Plasmodium falciparum merozoite surface protein 5 (PfMSP5) is an attractive blood stage vaccine candidate because it is both exposed to the immune system and well conserved. To evaluate its interest, we investigated the association of anti-PfMSP5 IgG levels, in the context of responses to two other conserved Ags PfMSP1p19 and R23, with protection from clinical episodes of malaria in cross-sectional prospective studies in two different transmission settings.Ndiop (mesoendemic) and Dielmo (holoendemic) are two Senegalese villages participating in an on-going long-term observational study of natural immunity to malaria. Blood samples were taken before the transmission season (Ndiop) or before peak transmission (Dielmo) and active clinical surveillance was carried out during the ensuing 5.5-month follow-up. IgG responses to recombinant PfMSP5, PfMSP1p19 and R23 were quantified by ELISA in samples from surveys carried out in Dielmo (186 subjects) and Ndiop (221 subjects) in 2002, and Ndiop in 2000 (204 subjects). In addition, 236 sera from the Dielmo and Ndiop-2002 surveys were analyzed for relationships between the magnitude of anti-PfMSP5 response and neutrophil antibody dependent respiratory burst (ADRB) activity.Anti-PfMSP5 antibodies predominantly IgG1 were detected in 60–74% of villagers, with generally higher levels in older age groups. PfMSP5 IgG responses were relatively stable for Ndiop subjects sampled both in 2000 and 2002. ADRB activity correlated with age and anti-PfMSP5 IgG levels. Importantly, PfMSP5 antibody levels were significantly associated with reduced incidence of clinical malaria in all three cohorts. Inclusion of IgG to PfMSP1p19 in the poisson regression model did not substantially modify results.These results indicate that MSP5 is recognized by naturally acquired Ab. The large seroprevalence and association with protection against clinical malaria in two settings with differing transmission conditions and stability over time demonstrated in Ndiop argue for further evaluation of baculovirus PfMSP5 as a vaccine candidate. Plasmodium falciparum malaria is one of the most important causes of morbidity and mortality worldwide, currently killing over 650,000 people annually, primarily African children under 5 years old. While scaled up control measures have decreased malaria morbidity and mortality in many areas of Africa via transfer of antibodies from hyperimmune African adults to P. falciparum patients The protective role of antibodies against blood stage malaria has been demonstrated using passive immunisation Clinical symptoms of malaria occur during the blood stage of the parasitic cycle, during which asexual merozoites invade red blood cells, multiply intra-cellularly and egress to reinvade new cells in a cyclical process. Erythrocyte invasion is a rapid, multi-step process involving a number of merozoite membrane proteins accessible to immune effectors such as antibodies and complement P. falciparum MSP1p19 are major contributors to invasion inhibitory activity present in the serum of immune adults The first identified and most studied MSP is merozoite surface protein 1 (MSP1), a 200 kDa protein proteolytically processed to a conserved C-terminal GPI anchored moiety of around 19 kDa called MSP1p19 composed of two adjacent EGF-domains P. falciparum MSP5. The msp5 gene codes for a 272-residue protein with a C-terminal EGF-like domain and a GPI attachment motif P. falciparum is unknown, it is apparently not critical for parasite survival since viable knock-out mutants can be isolated with no apparent growth defect, at least under in vitro culture conditions One such target of interest is P. falciparum MSP5 antibodies in sera from Senegalese endemic inhabitants using a baculovirus recombinant PfMSP5, and the protection status of the same individuals with regards to clinical malaria episodes and evaluate how this response vis a vis a response to other low polymorphic Ags already explored in this setting i.e. MSP1p19 and R23, an infected red blood cell associated repeat Ag in vitro assay based on neutrophil antibody-dependent respiratory bursts induced by opsonized merozoites (ADRB) The aim of the present study was to evaluate the relationship between The results provide a compelling case for the interest of baculovirus PfMSP5 as a blood stage malaria vaccine candidate.ad hoc Ethics Committee of the Ministry of Health. Each year, the project was reviewed by the Conseil de Perfectionnement de l'Institut Pasteur de Dakar and the assembled village population, and informed consent was individually renewed by all subjects. Individuals could withdraw from the study and the follow-up procedure at any time.Subjects were recruited in Ndiop and Dielmo, two Senegalese malaria endemic villages participating in a long-term observational study on the acquisition and maintenance of natural immunity to malaria The protocol and objectives were carefully explained to the assembled villagers, and informed consent was obtained from all participants or their parents or guardians in the presence of an independent witness and confirmed by signature or by thumbprint on a voluntary consent form written in both French and Wolof, the local language.ie before peak transmission in Dielmo, where transmission occurs year round due to a nearby stream, and before the transmission season in Ndiop (meso-endemic). These studies involved 186 and 221 healthy villagers, in Dielmo and Ndiop, respectively, i.e. 67% and 68% of the village population at that time. In Ndiop, Ab responses were also measured in 204 blood samples collected in July 2000, of which 141 were from villagers analysed in the 2002 survey. Characteristics of the three groups are summarized in Villagers were enrolled in the cross-sectional study in July 2002, before the rainy season Active clinical surveillance was carried out during the following 5.5-month transmission period. The protocol included notification of all febrile episodes and controlled use of anti-malarial drugs by the medical staff. Each villager was visited daily at home for clinical surveillance, and blood films were made in case of fever, and read extemporaneously, as described The cumulative entomological inoculation rate (EIR) was monitored as described Trichoplusia ni insect cells  with recombinant baculovirus, and purified by metalloaffinity chromatography using Talon, as previously described −1.A synthetic gene coding for PfMSP5  was constructed with a codon usage adapted to baculovirus expression (manuscript in preparation). The construct encoded a polypeptide of 247 amino acids from Met-1 to Ile-247 with the C-terminal 25 amino acids mediating GPI modification deleted to allow protein secretion and replaced with a hexa-histidine tag to facilitate purification. Serine codons at positions S-80, S-99 and S-124 were replaced by alanine codons to prevent N-glycosylation. Recombinant baculovirus was produced using the pVL1393 transfer vector as previously described IgG responses were quantified by ELISA in duplicate plasma samples diluted 1∶200 as previously described The Ags and ELISA test for IgG responses to PfMSP1p19 and to R23 was carried out as already described 4Cl, 0.8 g.L−1 buffer, PMN were washed twice with Hank's balanced salt solution (HBSS), enumerated using trypan blue, and resuspended in PBS at 1–5.107 cells mL−1.The ADRB assay has been detailed elsewhere −1 stock in DMSO) plate reading started and continued for 1 h. To facilitate rapid handling, only 40–50 wells per plate were used, with the hyperimmune serum (HIS) internal control systematically deposited in the first and last wells.Chemiluminescence was measured using opaque 96-well plates (Berthold), and a MicroLumat Plus 96 luminometer (Berthold). Merozoite pellets (40 µL) were incubated with 10 µL of test or control sera for at least 30 min at 37°C. Immediately following addition of PMN (100 µL) and isoluminol  and incidence of malaria attacks during the 5.5-month follow-up period. For the analysis, a P = 0.058). In Dielmo, prevalence was high even in the youngest age group (<7 y). There was no significant difference in overall levels of anti-PfMSP5 IgG response (mean OD/median and OD ratios) between the two villagesP<0.05). In Dielmo, the higher responses observed in the <7 y group compared to the 7–14 y old children is possibly relate to substantial and recent exposure of individuals , although this may reflect also a small sample size effect of the youngest age group (n = 19). A significant relationship between age and IgG response to PfMSP5 (P<0.01) was observed in Ndiop only, albeit with a low correlation coefficient (0.2–0.25).The age distribution of clinical accesses and anti-PfMSP5 IgG responses in the two 2002 follow up studies is shown in P<0.001). No correlations between antibody responses and gender or hemoglobin phenotype (P>0.05) were observed in either village.Asymptomatic parasitemia on the day of sampling was associated with higher anti-PfMSP5 IgG only for the individuals from Ndiop . IgG response to R23, was substantially lower in the 2 villages in 2002 (mean OD/ODratio of 0.22/2.3 and 0.21/2.7) with a prevalence of 24% and 30% in Dielmo and Ndiop, respectively. Ab responses to R23 correlated with age in Ndiop only .In vs 71%) and antibody levels (ODratios  = 4.1 vs 4.4) showed very similar results in the 2000 and 2002 surveys with no significant difference by the Wilcoxon rank test.In Ndiop, it was possible to retrospectively evaluate the IgG responses to PfMSP5 of 204 villagers in a previous sampling conducted in July 2000, including 141 villagers who participated in the 2002 study. It was thus possible to compare the response in this sub-group of 141 villagers participating in both the 2000 and 2002 studies. Both seroprevalence  having a large age-distribution (5.2 to 50.3 years old in 2000) were negative in both surveys, whereas 74 others (52%) scored positive at both time points, showing comparable elevated levels of Ab with mean ODratios of 5.9 and 6.5 in 2000 and 2002, respectively In The isotype of IgG antibodies to PfMSP5 was investigated using a set of 30 positive sera with a similar age-distribution as the cohorts . Anti-PfMSP5 antibodies were predominantly IgG1 with only low levels of other subclasses . IgG1 leThe antibody-dependent respiratory burst (ADRB) assay measures the capacity of antibodies to opsonize merozoites and induce a neutrophil respiratory burst, which is quantified by chemiluminescence. Because of its dependence on relatively intact merozoites P<0.001 rho = 0.53; 0.41 and 0.44 for Dielmo-2002, Ndiop-2002 and Ndiop-2000, respectively, and ADRB indexes were significantly lower in the youngest age group (P<0.05).In the three surveys, age and ADRB responses were significantly