Plasmodium falciparum clinical malaria episodes is over-dispersed among children in endemic areas, with more children experiencing multiple clinical episodes than would be expected based on a Poisson distribution. There is consistent evidence for micro-epidemiological variation in exposure to P. falciparum. The aim of the current study was to identify children with excess malaria episodes after controlling for malaria exposure.The distribution of We selected the model that best fit the data out of the models examined and included the following covariates: age, a weighted local prevalence of infection as an index of exposure, and calendar time to predict episodes of malaria on active surveillance malaria data from 2,463 children of under 15 years of age followed for between 5 and 15 years each. Using parameters from the zero-inflated negative binomial model which best fitted our data, we ran 100 simulations of the model based on our population to determine the variation that might be seen due to chance.th percentile of the simulations run from the model, hereafter referred to as “excess malaria (EM)”. We then identified exposure-matched controls with “average numbers of malaria” episodes, and found that the EM group had higher parasite densities when asymptomatically infected or during clinical malaria, and were less likely to be of haemoglobin AS genotype.We identified 212 out of 2,463 children who had a number of clinical episodes above the 95Of the models tested, the negative zero-inflated negative binomial distribution with exposure, calendar year, and age acting as independent predictors, fitted the distribution of clinical malaria the best. Despite accounting for these factors, a group of children suffer excess malaria episodes beyond those predicted by the model. An epidemiological framework for identifying these children will allow us to study factors that may explain excess malaria episodes.The online version of this article (doi:10.1186/s12916-015-0422-4) contains supplementary material, which is available to authorized users. Malaria remains a major public health problem with approximately 60 % of the world’s population at risk of infection . The curPlasmodium falciparum malaria. These include the sickle cell trait , thalasaemias, and blood group .Previously, we described a group of children who suffer multiple episodes of clinical malaria in Kenya . In the Approval for human participation in these cohort studies was given by Kenya Medical Research Institute Ethics Research Committee, and research was conducted according to the principles of the Declaration of Helsinki, which included the administration of informed consenting in the participant’s local language.P. falciparum parasite prevalence from cross-sectional surveys at 30 % during January to May , P = 0.0001; Fig. P = 0.0001; Fig. P. falciparum positive blood smears at pre-transmission cross-sectional surveys  vs 0.16 , P = 0.2, for EM and AM, respectively; Fig. Compared to AM, the EM children had 1) a higher geometric mean parasitaemia density during asymptomatic infections determined from cross-sectional surveys of blood smears in the dry season when  a higherBoth EM and the matched AM children were frequently found in the same geographical locations, further supporting the idea that having excess malaria is not always explained by environmental factors like increased exposure to malaria in the microenvironment Fig. . For exaAll these epidemiological markers point to EM as a group of children, who, over a sustained period of time, fail to acquire the ability to control parasite growth. Together, our analyses suggest that the factors responsible for increased susceptibility in the EM group are host dependent and not environmental, with sickle-cell trait being an obvious example tested.We compared the outputs of classification of EM based on the zero-inflated binomial regression model and the output of a simple cut-off, varying the cut-off from >5 to >10 clinical episodes irrespective of exposure and other variables (Additional file P. falciparum, similar levels of bed net usage, and similar ages but with some having suffered excessive numbers of clinical episodes. We also found EM and AM children frequently living close to each other or even sharing the same homestead.Previous studies demonstrated heterogeneous distributions of the numbers of accumulated malaria episodes for individuals, even amongst children of similar ages living in the same villages , 5, 23. Furthermore, EM children had increased parasite densities upon asymptomatic infections and during clinical malaria compared to AM controls, suggesting that they have reduced immunity to control malaria infections. Furthermore, the increased parasite densities and the higher proportion of EM children admitted to hospital with a diagnosis of malaria point to the possibility that clinical malaria in these children would have been more severe than malaria in AM children if left untreated, or with delayed treatments. In interpreting this result we should note that because the children in these cohorts are under active weekly surveillance for malaria, they receive more timely treatment for malaria upon diagnosis than the general population. Hence, we assume that the risk of hospital admission due to malaria would be higher in the general population.These findings are in agreement with our previous study which also described a group of children with increased susceptibility to clinical malaria in Kenya . In the A study in Senegal has also shown that asymptomatic parasite densities increased with age among a group of children with multiple episodes of malaria . In contIn the current study, we carefully controlled for variations in exposure, including use of EI, to predict the risk of a zero count, representing a sub-population of children with no exposure during a year of follow-up . HoweverP. falciparum transmission. We included the possibility of interactions between calendar time and site, and between calendar time and age, and discarded these because they were not significant.An additional finding was the