a study conducted at the Centro de Investigação em Saúde de Manhiça, Manhiça District, southern Mozambique, where transmission of P. falciparum malaria by microscopy after traveling to an African country. Blood samples from acute episodes (day 0) and convalescence after malaria treatment (days 7 and 28) were collected by venipuncture into one heparin vacutainer for infected erythrocyte (IE) pellet cryopreservation in glycerolyte solution, and one vacutainer without anticoagulant for serum cryopreservation at −80°C. Two blood drops from each sample were spotted onto filter paper for parasite PCR analysis.Travelers and expatriates were European adults without previous malaria exposure (22 travelers) or who have lived in a malaria endemic area for a minimum of one year (15 expatriates). They were recruited at the Tropical Medicine Unit in the Hospital Clínic de Barcelona, Spain, between 2005 and 2009, and diagnosed with P. falciparum were non-pregnant women and men recruited from patients attending the Manhiça District Hospital, Mozambique, with clinical malaria between 2004 and 2005 Malaria-exposed adults with life-long exposure to PfEMP-1 PfEMP-1 42 from 3D7 and FVO strains Apical membrane antigen 1 (AMA-1) from the 3D7 strain P. falciparum antigens using Luminex xMAPTM  and the Bio-Plex 100 platform  g for 5 min) and resuspended with 0.05% Tween 20-PBS. One hundred µL of biotinylated anti-human IgG  diluted 1/2,500 in assay buffer were applied and incubated for 45 min. The plate was washed as before, and 100 µL of streptavidin-conjugated R-phycoerythrin  diluted 1/1,000 in assay buffer were added and incubated for 25 min. All incubations were done at room temperature (RT) with plate agitation and protection from light. The plate was read using Bio-Plex Manager version 4.0, and at least 100 microspheres per analyte were acquired per sample. Mean fluorescent intensity (MFI) with background fluorescence subtracted was exported and arbitrary units (AU) concentration for each antigen was calculated by dividing MFI of each sample by the MFI of the positive control run in each plate.A multiplex suspension array technology panel was established to quantify IgG responses to Trav1, IETrav2, IETrav3), 2 pediatric isolates from Mozambican children, one with uncomplicated malaria (IECh1) and another with severe malaria (IECh2) and 1 isolate from a pregnant Mozambican woman (IEWoman), each of whom had the O blood group, were tested for recognition by IgG using flow cytometry. Study samples were tested blindly in a single assay against each parasite. A pool of plasma samples from immune Mozambican adults and a pool of plasma samples from non-exposed European adults were included as positive and negative controls, respectively. Cryopreserved ring-stage parasites were thawed in a sorbitol gradient and grown to late trophozoites. Cells were washed 3 times in PBS and resuspended at 1% hematocrit and 1–5% parasitemia in 1% BSA-PBS solution. Five μL of plasma were added to 95 μL/well of erythrocyte suspension, incubated for 30 min at RT and stained for 30 min with 100 μL of polyclonal rabbit anti-human IgG (DakoCytomation) at 1/200. Subsequently, cells were incubated with 100 μL of AlexaFluor®-conjugated donkey anti-rabbit IgG (Invitrogen) diluted at 1/1,000 and 10 µg/mL of ethidium bromide (Ecogen) for 30 min in darkness at RT. Samples were washed 3 times with PBS-BSA between incubations. Data from 1,000 positive events was acquired with a Becton-Dickinson (BD) FACSCalibur flow cytometer. Reactivity against IE surface antigens was expressed as the difference between the MFI of IEs and the MFI of uninfected erythrocytes.Two laboratory clones , 3 isolates collected from travelers to endemic regions (IEConcentrations (pg/mL) of interleukin (IL)-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1β, TNF and TNF-β in plasma and serum were measured using a commercial multiplex suspension array technology kit  and flow cytometry. Twenty-five µL of plasma or serum were tested following manufacturer’s instructions. MFI from microspheres was acquired with a BD FACSCanto II and analyzed in FlowCytomix Pro2.2.1 software (Bender MedSystems). Concentration of each analyte was obtained by interpolating fluorescence intensity to a 7-point dilution standard curve supplied by the manufacturer. Any value below the limits of detection was given a value of half the detection limit for that cytokine. TNF-β was excluded from the statistical