correlated: P<0.001 rho  = 0.33, 0.40 and 0.29 for Dielmo-2002, Ndiop-2002 and Ndiop-2000, respectively). When anti-PfMSP5 IgG OD ratios were stratified into negative (ODratio <2), median (ODratio 2–4) and high values (ODratio>4), they were positively associated with significantly different ADRB indexes in all cases (P = 0.01), suggesting a potential contribution of anti-PfMSP5 antibodies to this functional parameter , there was an inverse relationship with ADRB indexes (P<0.01) as well as in Ndiop-2000 (data not shown). This association was independent of age by the chi square test of independence (P<0.01).ADRB indexes were related to protection against clinical malaria during the 5.5 month follow-up. When stratifying into three clinical group, , but not hemoglobin phenotype or positive parasitemia at sampling. Dichotomization of the anti-PfMSP5 response based on negative vs positive sera was the best model for this cohort, showing a significant association with protection in the age-adjusted analysis .The relationship between the anti-PfMSP5 response and incidence of clinical malaria during the 5.5-month follow-up period was analyzed using an age-adjusted Poisson regression model for each survey. Ab responses to R23 did not correlate with protection and were not included in the final multivariate model. In Dielmo-2002, clinical episodes were significantly associated with age  and anti-PfMSP5 response , only the anti-PfMSP5 responses were correlated with clinical malaria (P<0.01).1 = 2.75, P = 0.004; 2–14 vs ≥30 y, RR2 = 13.10; P<0.001); (ii) hemoglobin phenotype ; (iii) positive parasitemia at sampling  and (iv) the continuous anti-PfMSP5 response (OD ratios) in an age-adjusted model . In an age-adjusted model including both anti-PfMSP1 response  and anti-PfMSP5 response , only the anti-PfMSP1 response was correlated with clinical malaria . Analysis of the Ndiop-2000 cohort showed similar results in age-adjusted model not including anti-PfMSP1 response. However, according with the age-adjusted model including both anti-PfMSP1  response and anti-PfMSP5 response , showed that both anti-PfMSP1 and anti-PfMSP5 responses were correlated with clinical malaria .For the Ndiop-2002 cohort, variables correlated with clinical malaria were: (i) age  where transmission level was higher than in 2002. The only exception is observed in the oldest age group in Ndiop 2000 where the very low incidence can explain the difference with the other results .P. falciparum malaria. PfMSP5 has been relatively neglected in the literature compared to other P. falciparum MSP vaccine candidates such as PfMSP1, PfMSP2 and MSP3 or AMA-1 all of which are polymorphic and moreover induce allele-specific responses The present study aimed to investigate the value of PfMSP5 as a blood stage malaria vaccine candidate by analyzing possible associations between naturally acquired antibodies to this antigen in endemic populations and susceptibility to clinical episodes of in vivo relevance of the results obtained. First, the recombinant PfMSP5 antigen used in the study was produced in the baculovirus expression system to optimize its resemblance to the native homologue, especially regarding conformational epitopes in the EGF domain and elsewhere in the molecule. Previous work with recombinant MSP1p19 has shown that this system appears to be particularly competent in achieving this objective E. coli recombinant antigen vs 60–74% here) and IgG isotype (IgG1 and/or IgG3 vs exclusively IgG1 here).Two features of the approach used here were particularly important for maximizing the Secondly, the study was carried out in two endemic Senegalese villages with well-defined seasonal (Ndiop) and year-round (Dielmo) transmission, which have participated in a long-term cross-sectional prospective study with intensive follow-up for over 15 years. The study thus could rely on access to antisera from three different large cohorts of endemic donors and the protocol involved active case detection over a long 5.5-month follow-up with daily medical visits to each participant and the use of efficient diagnostic criteria to distinguish fevers due to malaria from other potential causes. These various factors provided the statistical power underlying many of the findings presented here.IgG responses to PfMSP5 generally increased with age, although the youngest Dielmo age group had substantially high magnitudes, possibly due to the low number of group members and/or transient elevated responses by some susceptible individuals having experienced recent infection at this holoendemic site with frequent infective bites . The general trend associating older individuals with augmented antibody responses is a frequent observation with many plasmodial antigens in endemic areas, which likely reflects the probability of increased cumulative parasite exposure with age. It has been observed in this study in a very similar manner for IgG responses to PfMSP1p19 in all cohorts, and to a lesser extent for R23 in Ndiop only.Anti-PfMSP5 responses were surprisingly stable in prevalence and magnitude over two years for the group of 141 individuals who were members of both the 2000 and 2002 Ndiop studies, the main difference being the 26% who became sero-positive with low OD levels during this interval. Since sampling occurred prior to the transmission season before any boosting by exposure to parasites, it is likely that the observed anti-PfMSP5 antibody levels persist for at least 6–7 months between transmission seasons, and may not change appreciably in the overall population from one season to the next. Thus, anti-PfMSP5 antibodies do not appear to be short-lived as suggested elsewhere P. falciparum malaria in vitro, suggesting that PfMSP5 antibodies might be a component of this mechanism. In this study, a similar relationship between ADRB and IgG to baculovirus derived PfMSPp19 Ag was evidenced. We observed in 2002 a significant relationship of ADRB with clinical protection in an age-adjusted manner in the two villages in vitro potential capacity of anti PfMSP5 Ab could parallel /complement PfMSP1p19 IgG and warrants to be further explored.When a cohort of 30 positive anti-PfMSP5 responder sera was analyzed for IgG isotype levels, IgG1 was by far the predominant species with only small amounts of IgG2, IgG3 or IgG4. The previous study measuring anti-PfMSP5 IgG isotype responses seemed to implicate also IgG3 In two different transmission settings, anti-PfMSP5 IgG was correlated with protection from clinical episodes of malaria. This was particularly apparent in the youngest, most vulnerable age groups of each cohort experiencing the overwhelming majority of episodes. However, it was also evident in the intermediate 15–29 y group in the Ndiop-2000 study (data not shown), with a high incidence of clinical cases probably because transmission was particularly efficient that year. The older groups as expected generally had too few cases to permit reliable comparisons.P. falciparum vaccine candidate, one of very few with so little polymorphism, potentially offering the prospect of sustained protection against this accomplished antigenic chameleon.This is the first epidemiological study in which the PfMSP5 antibody response has been investigated in depth in two settings studied with the same active case detection system using a relatively long-term intensive follow-up protocol. The results provide significant support for the interest of PfMSP5 to be included in a multitarget


P.falciparum malaria infection in preschool children.Iron deficiency is a major public health concern. Globally, iron deficiency ranks number 9 and is responsible for about 60% of all anemia cases among preschool children. In Africa iron deficiency is 43-52% while in Kenya, children under 5 years constitute the largest burden with 69% of them being deficient. There is limited iron deficiency data in Kenya. This study determined haemoglobin levels, serum ferritin levels, nutritional status and A household cross sectional study was undertaken among 125 preschoolers in Western province, drawn from 37 clusters. Systematic random sampling was used for sample selection. Data was collected using pretested structured questionnaires, entered in Microsoft package. Data analysis was done in Statistical package for social science (SPSS) version 20 using bivariate and multivariate logistic regression and differences were considered significant at P < 0.05.plasmodium falciparum malaria were 20.8%, 25% and 6.8% respectively. There was a significant association between iron deficiency and anaemia . A preschool child with anaemia was 3.43 times likely to be iron deficient compared to a preschool child who was not anaemic.The prevalence of iron deficiency (Serum ferritin <12mg/l), anaemia (Hb < 110g/l) and plasmodium falciparum malaria was prevalent among preschool children. The findings revealed a significant association between iron deficiency and anaemia. Therefore effective interventions to improve iron status will have large health benefits by greatly reducing anaemia in preschool children.Iron deficiency, anaemia and  Globally, iron deficiency ranks number 9 and is responsible for about 60% of all anemia cases among preschool children. The global burden of iron deficiency has been estimated from anaemia prevalence surveys and is undoubtedly large. Iron deficiency is a significant public health problem in Kenya among preschool children because their bodies need iron to grow and develop. Its control is a global priority in public health . Anemia In the developing world, 42% of children less than five years of age are highly affected . The larP.falciparum malaria infection rate and factors affecting iron deficiency among preschool children in Western Province. The objective was to describe iron deficiency by anaemia, age, sex, residence, wealth index, anthropometric indicators and malaria infection.The aim of this study was to determine iron status, nutritional status and Study site: The study was carried out in Western Province of Kenya outside Nairobi, West of the Eastern Rift Valley and is inhabited mainly by the Luhya people. It harbours 3,358,776 inhabitants within an area of 8,361 km2. The main economic activity is farming with maize as the staple food. This area is a stable malaria endemic area.Study population: The study population consisted of preschool children aged between 6-59 months. The inclusion of the study subjects was based on consenting parents/ guardians of children aged 6-59 months and without physical disability that would affect height measurement but those on iron supplementation, blood transfusion and had physical disability were excluded from the study.Study design and sampling procedure: The study