increased risk of clinical malaria with calendar year between 2005 and 2013. This is consistent with wider trends in malaria transmission described on the Kenyan Coast , and couWe might expect that children of the same age and living with the same amount of exposure to malaria or the in the same geographical location would be similarly susceptible to clinical malaria. However, this study indicates that there may be a skewed distribution, with some children experiencing excessive numbers of episodes, perhaps indicating that these children fail to acquire natural immunity to malaria. However, there may be other inter-individual variations in exposure or behaviour patterns between children or their parents that are poorly accounted for by the model and could also explain the skewed distribution of children with excess episodes. Further studies of immunological parameters will be required to provide further evidence for the hypothesis that host immunity contributes to the variation observed. Furthermore, such studies will also reveal biomarkers for identifying such children with EM, which could in theory be used for targeting interventions to a small group of children that are responsible for a disproportionate amount of malaria-driven morbidity and mortality. Moreover, if these children represent the 20 % predicted to be responsible for the majority of malaria transmission , targeti


Early diagnosis is important in preventing mortality from malaria. The hypothesis that guardians’ fear of covert human immunodeficiency virus (HIV) testing delays presentation of children with suspected malaria was tested.The study design is a cross-sectional survey. The study population consisted of guardians of children with suspected malaria who presented to health centres in Oromia Region, Ethiopia. Data were collected on attitudes to HIV testing and the duration of children’s symptoms using interview administered questionnaires.Some 830 individuals provided data representing a response rate of 99% of eligible participants. Of these, 423 (51%) guardians perceived that HIV testing was routinely done on blood donated for malaria diagnosis, and 353 (43%) were aware of community members who delayed seeking medical advice because of these concerns. Children whose guardians suspected that blood was covertly tested for HIV had longer median delay to presentation for evaluation at health centres compared to those children whose guardians did not hold this belief . Children whose guardians were concerned about covert HIV testing were at a higher odds of a prolonged delay before being seen at a health centre .Children whose guardians believed that covert testing for HIV was routine clinical practice presented later for investigation of suspected malaria. This may account for up to 14% of the delay in presentation and represents a reversible risk factor for suboptimal management of malaria. Malaria constitutes an important treatable cause of global morbidity and mortality, with 3.4 billion people estimated to be at risk of infection in 2012, resulting in approximately 200 million cases of malarial infection and 600,000 deaths, predominantly in young children , althougThe prevalence of human immunodeficiency virus (HIV) infection in Ethiopia in 2011 was estimated to be 4.2% in urban areas and 0.6% in rural areas . There iDespite the requirement for informed consent before requesting an HIV test on any blood sample, there may be a perception that having a blood test for malaria is synonymous with being tested for HIV infection, as previously reported in qualitative studies elsewhere -16. ThisThe study design is a cross-sectional survey. This study was conducted in East Shewa Zone of Oromia Regional State in Ethiopia. The total population of the Zone in 2010/11, as projected based on the 2007 census, was 1,519,103 . The Zone is located in the Great Rift Valley of East Africa and malaria is a major health problem in the area. Five health centres, namely Modjo, Meki, Batu, Bulbula, and Shashemenne, were included in the study and data were collected in October and November 2012. All children under the age of 16 years who presented with symptoms consistent with malaria and gave finger-prick blood for microscopic examination at the health centres and their guardians constituted the study population. Data were collected opportunistically, when the guardian was available for interview. The study protocol was approved by the University of Nottingham Ethics Committee and the Addis Ababa University School of Public Health Research and Ethics Committee. All participants gave informed consent and received standard treatment for their presenting condition.Afan Oromo) was used for data collection  and ‘late’ (≤3 days) as used in previous studies of this topic ,17-19 anThe aim was to recruit 850 individuals. All children under the age of 16 years who presented at the health centre with their guardian and were clinically suspected of potentially having malaria (defined as having a blood film test requested) were eligible for inclusion in the study. The total sample size allocated to each health centre was proportional to the total patients tested for malaria in the previous 3 months  and individuals were recruited consecutively until the estimated sample size was achieved. Assuming that 20% of guardians had concerns about HIV testing in population and 50% of children have had symptoms for 5 days or less, it was estimated that the study would have over 90% power (5% risk of type I error) to detect an absolute increase of 20% in the guardians who had concerns about HIV testing in the children who experienced malaria symptoms for five days or more compared to those whose guardians had no such concerns.The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to the data in the study and had full responsibility for the decision to submit.A total of 836 guardians were invited to participate in the study and of these, 830 (99%) provided data for analysis. The demographics of the study participants are reported in Table Four-hundred and twenty-three (51%) individuals considered that