analysis since levels in most samples  were below the limit of detection.42 3D7; 638.33 AU for MSP-142 FVO; 3110.36 AU for EBA-175; 1572.55 AU for DBL-α; and 217.44 AU for DBL3X. Comparisons between groups for categorical variables were done using χ2test or Fisher’s exact test. Continuous variables were analyzed using the non-parametric Kruskal Wallis test or the Mann-Whitney U test. Correlations within groups were assessed by Spearman’s rank coefficient. All p-values were two-sided and considered statistically significant when <0.05. All data collected were analyzed using Stata version 11.0 .Recognition of recombinant proteins and parasites by antibodies was considered positive if AU or MFI values were above the mean of the negative controls plus 2 standard deviations for each antigen. Threshold values for seroprevalence were: 238.02 AU for AMA-1 3D7; 1134.73 AU for AMA-1 FVO; 921.18 AU for MSP-1P. falciparum malaria endemic regions presenting with an episode of clinical malaria. There were no differences in parasite densities between travelers and expatriates and between children and malaria-exposed adults. Some children were born to mothers with placental malaria infection defined as having malaria pigment or parasites in the placenta  and 3 children had cord blood that was PCR positive for P. falciparum.P. falciparum IEs were measured in children and travelers presenting with a first malaria episode on the day of malaria diagnosis and at convalescence. Merozoite proteins AMA-1, MSP-142, EBA-175 were chosen because they are involved in essential parasite functions (erythrocyte invasion) and are targets of naturally acquired antibodies associated with malaria immunity and thus are leading vaccine candidates PfEMP-1 PfEMP-1 domain cloned from the R29 lab isolate that is involved in rosetting through adhesion to complement receptor 1 . Similarly, there were no differences in antibody prevalences or in breadth of response (data not shown) with the exception of IgG prevalence against MSP-142 3D7 that was higher in children than travelers (P = 0.027)  .At convalescence, there were no significant differences in IgG levels or prevalences to recombinant proteins . ChildreP = 0.020). During convalescence children had significantly higher levels of IL-10  compared to travelers . The day 0/day 28 ratio was significantly higher for IL-12 in children compared to travelers  and significantly lower for IL-10   .IL-10 levels correlated moderately with parasite density in both groups whereas IL-8, IL-6 and IL-1β concentrations correlated only in travelers (Table S2). There were no correlations between cytokine and antibody levels (data not shown). In children, no association was found between cytokine levels and being born from a mother with placental malaria (data not shown).P<0.001) regardless of the antigen. Expatriates with previous few exposures to malaria still had significantly lower levels of antibodies compared to life-long malaria-exposed individuals although the levels were higher than naïve subjects with the exceptions of IgG to MSP-142 FVO, EBA-175 and DBL-α. Breadth of response was also significantly lower in primo-infected individuals or expatriates compared to malaria-exposed adults (data not shown).To study how exposure to malaria is affecting the IgG antibody responses to different antigens, we compared IgG levels  and prevP<0.0001, P<0.0001, P = 0.006, respectively) . Children, travelers and expatriates also had significantly higher concentrations of IL-8  in comparison with malaria-exposed adults . Compared to malaria-exposed adults, children also had higher levels of IL-12 , IL-4 , IL-1β  and TNF .Cytokine measurements in individuals with an acute episode showed that children, travelers and expatriates had significantly higher levels of IFN-γ than malaria-exposed individuals  . In addiIn addition to the correlations between cytokines and parasitemia in children and travelers described above, IFN-γ, IL-4 and IL-1β correlated significantly with parasitemia only in the expatriates group . None ofNo correlations between cytokines and antibodies were observed in any study group (data not shown).42 3D7 and to a parasite from a pregnant woman, and in IL-12 concentrations in serum/plasma. All three responses were higher in children. In addition, at convalescence children had higher levels of IL-10 and there were no differences in antibody levels or seroprevalences, although for travelers only 6 samples out