was a cross-sectional that used a two-stage stratified cluster sample. The households were clustered using National Sample Survey & Evaluation Programme (NASSEP IV). The province was stratified into rural and urban EAs (enumeration areas). The first stage involved selection of Primary Sampling Units (PSUs) which were the EAs using probability proportional to measure of size (PPMOS) method. The second stage involved the selection of households and EAs were selected with a basis of one measure of size (MOS) defined as the ultimate cluster with an average of 100 households and constituted of one (or more) EAs. The household and structure were listed through a quick count, amalgamation /segmentation of EAs to form clusters, physical numbering of the structure of the dwelling unit.The sample was selected using a stratified two-stage cluster design consisting of 37 clusters, 18 in the urban and 19 in the rural areas. For each cluster a total of 10 households were selected using systematic simple random sampling.Clinical history, demographic and socioeconomic data: A structured questionnaire was used to record data. It captured demographic, socioeconomic, anthropometric measurements, health history, knowledge and behaviours on children aged 6-59 months.Sample collection and transportation: Venous blood sample was drawn into heparin and EDTA tubes for determination of Haemoglobin, malaria and serum ferritin concentration and stored in a cool box containing gel packs, then transported to cluster lab and processed. Finger stick or heels stick blood collection procedures was only used in cases of collapsed veins or very small veins. The blood collected in the heparin tube was centrifuged at 3000 rpm for 10 minutes to obtain serum at a central field lab site. The serum was frozen at -200c for transportation to the central laboratory and analyzed within 1 month of blood collection.Iron status indexes: Haemoglobin concentration Haemoglobin was determined from venous blood sample in EDTA tube using "Hemocue globinometer" (Hemocue HB-301). Anemia was defined as Hb below 11.0g/dl. Serum FerritinThe serum ferritin concentration was determined with Elegance Amplified Enzyme Linked Immunosorbent Assay (ELISA) system. Iron deficiency (ID) was defined as serum ferritin below 12mg/dl [ 12mg/dl  but a liMalaria screening: Malaria rapid diagnostic kits (RDKs) were used at the household for malaria test using blood collected in EDTA tubes. The RDKs used were P. falciparum only (HRP2) to capture P. falciparum malaria. Thick blood smears was prepared on glass slides within 2 hours of blood collection. The slides were fixed and stained with Giemsa stain and allowed to dry and observed under a microscope using oil immersion objectives (x100). The presence or absence of malaria was reported as any parasiteamia detected in blood smearAnthropometric: Height and weight were measured among children who were in light clothing to determine their nutritional status. The weight measurement was taken using a Seca scale (Hanson mode) to the nearest 0.1 kg and height/ length portable wooden constructed scale calibrated for height measurement to the nearest 0.1 cm. Height for age (stunting), weight for age (underweight) and weight for height were calculated using = 2D NCHS  reference data. The height for age Z-score (HAZ) of <-2 was classified as stunted and Z-score cut off point of <-2SD was used to classify low weight for age, low height for age and low weight for height.The data was coded and double entered into a computer database using Ms-Access and Ms-Excel and analysis was performed using SPSS. In order to maintain the assumption of an equal probability sample, weighting was used to adjust for unequal cluster size due to variation in the number of absentees or refusals between clusters. The main predictor, iron deficiency, was defined as serum ferritin <12 µg/L. Possible confounders considered for inclusion were: sex, age, area of residence, socio economic status, malaria, nutritional status and anemia. Bivariate analysis was performed using Chi-square to determine the association between dependent variable and independent variables. A multivariate logistic regression using backward method was used to explore determinants of iron deficiency and P-values <0.05 were considered statistically significant.Ethical clearance was sought from scientific steering committee (SSC) and ethical review committee (ERC) of Kenya Medical Research Institute (KEMRI) for approval. Prior consent was sought from parents/guardians of the preschoolers who participated in the study. During the interview privacy and confidentiality was observed.The surveys produced the province estimates, and for a subset of indicators, cluster estimates that could not be generalized. This consideration was based on the prohibitive costs of taking a representative sample within the province. In specific population certain indicators would not be assessed, such as hook worm infestation and other infections in preschoolers due to feasibility and cost. The cross-sectional nature of this survey did not allow for measurement of iron level indicators over time, and might have been influenced by seasonality, illness and other external factors present at the time of survey collection. Socio-cultural beliefs about taking of blood samples still prevailed in some areas of the study.A total of 125 preschoolers aged 6-59 months were enrolled in the study and consisted of males 72 (57.6%) and 53 (42.4%) females with a mean age of 35+ (10 SD) ranging between 6-59 months. A high proportion (29.6%) was aged between 24-35 months. The majority (65.6%) of the participants resided in rural areas and a substantive proportion (34.4%) in urban areas as shown in The wealth was defined by the type of house, roofing material and number of sleeping rooms as revealed in The prevalence of iron deficiency in children with serum ferritin concentrations <12 mg/L was 20.8% . StuntinThe selected demographic characteristics were age in months, sex and residence that were not significantly associated with iron deficiency. Using children aged 48-59 months as the reference, males in all the age groups were at a higher risk of iron deficiency than females . The risk of iron deficiency decreased with age increase among the subjects, progressively lowering with each age group . Using fMultivariate analysis was performed to identify independent variables. The four variables included (1) Sex, (2) Consumed juice or juice drinks in the last 24hrs, (3) Consumed sour milk, cheese, yoghurt or other food made from milk in the last 24hrs and (4) anaemia status. Upon fitting the factors using Binary logistic regression and specifying ‘backward conditional’ method with removal at P < 0.05, One factor was retained in the final model as shown in -1) was taken into account . The prevalence of ID is same in both sexes. The findings revealed a significant association between iron deficiency and anemia. Therefore effective interventions to improve iron status will have large health benefits by greatly reducing anemia in preschool children.Iron deficiency, anemia and


Plasmodium parasites are very high. Both positive and negative interactions between malaria and other diseases caused by parasites belonging to numerous taxa have been described and in some cases, malaria may modify the process of another disease without being affected itself. Interactions include those observed during voluntary malarial infections intended to cure neuro-syphilis or during the enhanced activations of bacterial gastro-intestinal diseases and HIV infections. Complex relationships with multiple effects should also be considered, such as those observed during helminth infections. Moreover, reports dating back over 2000 years suggested that co- and multiple infections have generally deleterious consequences and analyses of historical texts indicated that malaria might exacerbate both plague and cholera, among other diseases. Possible biases affecting the research of etiological agents caused by the protean manifestations of malaria are discussed. A better understanding of the manner by which pathogens, particularly Plasmodium, modulate immune responses is particularly important for the diagnosis, cure, and control of diseases in human populations.In nature, organisms are commonly infected by an assemblage of different parasite species or by genetically distinct parasite strains that interact in complex ways. Linked to co-infections, pathocoenosis, a term proposed by M. Grmek in 1969, refers to a pathological state arising from the interactions of diseases within a population and to the temporal and spatial dynamics of all of the diseases. In the long run, malaria was certainly one of the most important component of past pathocoenoses. Today this disease, which affects hundreds of millions of individuals and results in approximately one million deaths each year, is always highly endemic in over 20% of the world and is thus co-endemic with many other diseases. Therefore, the incidences of co-infections and possible direct and indirect interactions with  More than 1400 parasite species, including viruses, bacteria, fungi, protozoa and helminths, infect humans  cause benign tertian malaria, P. malariae causes benign quartan disease, and P. knowlesi primarily infects non-human primates. Repeated bouts of tertian or quartan fevers are caused by the cyclic release of merozoite parasites from lysed erythrocytes every 2 or 3 days, respectively  and commonly result in body temperatures as high as 41°C; however, malarial fevers can also be irregular, continuous or low-grade, as in children or during primary infections and co-infections  than to forsake the possibility of a way to fight against the parasite, and shows evidence of the high level of endemicity and continual selection pressure exerted by malaria in the past.In several areas of the past Afro–Eurasia (and later America), the levels of malarial endemicity led to the infection of a great number of humans every year, which may have reduced life expectancy . Misdiagnoses with other diseases with similar signs and symptoms are frequent: diseases with non-specific periodic fever such as gastroenteritis, typhoid fever, hepatitis A, influenza, pneumonia or bacterial meningitis have been diagnosed although the implication of malarial infections alone were subsequently validated  would induce a breakdown in pathocoenosis, causing a shift toward a supposedly novel state of equilibrium. Indeed, pathocoenoses are constantly evolving, but these dynamic states are difficult to comprehend.The concept of pathocoenosis was introduced by Grmek , 1991, wSallares  considerThe impacts of infectious agents not only depend on their