HIV testing was applied to blood given for malaria testing at the health centre Table ; 353 . After adjustment for clustering by health centre, those children whose guardians were concerned about covert HIV testing had a higher odds of a prolonged delay before being seen at a health centre . The age and sex of the child, residence, age of the guardian, main material of the household’s roof, presence of electricity, time to travel to the health centre and health centre location were not confounding factors for this association between HIV beliefs and a delay in presentation with suspected malaria. This resulted in a population attributable fraction for the association of concerns about covert HIV testing on delayed presentation with malarial symptoms in children of 14% (95% CI: 3 to 23%).This is the first study to specifically assess the impact of concerns about HIV testing on the time to attendance at a health centre among children with suspected malaria. The data demonstrate that in this population, over half of guardians of children with suspected malaria considered that HIV testing was implemented on all blood donated for malaria testing and that 43% of respondents knew of others in their community who had delayed seeking medical care because of these concerns. These beliefs appear to impact not only on the individual, but their children as guardians who were concerned about HIV testing were more likely to bring their children for medical assessment later than those who had no such concerns.a priori hypothesis from a population of the guardians of children with suspected malaria, who had no knowledge of the hypothesis being tested. The response rate was high at 99%, giving confidence in the generalizability of the findings to this population and the minimization of bias, and data were collected on co-variates permitting adjustment for other demographic factors that may have been important.The study has a number of strengths including the prospective assessment of an The main limitation of these data is that while they may be generalizable to the community studied during the period of high malarial prevalence, these attitudes to HIV testing and accessing health care in general are not necessarily applicable to other communities and cultures or at other times of the year. Hence, further studies are required to determine how prevalent the belief that covert HIV testing may accompany testing for malaria actually is in the many countries with a high prevalence of HIV. A delay of over two days before obtaining medical assessment after symptoms of suspected malaria started was seen in 49% of the study population, and similar delays have been observed from other observational studies from Nigeria , SenegalThe prevalence of refusal for HIV testing among women attending for antenatal care in the Gambella Region of Ethiopia in 2008 is 25%, suggesting that in this population there was a sizeable minority who did not want to be aware of their HIV status . The datThe observation that concerns about HIV testing may delay malaria diagnosis in children is important for public health policy makers as firstly malaria is a common disease that infects approximately 200 million individuals annually , and thaThese data are consistent with the hypothesis that guardians’ concerns about covert HIV testing without informed consent is an independent risk factor for delayed presentation to health centres for children with suspected malaria. This is important, as it is a belief that is open to modification by appropriate intervention programmes, hence improving the early delivery of appropriate anti-malarial treatments to patients and hence improving clinical outcomes.The authors have declared that there are no competing interests.YH was involved in proposal writing, designed the study and participated in coordination, supervision and the overall implementation of the project, analysed the data, drafted and finalized the manuscript. WD, AWF and GD conceived the study and participated in all stages of the study and revision of the manuscript. AWF obtained funding for the study and checked the statistical analyses. All authors read and approved the final version of the manuscript.Study Questionnaire.Click here for fileStrobe Statement.Click here for file


Prompt treatment of malaria following adequate diagnosis helps to reduce morbidity and mortality in children. Lack of resources and adequate manpower in developing countries make malaria microscopy difficult. The Rapid Diagnostic Test kit (RDT) remains unpopular despite its availability and ease of use because of lack of local research on its effectiveness leading to over-prescription of Artemisinin Combination Therapy (ACT).A total of 132 children age range 1-9 years were screened for malaria routinely with rapid diagnostic test kits (RDTs HRP 2) and malaria microscopy at the general outpatient department of OAU Teaching Hospital as well as at a comprehensive health centre at Imesi Ile, South West Nigeria while marking the 2013 World Malaria Day. Needle prick was used to collect blood sample for thick and thin smear. Giemsa stain was used to prepare the slide for microscopic examination.Plasmodium falciparum. In the Microscopy category, 35 (26.5%) children tested positive while 97 (73.5%) tested negative for malaria parasite. Out of the 35 children tested positive, RDT picked 33 as positive and 2 as negative, (sensitivity = 94.3%). While out of the 97 that tested negative for microscopy, RDT picked 94 as negative and 3 as positive (specificity = 96.9%). The positive predictive value and negative predictive values are 91.7% and 97.9% respectively.A total of 132 children were recruited into the study. There were more females (59.1%) than males (40.9%). 35 (26.5%) children tested positive while 97 (73.5%) children tested negative for Rapid Diagnostic Test is an effective diagnostic tool for malaria amongst children in the study population. Primary and secondary health centres in the region should adopt Rapid Diagnostic Test in malaria diagnosis before administration of ACTs to avoid unnecessary treatment.


Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections.Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells.Apoptosis of nRBC was evaluated in blood samples of P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.Apoptosis of normal RBC can be induced by plasma from individuals with  It is currently known that the physiological processes of apoptotic cell death are not restricted to nucleated cells, occurring also in cells lacking a nucleus and organelles, such as the red blood cells (RBC) . SimilarP. yoelii 17XL [P. berghei ANKA [in vitro P. falciparum culture [P. yoelii infection, pointing to the putative role of erythrocytic apoptosis in the pathogenesis of malaria [In malaria, pRBC apoptosis was detected in experimental lii 17XL  and P. bhei ANKA  rodent m malaria . Since aP. vivax – the species that accounts annually for around 82% of malaria cases in Brazil [g for 10 min; plasma was stored in liquid nitrogen and RBC were used for apoptosis assays. Blood samples from 10 clinically healthy individuals living in the same area and presenting as negative by thick blood smear and no history of previous malaria episodes were used as controls. During the blood collection no cases of P. falciparum malaria or complicated P. vivax malaria attended the Tropical Medicine Foundation. The study was approved by the Tropical Medicine Foundation Ethical Committee.For this, the levels of nRBC apoptosis were examined in peripheral blood of 20 malarious patients infected by n Brazil ,10. PatiP. vivax patients (n = 11) presented parasitemia ranging from 501 to 10,000 parasites/μL. In two patients parasitemia was between 301 and 500 parasites/μL and in seven it was less than 300.Parasitemia was estimated by examination of thick blood smears using a semi-quantitative method, as described previously . As showex vivo and after RBC incubation for 24 h at 37°C at a hematocrit of 0.5% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, and 1 CaCl2 (pH 7.4). Apoptotic nRBC was detected through flow cytometry using Syto 16 and annexin V-PE double staining, as performed in our previous studies with P. yoelii 17XL [ex-vivo and after 24 h incubation, no significant alteration of nRBC apoptosis rates was detected in P. vivax patients  when compared with non-infected control individuals  . The same was true when P. vivax patients were grouped according to parasitemia density (data not shown).Apoptosis was assayed both lii 17XL . HoweverP. vivax. Although some reports have considered that P. vivax can induce severe manifestations, including acute anemia [P. falciparum, it has been believed that a simple infection by P. vivax is per se slightly competent to induce complications and that severe cases of vivax malaria are attributed to co-morbidities as well as high rates of P. vivax recurrence in areas of intense transmission as a result of drug resistance, relapse from hypnozoites or reinfection with heterologous strains [P. vivax is the predominant human malaria parasite and the intensity of transmission is low, severe malaria is rarely registered [This lack of erythrocytic apoptosis seems to be in consonance with the pathogenic profile of e anemia –14, it i strains ,16. In Bgistered ,10.P. vivax [P. falciparum malaria also from Manaus, whose parasitemias are shown in Table P. vivax malaria previously studied here. A pool of plasma was tested in parallel, it was obtained from BALB/c mice either at early (4 day) or at late (7 day) stages of lethal P. yoelii 17XL infection [+) from a clinically healthy individual living in a non-endemic malaria area. Normal RBC were incubated for 24 h and 48 h at 37°C in 5% CO2 in a 5% hematocrit with the plasma samples. Erythrocytic apoptosis was detected by flow cytometry using annexin V single staining, as previously described [Since the classically known lower pathogenic potential of P. vivax  could benfection , when meescribed .P. vivax malarious patients, plasma from these subjects did not show any proapoptotic effects on RBC from the clinically healthy individual . Similar to the plasma from P. falciparum patients, plasma from P. yoelii 17XL-infected BALB/c mice obtained at the stage of infection related to increased induction of nRBC apoptosis in vivo (late stage) [P. falciparum infection. However, it was not possible to statistically infer the relationship between parasite load and apoptotic levels during falciparum malaria, because the majority of P. falciparum patients