of 22 were available at day 28 of convalescence.Results comparing naïve adults and children indicate that age has no prominent role in the immune responses induced during a first malaria episode. Differences were found only in IgG responses to MSP-1H1 cytokine produced mainly by dendritic cells (DC) in response to Toll-Like-Receptor (TLR) stimulation. It induces and regulates DC maturation and function and promotes IFN-γ production by T cells and NK cells as well as their activation IL-12 has been associated with protection against malaria P. falciparum PCR positive at day 28 (data not shown), although they were treated with anti-malarial drugs and they did not have fever or microscopic parasitemia. Conversely, none of the adults were PCR positive at convalescence. It is possible that the higher IL-10 levels at convalescence were reflecting a response to submicroscopic parasitemia from uncleared/recrudescent or new infections. However, the most plausible explanation is that PCR positives were due to residual parasite DNA in dead IEs remaining in circulation At convalescence, children had higher levels of the anti-inflammatory cytokine IL-10 than naïve adults. Therefore, the total change in magnitude of this cytokine in acute vs. convalescence was significantly lower in children compared to adults, suggesting different capacity of regulating the pro-inflammatory response. The observation that children had higher levels of TNF and IL-1β in acute phase compared to life-long malaria-exposed adults also suggests a different ability to regulate pro-inflammatory and anti-inflammatory responses according to age and/or exposure. Nevertheless, most children were P. falciparum allowed us to identify IFN-γ, IL-2 and IL-8 as cytokines/chemokines involved in cellular immune responses to P. falciparum. As expected, increasing exposure showed to be associated to an increase of antibody levels against any antigen, but in this study we were unable to evaluate its role in immunity. Compared to antibodies, the more moderate cytokine responses may reflect naturally acquired immunity that allows a better control of pro-inflammatory responses rather than mere exposure. Although IFN-γ has been associated with malaria protection H1 and inflammatory responses detected reflect innate rather than protective adaptive immune responses. Moreover, levels of IL-1β and TNF were higher in children compared to malaria-exposed adults during acute infection despite having a trend to lower parasite density than malaria-exposed adults mounting a higher antibody response to infection. This reinforces the idea that naïve individuals have stronger pro-inflammatory responses that would be independent from the parasite biomass and may be key factors to the prognosis of clinical episodes. Children also presented significantly higher IL-4 levels compared to malaria-exposed adults. It has been described that IL-4 promotes IL-12 DC secretion H1/pro-inflammatory responses observed in naïve individuals were not caused by higher parasite densities. Although we could not compare parasitemias between all groups because of different methods used to assess parasite density, there were no significant differences between children and malaria-exposed adults or between travelers and expatriates. Additionally, correlations between cytokines and parasitemia showed that cytokines differently expressed in naïve versus malaria-exposed individuals  did not show to be associated with parasitemia with the exception of IL-8 in naïve adults. These results suggest that these cytokines are not directly affected by parasitemia and the differences observed in cytokine concentrations may not be a direct effect of differences in parasite densities among the groups. Moreover, the fact that the correlations between cytokines and parasite densities were different among groups might be a reflection of different mechanisms of cytokine regulation depending on age and immunity.Comparisons between individuals with different levels of exposure to P. falciparumHence, our data may be reflecting development of tolerance, a recognized mechanism of acquisition of immunity to P. falciparum infection  of IgG antibody responses in a malaria acute episode against recombinant proteins and IEs surface antigens in differently exposed individuals.(PDF)Click here for additional data file.Table S2Spearman correlations between cytokines/chemokines and parasitemia.(PDF)Click here for additional data file.