associated morbidity but also on the general health of the affected population. Many epidemics due to malaria or other diseases have coincided with periods of famine, affecting underprivileged populations  with each other and with their host. With regard to ecology in general and the microbial world in particular, one population can influence a second one in different ways, and these effects can be positive, negative or neutral  was performed using infected blood or more rarely by the bite of infected mosquitoes. Malaria was cured using quinine only after some fits of fever. Some patients were effectively cured of syphilis, and many went into partial remission or observed a halt in the progression of their symptoms. However, the Hippocratic principle primum non nocere was not pre-eminent; e.g., some patients with syphilis who were treated using P. vivax subsequently died, and higher mortality rates (up to 15%) were observed in individuals with additional comorbidities  was rare or absent in highly malarial endemic areas because of an antagonism between tuberculosis and intermittent fevers Bidlot, . HoweverPlasmodium and co-infections are numerous: malaria co-occurs with 80–90% of all soil-transmitted helminth cases worldwide  may be considerable .Several malaria-endemic areas are also highly endemic for HBV infection  is a gamma-herpesvirus. Epstein–Barr virus infection generally has a benign clinical course; however, this agent is linked to several B-cell  malignancies, including endemic Burkitt's lymphoma (eBL), especially in immunosuppressed hosts  correlations between malaria and gastro-intestinal diseases have been mentioned by past physicians  and malaria have different etiologies, some of their clinical features overlap, including hepatic, pneumonial, encephalopathic and gastro-intestinal disorders continuous fevers, which are typical of enteric infections , which cause cystic fibrosis in homozygotes, could led to a survival advantage following infections of typhoid, but also secretory diarrhea (including cholera) and tuberculosis in heterozygotes  and not by Vibrio. Indeed, it is also known that cholera symptoms can be caused by Plasmodium and not by Vibrio, as observed in a malarious area of Italy where autopsies revealed high levels of P. falciparum parasites in the blood vessels of the mucous membranes of the stomach and the small intestine, with relatively few parasites in the rest of the body  even if the strains may have varied  may be debilitating, and individuals suffering from repeated attacks of malaria may be less able to resist other infections  and indirectly because it leads to the enhancements of several immunological processes , immune-mediated, or potentially, interference competition between strains results in the suppressions of parasite densities  relatively rarely affect humans, and thus, direct competition with plasmodia rarely occurs. Parasites causing hemorrhaging and anemia can impact the age profiles of the red blood cells available for plasmodia (negatively or positively). The case of Plasmodium/HBV co-infection is particularly interesting because interactions between these two parasites may be both direct, involving competition for the same attachment sites on hepatic cells, and indirect, via the host's immune system, which has antagonistic consequences  or by chemical means, and indirectly via “bottom-up” processes  or “top-down” processes   and tuberculosis co-infections in mummies from Ancient Egypt (Lalremruata et al., Despite the very limited medical knowledge available over 2000 years, ancient authors were aware of the often deleterious consequences of co-infections on human populations, and they have also cited examples of the supposed antagonistic interactions between concurrent diseases (Sallares, Pinello, . In the To conclude, the improved understanding of the prevalence of co-infection is greatly needed, in part because co-infections—including those with malaria—are often associated with worse host health symptoms and higher parasite abundances compared with hosts with single infections, and they are associated with reduced treatment efficacies and increased treatment costs (Griffiths et al., The Guest Associate Editor Anaïs Baudot declares that, despite being affiliated to the same institution as the author, the review process was handled objectively and no conflict of interest exists. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malaria is an acute and chronic illness characterized by paroxysms of fever, chills, sweating, fatigue, anemia, and splenomegaly. Most malarial deaths occur in infants and young children.Anemia in malaria has diverse pathophysiologic mechanisms such as direct invasion of Red cells, In the following, we presented comparison of hematological aspects: children with Malaria and healthy children.This was a lab trial study. Patients were referred and admitted to the pathobiology laboratory along with physical examination. Then, they underwent a complete blood count and the result of complete blood count was compared with healthy person in the same age. Out of 30 patients, with equally falciparum, vivax, and healthy .The hematological examination was performed. Finally, the data was analyzed using SPSS version 19 software. The levels of HGB (P=0.001), HCT (P=0.001), MCV (P= 0.001), MCH (P=0.001), WBC (P=0.001), and Plt (P= 0.02) decreased significantly in children with falciparum and vivax malaria compared to healthy controls. The levels of RBC (P=0.49) increased significantly in children with falciparum and vivax malaria compared to controls. Blood culture at two times. To investigate malaria; blood smears taken after microscopic study of Plasmodium falciparum ring was observed. Malaria is a multisystem disorder which can lead to many diseases. Physicians, especially those in endemic areas, should be aware of the varied manifestations and maintain in a high index of suspicion for the disease in order to that diagnose and treat timely and, morbidity and mortality. Malaria is an acute and chronic illness characterized by paroxysms of fever, chills, sweating, fatigue, anemia, and splenomegaly. Most malarial deaths occur in infants and young children. In spite of intensive worldwide efforts to reduce its transmission, malaria remains the most serious and widespread protozoal infection of humans. Over 40% of the world’s populations are at risk of malaria, which is endemic in 91 countries, mostly developing. Most of the systemic complications from malaria results from heavy parasitemia. Hematological changes are the most common complications encountered in malaria and play major role in the fatality. Plasmodium falciparum causes the most severe form of malaria and is associated with more intense parasitemia. It is one of the most prevalent human infections in the world. It is estimated that 300-500 million cases and 1.5-2.7 million deaths occur each year. Mortality rate is usually high (20%) in severe malaria (parasitemia >5%). Over 40% of world population live in malaria endemic area including Southeast Asia, India, Africa and areas of Middle East, Central and South America , and 7. This was a lab trial study. Patients were referred and admitted to the pathobiology laboratory along with physical examination. Out of 30 patients, with equally falciparum, vivax, and healthy.Then, they underwent a complete blood count (CBC) Based on the following procedures: The methods used to derive CBC parameters are based on the Beckman Coulter measurements of these three parameters. Result of method of counting and sizing, in combination with an automatic diluting and mixing device for sample processing, and a single beam photometer for. The WBC differential uses VCS technology. Analysis and classification of WBCs use three simultaneous measurements of individual cell volume (V), high frequency conductivity (C), and laser light scatter (S). The scatter gram plots the cells based upon the complete blood count was compared with healthy person in the same age. The hematological was performed for children with Malaria and as well as healthy children. Serum levels of Hemoglobin, Hematocrit, HGB, MCV, MCH, RBC, WBC and, platelet were measured.Statistical Analysis The data was analyzed using SPSS version 19 statistical software with appropriate statistical methods. Differences were considered significant at P-values of less than 0.05. Informed consent was taken from patients and parents.Of the total 30 cases of malaria falciparum, vivax and healthy choices were equally, the levels of HGB (P=0.001), HCT (P=0.001), MCV (P= 0.001), MCH (P=0.001), WBC (P=0.001), and Plt (P= 0.02) decreased significantly in children with falciparum and vivax malaria compared to healthy controls. The levels of RBC (P=0.49) increased significantly in children with falciparum and vivax malaria compared to controls. Blood culture at two times. To investigate malaria; blood smears were taken after microscopic study of Plasmodium falciparum ring was observed . Fortunately, Plasmodium falciparum chloroquine-sensitive and the patient were discharged in good condition and pancytopenias were also on hand .Hematological abnormalities are thought-outed a hallmark of malaria, and reported to be most in Plasmodium falciparum infection, probably as a result of the higher levels of parasitemia found in these patients. Malaria is a multisystem disorder which can cause many diseases. Physicians, especially those in endemic areas, should be aware of the varied manifestations and maintain a high index of suspicion for the disease in order to diagnose and treat timely and morbidity and mortality minimized. Hemophagocytic syndrome and erythrophagocytosis, dyserythropoirsis, immune haemolysis and cytokine dysregulation Anemia of chronic disorder is characterized by moderate to mild normocytic normochromic anemia along with microcytic hypochromic cells. Evaluation of Malaria infection especially in malaria- borne areas is highly recommended for consideration and further therapy  and 14. In children, Plasmodium parasitaemia has a significant impact on the packed cell Volume, hemoglobin, and platelet. Preventative strategies including intermittent preventative, regular chemoprophylaxis, Treatment with antimalarials, provision of iron supplementation, and insecticide-treated bed nets should be implemented urgently to prevent the negative impact of malaria Parasitaemia on the hematological parameters of children in the area. There is a necessity for community and peer-based Education initiatives to strengthen the malaria Prevention programme by educating parents on the benefits of effective environmental sanitation to destroy the breeding sites of Anopheles mosquito –the vector of malaria and awareness.


The East African highlands are fringe regions between stable and unstable malaria transmission. What factors contribute to the heterogeneity of malaria exposure on different spatial scales within larger foci has not been extensively studied. In a comprehensive, community-based cross-sectional survey an attempt was made to identify factors that drive the macro- and micro epidemiology of malaria in a fringe region using parasitological and serological outcomes.2 area in the Western Kenyan highlands of Rachuonyo South district. Households were geo-located and prevalence of malaria parasites and malaria-specific antibodies were determined by PCR and ELISA. Household and individual risk-factors were recorded. Geographical characteristics of the study area were digitally derived using high-resolution satellite images.A large cross-sectional survey including 17,503 individuals was conducted across all age groups in a 100 kmMalaria antibody prevalence strongly related to altitude . A strong negative association with increasing altitude and PCR parasite prevalence was found. Parasite carriage was detected at all altitudes and in all age groups; 93.2 % (2481/2663) of malaria infections were apparently asymptomatic. Malaria parasite prevalence was associated with age, bed net use, house construction features, altitude and topographical wetness index. Antibody prevalence was associated with all these factors and distance to the nearest water body.Altitude was a major driver of malaria transmission in this study area, even across narrow altitude bands. The large proportion of asymptomatic parasite carriers at all altitudes and the age-dependent acquisition of malaria antibodies indicate stable malaria transmission; the strong correlation between current parasite carriage and serological markers of malaria exposure indicate temporal stability of spatially heterogeneous transmission. Plasmodium falciparum transmission is driven by temperature and aridity that limit the distribution and competence of Anopheles vectors  using contemporaneous high-resolution satellite data . Where possible, a maximum of 16 compounds were chosen from each 500 × 500 m cell. The aim was to obtain measurements from ≥50 individuals per 500 × 500 m and sampling of individuals was guided by pre-defined age strata  to maximize the discriminative power of serological markers of exposure  detecting P. falciparum-specific histidine rich protein-2 was performed. A single finger prick sample was taken for haemoglobin (Hb) measurement using a HemoCue photometer  and three droplets of blood transferred onto a filter paper  for serum and DNA ; women of child bearing age who were RDT positive were assessed for pregnancy and offered a pregnancy test if deemed appropriate. Febrile children below 6 months of age and pregnant women with malaria were referred to the nearest health facility for full assessment and treatment.The survey was carried out in 2011, during what is considered to be the main malaria transmission season, between June and July. After initial consent during enumeration, participating compounds were visited and the name, gender, age, residency and travel history, use of insecticide-treated nets (ITNs), indoor residual spraying (IRS) in the past 12 months and sleeping times of each compound member were recorded. All compounds where at least one adult (>20 years) and one child (<15 years) were permanent residents (defined as sleeping regularly in the structure) qualified for enrolment during the survey. The axillary temperature of each compound member was measured by digital thermometer. For all febrile individuals a rapid diagnostic test .Broad patterns of transmission intensity were described by fitting age-seroprevalence curves , 36 to sThis study was approved by the ethical committees of the London School of Hygiene and Tropical Medicine (LSHTM 5721) and the Kenya Medical Research Institute (SSC 1802 & SSC2163). Approval was sought from district medical officers, local chiefs and communities. Individual informed consent was sought from all participants or guardians of those less than 18 years old by signature or a thumbprint accompanied with the signature of an independent witness. Assent was also sought from children above 13 years of age. As defined in the Kenya national guidelines, participants below 18 years of age who were pregnant, married, or a parent were considered “mature minors” and consented for themselves.2 study area. The majority of individuals resided within a narrow altitude band of 1400–1550 m . The median number of individuals per compound was 5 . As reported recently, PCR detected parasite prevalence was 21.2 % (738/3476) in children ≤5 years of age, 26.1 % (609/2337) in children aged 6–10 years, 24.8 % (462/1403) in children aged 11–15 years, 19.2 % (374/1574) in individuals aged 16–25 years and 14.6 % (480/3286) in individuals aged >25 years [In total 17,503 individuals were sampled, coming from 3213 compounds across a 100 km % 609/23 in child19 and/or AMA-1 for the four altitude bands and indicated a clear gradual decline of the seroconversion rate with increasing altitude , parasites detected by PCR, malaria-specific antibody responses Fig.  and anaeThere was considerable inter-compound variation in antibody prevalence and PCR parasite prevalence. In some compounds no members were antibody positive  or PCR parasite positive  while for other compounds ≥80 % of all compound members were antibody  or parasite positive . Malaria antibody prevalence and parasite prevalence were strongly correlated at an individual level  . This asIn univariate analysis, individual-level parasite prevalence and antibody prevalence was associated with age. When adjusting for age, the following factors were associated with parasite prevalence and antibody prevalence: the presence of eaves in the sleeping room, reported bed net use, construction of the sleeping room walls with mud, distance to water, altitude, the proportion of parasite positive individuals within a 500 m radius of the compound and the proportion of malaria antibody positive individuals within a 500 m radius of the compound Table . In a muP. falciparum infections in all age groups, suggests that malaria transmission is relatively stable in the study setting.This study, conducted in the Kenyan highlands, shows the occurrence of ongoing stable malaria transmission across an altitudinal range of 1350–1600 m, characterized by marked spatial heterogeneity. The age-dependent acquisition of malaria antibodies, strong correlation antibody prevalence and current parasite prevalence, along with the considerable asymptomatic reservoir of P. falciparum infections were very common in this highland setting, apparently asymptomatic and negatively correlated with altitude. Parasite prevalence by PCR was 27.2 % in the population residing at 1350–1449 m, 19.6 % at 1450–1499 m, 16.7 % at 1450–1549 m and 8.1 % at 1550–1650 m. Using an equation model fitted to 86 surveys that determined parasite prevalence by microscopy and PCR [P. falciparum infections resulted in fever at the time of sampling and apparently asymptomatic parasite carriage was prevalent at all altitudes [P. falciparum antigens at different altitudes suggests stable local malaria transmission in the area.Much of the highlands of East Africa represent fringe regions between stable and unstable malaria transmission; seasonal and spatial patterns in malaria transmission are affected to some degree by annual variations in rainfall but primarily by ambient temperature . The not and PCR , the corltitudes . Recentlltitudes . Since tltitudes , the higWhilst travel, a known risk factor for malaria in highland areas , 44, wasEvidence of relatively stable malaria transmission in this site at 1350–1600 m altitude was observed. Altitude was a major driver of malaria transmission in the study area, even across narrow altitude bands. Although malaria risk was spatially heterogeneous, the strong correlation between current parasite carriage and serological markers of malaria exposure and other established risk factors for malaria indicate temporal stability of geographical patterns in malaria exposure. The fine scale heterogeneity in this low-endemic setting may reflect a likely scenario for more endemic areas with active and effective control programmes as they reduce transmission to increasingly low levels. A priori knowledge of the factors that influence residual malaria in foci of low transmission is likely to further expedite control and elimination attempts.


The only malaria vaccine to have been tested in Phase III clinical vaccine trials  induces Antibody titres were measured in 138 malaria-exposed children vaccinated with ChAd63 MVA ME-TRAP in three Phase I studies in The Gambia and Burkina Faso. Age groups at first immunisation were 2-6 years, 5-12 months and 10 weeks in The Gambia and 5-17 months in Burkina Faso. Avidity and isotype profiles were also analyzed.in vitro using a sporozoite invasion inhibition assay.Antibody responses to TRAP were significantly higher in 10 week old and 5-12 month old infants in The Gambia and 5-17 month old infants in Burkina Faso compared to 2-6 year old children and adults in The Gambia and malaria-naïve UK adults. IgG isotype responses were predominantly IgG1 and IgG3 and we also detected IgA and IgM. TRAP-specific IgG avidity was significantly higher in Burkinabe infants aged 5-17 months and Gambian infants aged 5-12 months compared to Gambian adults and 2-6 year old children. TRAP-specific IgG1 avidity significantly correlated with age at vaccination in 5-17 month old Burkinabe infants and was significantly higher than in 10 week old Gambian infants. Functional activity of anti-TRAP antibodies will be analysed We demonstrate excellent humoral immunogenicity in key target populations vaccinated with a pre-erythrocytic malaria vaccination regime, exceeding that seen in UK malaria-naïve adults.


Debate for a greater role of mid-upper arm circumference (MUAC) measures in nutritional programming continues, but a shift from therapeutic feeding programs admitting children using MUAC and/or weight-for-height Z (WHZ) to a new model admitting children using MUAC only remains complicated by limited information regarding the clinical profile and response to treatment of children selected by MUAC vs. WHZ. To broaden our understanding of how children identified for therapeutic feeding by MUAC and/or WHZ may differ, we aimed to investigate differences between children identified for therapeutic feeding by MUAC and/or WHZ in terms of demographic, anthropometric, clinical, and laboratory and treatment response characteristics.Using secondary data from a randomized trial in rural Niger among children with uncomplicated severe acute malnutrition, we compared children that would be admitted to a therapeutic feeding program that used a single anthropometric criterion of MUAC< 115 mm vs. children that are admitted under current admission criteria (WHZ< -3 and/or MUAC< 115 mm) but would be excluded from a program that used a single MUAC< 115 mm admission criterion. We assessed differences between groups using multivariate regression, employing linear regression for continuous outcomes and log-binomial regression for dichotomous outcomes.We found no difference in terms of clinical and laboratory characteristics and discharge outcomes evaluated between children that would be included in a MUAC< 115 mm therapeutic feeding program vs. children that are currently eligible for therapeutic feeding but would be excluded from a MUAC-only program.A single anthropometric admission criterion of MUAC < 115 mm did not differentiate well between children in terms of clinical or laboratory measures or program outcomes in this context. If nutritional programming is to use a single MUAC-based criterion for admission to treatment, further research and program experience can help to identify the most appropriate criterion in a broad range of contexts to target children in most urgent need of treatment. It is estimated that 34 million children under the age of 5 each year are affected by severe acute malnutrition (SAM), a condition associated with significant increased risks of mortality and morbidity , 2. In 2Since 2009, the World Health Organization (WHO) has recommended using weight-for-height Z-score (WHZ) < -3 and/or mid-upper arm circumference (MUAC) < 115 mm as anthropometric criteria for admission to therapeutic feeding programs . In receTo inform decision making regarding the use of MUAC as a standalone admission criterion in nutrition programming, more information on the programmatic implications of using MUAC alone is urgently needed. Preliminary reports demonstrate demographic and anthropometric differences among children identified by WHZ and MUAC: MUAC is more likely to identify children that are younger, female and more stunted –21. ThesTo broaden our understanding of how children identified for therapeutic feeding by MUAC and/or WHZ may differ, and to inform future program planning if MUAC-based programming is to occur, we analyzed data from a randomized trial of children aged 6 to 59 months with uncomplicated SAM in Niger. The objective of this analysis was to compare children that would be admitted for outpatient SAM treatment in a MUAC-based program vs. children that are admitted under current admission criteria of low WHZ and/or low MUAC but would be excluded if using MUAC-based admission criteria. We aimed to specifically investigate differences in demographic and anthropometric characteristics between groups previously reported, as well as extend our understanding on other potential differences in vulnerability in terms of clinical and laboratory characteristics and treatment response. These characteristics were also used to explore possible variability within a potential MUAC-only program, comparing children admitted by MUAC < 115 mm admission criteria, with vs. without WHZ < -3. These findings will be useful to describe the potential implications of MUAC-based programming in terms of the clinical needs of the expected patient population and can inform planning if such programs are to be implemented.ClinicalTrials.gov number, NCT01613547). The trial protocol was approved by the Comité Consultatif National d’Ethique of the Ministry of Public Health of Niger and the Comité de Protection des Personnes, Ile-de-France and parental informed consent was obtained for all trial participants  score < -3 according to the 2006 World Health Organization Growth Standards and/or MUAC < 115 mm; sufficient appetite according to a test feeding of RUTF; and absence of clinical complications requiring hospitalization, including bipedal edema. All study children were randomized to amoxicillin (80 mg/kg/day) or placebo for 7 days and received standard nutritional and medical care according to guidelines of the Government of Niger and Médecins Sans Frontières (MSF). At inclusion, children received RUTF , routine medicines  to treat and prevent complications and a comprehensive physical exam with systematic assessment of hemoglobin, HIV status and malaria infection by rapid test. Stool, urine and blood samples were collected at admission for bacteriological analysis among a sub-sample of children of approximately 1000 children between October 2012 to October 2013. Weekly follow-up was conducted at the health facility until nutritional recovery or transfer to inpatient care for a maximum of 8 weeks. At each weekly visit, weight to the nearest 100 g; length (children <24 months of age) or standing height (children ≥ 24 months of age) to the nearest 0.1 cm; and MUAC to the nearest 0.1 cm were measured using standard techniques, a physical exam was conducted, and the next weekly ration of RUTF was provided until discharge. Nutritional recovery was defined at or after 3 weeks with WHZ ≥ -2 and MUAC ≥ 115 mm on two consecutive visits; no acute complication or edema for at least 7 days; and completion of all antibiotic and anti-malaria treatment at the time of discharge. Transfer to inpatient care was initiated in case of weight loss > 5% between two consecutive visits or lack of weight gain after 2 weeks and/or clinical complication requiring inpatient management. Non-response was defined as not meeting the criteria for nutritional recovery at 8 weeks.Demographic characteristics included child age (months) and sex. Weight-for-height (WHZ) and height-for-age (HAZ) were calculated using the 2006 WHO Growth Standards , with Z In addition, we considered 4 types of program outcomes: recovery, transfer to inpatient care, default, and death. Among all children and those who recovered, we calculated weight gain (g/kg/day) and MUAC gain (mm/day) from admission to week 1 and week 2. Among children who recovered, we calculated length of stay, defined as the number of days between the date of discharge and admission.The first comparison presents children that would be included in a MUAC < 115 mm-only therapeutic feeding program (hereafter “MUAC < 115 mm” children) vs. children that are currently eligible for therapeutic feeding using WHZ < -3 and/or MUAC < 115 mm criteria but would be excluded from a MUAC-only program using MUAC < 115 mm for admission (hereafter “MUAC ≥ 115 mm and WHZ < -3” children). The second comparison looks within the hypothetical program population identified by MUAC < 115 mm and compares children with and without WHZ < -3 .Differences between groups adjusting for the parent trial regimen (amoxicillin vs. placebo) were assessed using multivariate regression, employing linear regression for continuous characteristics, ordinal logistic regression for ordinal characteristics, and log-binomial regression for dichotomous. In secondary analyses, we additionally adjusted for age (± 24 months), sex, stunting (HAZ ± -2) at admission. We hypothesized that differences between groups may be limited to certain subgroups and explored possible effect modification by age (± 24 months), sex and stunting (HAZ ± -2). Statistical interactions were therefore assessed using linear regression for continuous outcomes and log-binomial regression for dichotomous outcomes. If a significant interaction was found, the appropriate stratified estimates are presented. Stata 12  was used for statistical analysis. A P-value < 0.05 was considered statistically significant.2,399 children aged 6–59 months with MUAC < 115 mm and/or WHZ < -3 were included in the analysis. Overall, children had a median age of 15 months (IQR: 10–22 months), and there was a nearly equal proportion of girls and boys. The distribution of children across anthropometric groups was as follows: 929 (39%) children with MUAC < 115 mm only, 530 (22%) children with WHZ < -3 only, and 940 (39%) children with both MUAC < 115 mm and WHZ < -3. One (<0.1%) child was confirmed to be HIV-positive.Table 1). Children with MUAC < 115 mm were more likely to be younger, female (57% vs. 25%) and stunted (81% vs. 73%), compared to children with MUAC ≥ 115 mm and WHZ < -3. Clinical characteristics on admission and program outcomes did not significantly differ between groups. The prevalence of bacterial infection was low in this population and did not differ between groups. Immediate response to treatment, as evidenced by the rate of weight gain from admission to week 1 and week 2, was greater among children with MUAC ≥ 115 mm and WHZ < -3, compared to MUAC < 115 mm children though ultimately there was no difference in length of stay between groups. Results remained largely consistent with adjustment for age, sex and stunting.To describe potential differences among children who would be included in a MUAC < 115 mm program vs. those who are currently eligible but who would be excluded using MUAC < 115 mm alone for admission, the first comparison presents children with MUAC < 115 mm (n = 1869) vs. children who would be excluded from a MUAC-based program having MUAC ≥ 115 mm and WHZ < -3(n = 530) (Table 2). Children with MUAC < 115 mm and WHZ < -3 were older and male; both groups were similar in terms of clinical characteristics on admission, except for a greater prevalence of diarrhea (34% vs. 29%) and bacteremia (5% vs. 2%) among children with MUAC < 115 mm and WHZ < -3. Compared to children with MUAC < 115 mm and WHZ ≥ -3, children with both MUAC < 115 mm and WHZ < -3 were less likely to recover, particularly children < 24 months of age (71% vs. 53%), and were more likely to be transferred to inpatient care due to lack of weight gain or weight loss. Immediate weight gain was greater among children with both MUAC < 115 mm and WHZ < -3 compared to children with MUAC < 115 mm and WHZ ≥ -3, though length of stay was greater.To describe potential heterogeneity within children who would be included in a MUAC < 115 mm program, the second analysis compared children with both MUAC < 115 mm and WHZ < -3 (n = 940) vs. children with MUAC < 115 mm and WHZ ≥ -3 (n = 929)  is more likely to identify SAM children who are younger, female and stunted –21. It hWe noted significantly greater initial weight gain (e.g. at week 1 and week 2) among children with MUAC ≥ 115 mm and WHZ <- 3 children compared to children with MUAC < 115 mm and considered several potential mechanisms or explanations of this finding. First, greater initial weight gain may reflect a greater need for nutritional therapy among children with MUAC ≥ 115 mm and WHZ <- 3. The rate of weight gains observed in this group is not inconsistent with mean weight gain observed during recovery of severely malnourished children in inpatient settings  and earWith the hypothesis that differences among children selected by MUAC < 115 mm vs. MUAC ≥ 115 mm and WHZ <- 3 may further differ by age, sex and stunting, we investigated potential effect modification by these factors. A recent study from McDonald et al. importantly highlighted an increased risk of mortality with multiple anthropometric deficits , compared to single or no anthropometric deficits . The conPublished data on potential variability among children selected by different anthropometric criteria remains very limited to date. The most detailed study available was done in 1999–2002 among 8,190 children hospitalized in rural Kenya [Routine data collected from two Médecins Sans Frontières (MSF) therapeutic feeding programs have recently been analyzed to explore this subject. In Burkina Faso, Goossens et al. analyzed data from 24,792 children admitted to a therapeutic feeding program using MUAC ≤ 118 mm as the sole anthropometric admission criterion. This study showed greater weight gain associated with lower MUAC at admission . ProgramThis study is the first to date to compare the clinical and laboratory characteristics associated with the type of anthropometric diagnosis among a large population of children with uncomplicated SAM children admitted to an outpatient nutritional program. Characterization of individual vulnerability was informed by the reliable assessment of clinical and laboratory characteristics at admission, as well as through a careful follow-up over the course of the treatment. This study offers new information to extend current evidence on the potential implications of moving towards MUAC as the sole anthropometric admission criterion for therapeutic feeding.This study does, however, have some limitations. In particular, laboratory analyses did not include parasitological or virological exam or confirmation of malaria infection. The prevalence of bacteriological infection and mortality were low, limiting the interpretation of these results. Additionally, this analysis draws from data collected during a randomized trial among children presenting to a health facility with SAM and receiving treatment. The study population does not reflect an untreated or general population, or a nutritional program that draws its participants from community-based screening and referral. These data therefore do not allow a clear conclusion regarding the use of using MUAC-only based criterion among untreated children in the community.Therapeutic feeding programs must aim to target and treat malnourished children that are the most vulnerable and at the highest risk of death, but programs are often challenged to do so in highly resource-constrained settings. It was believed that a single MUAC-based anthropometric admission criterion, in addition to its operational advantages, would identify the most vulnerable children in need of treatment. Our study suggests that a single anthropometric admission criterion of MUAC < 115 mm identified younger and more female children but did not well differentiate children in terms of other measures of vulnerability. Children with MUAC < 115 mm and children with MUAC ≥ 115 mm and WHZ < -3 in rural Niger displayed similar characteristics in terms of their clinical and laboratory profile on admission. An upward adjustment of the MUAC threshold could yield a more sensitive criterion for admission to select children at high risk but would have important programmatic implications. For MUAC to be considered as a single anthropometric admission criterion for therapeutic feeding, experience with various MUAC-based thresholds and supporting evidence on the clinical characteristics and needs of children selected with each should be further evaluated in a broad range of contexts.S1 Trial Protocol(PDF)Click here for additional data file.


Plasmodium falciparum infection. It results in high mortality and post-recovery neuro-cognitive disorders in children, even after appropriate treatment with effective anti-parasitic drugs. While the complete landscape of the pathogenesis of cerebral malaria still remains to be elucidated, numerous innovative approaches have been developed in recent years in order to improve the early detection of this neurological syndrome and, subsequently, the clinical care of affected patients. In this review, we briefly summarize the current understanding of cerebral malaria pathogenesis, compile the array of new biomarkers and tools available for diagnosis and research, and describe the emerging therapeutic approaches to tackle this pathology effectively.Cerebral malaria is a severe neuropathological complication of  Plasmodium falciparum malaria continues to be the predominant infectious disease in tropical and sub-tropical countries, with an estimated global incidence of 207 million cases and 627,000 deaths reported in 2012  mice. Oxidative stress in the brain can be visualized in these animals after injection of luciferin, and an elevated bioluminescent signal was associated with the development of the pathology . The analysis revealed diffuse and heterogeneous reduction of metabolism in the cortex during the acute phase of infection  in the cerebral microvasculature is the hallmark of CM. In pediatric patients, retinal microvessels have been shown to sustain damage comparable to the ones occurring in the brain, making them an easily observable surrogate marker to assess the severity of cerebral pathology during CM .One major limitation of the EEG studies is the serial post-discharge follow-up assessments, which involve multiple patient visits to the hospital, or home-based visits by nurses. These are not always possible and often present a logistical hindrance to EEG studies in the field. However, a miniature version of the EEG equipment is now available as a portable headgear that can accommodate up to 32 electrodes and connects via Bluetooth technology to a small monitoring machine. The micro-EEG diagnostic accuracy of Biomarkers include tools and technologies that can facilitate the prediction, cause, diagnosis, progression, regression, or outcome of treatment of disease. For pathologies of the nervous system, there is a wide range of techniques used to gain information about the brain in both the healthy and diseased state. These involve measurements directly on biological media such as blood or cerebrospinal fluid (CSF) from the patients; or measurements via brain imaging, which do not involve direct sampling of tissue but measure changes in the composition or function of the nervous system. While biomarkers have been used to diagnose and prognosticate the progress and outcome of many chronic diseases, the field of malaria research only recently moved in the direction of actively identifying biomarkers that can accurately discriminate the severe forms of malaria, and in particular, CM. Such biomarkers, once identified, validated, and integrated into rapid diagnostic tests, could allow the accurate and early identification of CM patients and their subsequent referral to tertiary healthcare facilities for prompt intervention. Recent clinical studies have identified serological factors that have the potential of being biomarkers. Based on their function and stage(s) of usage, these can be classified as early screening and diagnosis biomarkers, as well as prognostic biomarkers Table .Plasmodium falciparum histidine rich protein 2 (PfHRP2) in the plasma samples has also been shown to be an accurate diagnostic tool to ascertain the parasite biomass in severe malaria patients, and allowed the distinction between severe and uncomplicated malaria  are submicron elements (100 nm–1 μM), which originate from extracellular vesiculation processes during host cell activation and/or apoptotic events. High MP numbers were shown to be significantly higher in the plasma of patients with CM compared to patients with uncomplicated malaria or severe anemia in several separate analyses , the recommended first-line therapy for infected patients, is now prevalent across mainland Southeast Asia . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models.Plasmodium berghei ANKA luciferase parasite.The cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation  was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units.The pLDH antigen was detected in all four murine This high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden. Malaria continues to be a leading cause of mortality in endemic countries, despite control and eradication initiatives, such as the use of insecticide-treated bed nets and anti-malaria drug treatment . ClinicaPlasmodium enzymes, are under development. The most promising antigens explored so far include: histidine rich protein-2 (HRP-2)  as the reference revealed the following per cent identity in different species of murine Plasmodium: 86 % identity with P. yoelii 17XNL [Accession ID XM_719008.1]; 85 % with Plasmodium chabaudi chabaudi [Accession ID XM_740087.1]; 85 % with P. berghei ANKA [Accession ID XM_674309.1]; and 83 % with Plasmodium vinckei [Accession ID XM_008624100.1]. The high degree of sequence similarity could potentially be exploited for use in diagnostics for rodent malaria parasites   and maintained under appropriate ARC and Griffith University conditions. This study was carried out in strict accordance with the National Health and Medical Research Council of Australia guidelines, as detailed in the document, , 8th ed . The GriP. chabaudi AS, P. yoelii 17XNL, P. yoelii YM, P. berghei and P. vinckei were used . Stabilates were maintained by intra-venous (IV) and intra-peritoneal (IP) passaging of 106 parasitized red blood cells (pRBC) into naïve BALB/c mice. Plasmodium berghei ANKA luc lines  were used in the bio-luminescent experiments for in vivo imaging after one in vivo passage in mice.Cloned lines of Thin blood smears were prepared, air dried, fixed in methanol and stained with Giemsa. Slides were examined using bright field microscopy and counts were performed with 100× magnification. For negative or low parasitaemia films (<1 %), at least 20 fields on the slide were counted. For high parasitaemia, at least 500 RBCs were counted. Percent parasitaemia was calculated as the number of pRBCs multiplied by 100 divided by the number of total RBCs.P. chabaudi AS, P. yoelii 17XNL, P. yoelii YM, P. berghei ANKA, and P. vinckei were determined using the pLDH SD Malaria Antigen ELISA kit (Standard Diagnostics), a sandwich ELISA for the qualitative detection of human Plasmodium pLDH. For quantitative assessment, blood was collected on the following days for each of the rodent malaria parasites: P. chabaudi AS: Day 7 (29 %), P. berghei ANKA: Day 4 (34 %), P. vinckei: Day 4 (32 %), and P. yoelii YM: Day 5 (24 %). Recombinant pLDH supplied as the positive control in the kit was used to generate a standard curve dilution series with a starting concentration of 1320 ng/mL in PBS or naïve mouse blood. Fifty microlitres of the samples  were tested in triplicate and the manufacturer’s instructions were followed. Plates were read on an xMark microplate spectrophotometer (Biorad) at 450 nm, with a reference wavelength of 620 nm. The reading was completed within 30 min of the stop solution being added. The linear range was then logarithmically plotted to generate a linear equation to quantify levels of pLDH units in test samples. Positive wells were used to measure the concentration of pLDH unit, with the cut-off for positivity calculated as the mean + three standard deviations of the wells containing naïve mouse blood or PBS alone. The mean optical densities (ODs) were calculated for standards and used to plot a standard curve OD vs ng/mL recombinant pLDH antigen. The concentration of pLDH units in the samples was calculated from the optical density reading from the linear range of the standard curve  video camera of the in vivo Imaging System  [d-luciferin dissolved in PBS  and measurements were performed within 5 min of injection. Bio-luminescence imaging was acquired with an exposure time of 60 s. Imaging data were analysed using the living image (Xenogen) program. In asynchronous infections, measurements were performed on different days at the same time.Luciferase activity in Xenogen) . Mice wer value greater than 0.6 was considered as a strong correlation and shown in the appropriate figures. P values less than 0.05 were deemed significant for all statistical analyses.Graphical and statistical analysis was performed using GraphPad Prism 6. All the datasets are presented as mean ± SE, unless stated otherwise. Non-linear standard curves were generated and the linear part of the curve was used for re-plotting three or more points in logarithmic scale. Logarithmic and arithmetic curves were both used for fitting a trend-line and obtaining an equation, which was used to calculate the concentration of pLDH units from the mean absorbance, for each test sample as described above. The limit of detection (LOD) was calculated as the lowest value in the dilution series that was significantly different from the negative control values. An ANOVA was performed to estimate the LOD. One-way ANOVA was used for the analyses presented in Figs. Plasmodium LDH genes [Plasmodium pLDH ELISA kit to assess its ability to detect murine Plasmodium pLDH. In a pilot study, pLDH was detected in both whole blood and serum obtained from a P. chabaudi AS infected mouse on day 7 post infection at 31 % parasitaemia  , did not have significant detectable pLDH units  from the commercial ELISA kit, serial dilutions were performed in both PBS and naïve mouse blood to establish standard curves. A representative standard curve from which the linear range was derived is shown in Fig. Plasmodium species  were established by serially diluting whole parasitized blood in naïve mouse blood. The LOD was approximately 0.7 % parasitaemia for P. chabaudi AS and 0.05 % for P. berghei ANKA  were infected and divided into two cohorts. Blood from each cohort was collected on alternate days. A strong correlation (r = 0.9889) was observed  was observed between parasitaemia and pLDH units  was observed between the levels of pLDH units and log average radiance  were infected with tor see “” ; therefore, pLDH ‘units’ rather than amount of pLDH per se has been referred to when assessing rodent parasites, with one unit being defined as the OD equivalent of 1 ng of Pf LDH. pLDH units were detected at lower levels in P. vinckei-infected blood compared to mice infected with other parasites. The assay was more sensitive for P. berghei ANKA, which could be due to less sequence variation in the region of the antibody-binding site compared to other parasites. However, as the binding site of the antibody is currently unknown, this could not be confirmed.This study investigated the use of a commercial human malaria ELISA kit to detect levels of pLDH units in the blood and serum of infected mice. Some differences are observed between P. berghei peripheral parasitaemia and pLDH levels was not detected, which may be due to the presence of sequestered parasites as shown in the bio-luminescent images (Fig. P. chabaudi peripheral parasitaemia and pLDH levels (Fig. This study compared the levels of pLDH units to an established bio-burden assay with luciferase transgenic parasites , 13 to eges Fig. . The strels Fig. c likely Plasmodium. This assay permitted the estimation of pLDH in different components of infected blood, thus, accounting for both circulating and sequestered parasites. This is confirmed by the strong correlation between the bio-luminescence and pLDH units during the initial parasite growth. Therefore, this assay provides an accurate assessment of parasite bio-burden in rodent malaria species. Furthermore, these results suggest that the pLDH assay can provide a valid estimate of total parasite bio-burden in malaria-infected patients.This study demonstrates the use of an established human malaria pLDH kit for detecting murine malaria parasite bio-burden. pLDH was detected in four strains of murine