presented similar parasitemia levels (Table Consistent with the data herein observed with nRBC from the  Figures  and 2, i Figures  and 2. OP. falciparum [in vitro adhesiveness of apoptotic RBC to endothelial cells [P. yoelli 17XL in BALB/c mice [P. falciparum patients does not usually reflect actual parasite load due to sequestration phenomenon of mature forms of the parasite [falciparum patients was higher and the induction of apoptosis could be related to parasite density. Moreover, the strong proinflammatory imbalance maintained during P. falciparum, but not during P. vivax infection [If the proapoptotic properties of plasma from malarious patients do reflect the aptness of malaria parasites to induce apoptosis of normal RBC, the observation reported here seems to match the different pathogenic potentials displayed between them. In this context, it would not be surprising that the most deadly human malaria parasite, lciparum , stimulaal cells ,20. SimiB/c mice , in whicB/c mice . In the parasite . Thus, anfection , could aP. falciparum patients was related to apoptosis (Figure Taking into account that labeling of PS by annexin V also occurs in non-apoptotic cells that suffered plasma membrane damage ,25, cells Figure .Figure 3P. falciparum can induce apoptosis of normal erythrocytes in vitro, pointing to the possibility that this phenomenon does exist in vivo. This property was not recorded when plasma from patients with malaria by a less pathogenic human malaria parasite, P. vivax was studied. Further ex vivo studies are required to confirm the existence of apoptosis of nRBC during P. falciparum malaria to better address its clinical significance in malaria pathogenesis.In conclusion, the present study showed that plasma from patients with malaria caused by


Plasmodium infections within asymptomatic carriers and that PCR assays are currently recognized as the most sensitive methods for Plasmodium identification, this study was conducted to weigh the asymptomatic carriage in DRC by a molecular method. Six provinces were randomly selected for blood collection in which 80 to 100 individuals were included in the study. Five hundred and eighty blood samples were collected and molecular diagnosis was performed. Globally, almost half of the samples collected from asymptomatic individuals  had Plasmodium infections and the most species identified was P. falciparum alone in combination with P. malariae. The high prevalence reported here should interpellate the bodies involved in malaria control in DR Congo to take into account asymptomatic carriers in actions taken and consider asymptomatic malaria as a major hurdle for malaria elimination. Malaria remains a major public health problem in the Democratic Republic of Congo (DRC) with 14 million cases reported by the WHO Malaria Report in 2014. Asymptomatic malaria cases are known to be prevalent in endemic areas and are generally untreated, resulting in a significant source of gametocytes that may serve as reservoir of disease transmission. Considering that microscopy certainly underestimates the prevalence of Plasmodium species transmitted to man by a mosquito bite [ Plasmodium species infect human but Plasmodium falciparum is responsible for the major morbimortality. Infection with P. falciparum can result in a simply asymptomatic carriage, uncomplicated or severe malaria. In fact, there is an exposure-related immunity in malaria that could explain these different expressions [Malaria is a parasitic disease caused byito bite . Four Plressions .In high transmission settings, symptomatic malaria is often concerning children below five years as they have not been for long time exposed to the parasite and asymptomatic infections generally concern adults that acquired an antidisease and/or an antiparasite immunity during their exposure time –6. Asymp Plasmodium carriers. Matangila et al. reported a prevalence of 21.6%, 27.4%, and 29.5% of asymptomatic P. falciparum infection in pregnant women in Kinshasa, respectively, by microscopy, Rapid Diagnostic Tests (RDTs), and PCR [ Plasmodium infection in asymptomatic children of 30.9% and 14.3% in two areas of Kinshasa, based on microscopic identification of thick blood smears [ Plasmodium infection, in asymptomatic children less than five years, of 23% and 31%, respectively, determined by microscopy and RDT [ P. falciparum [The Democratic Republic of the Congo (DRC) is the second largest country in Africa and has a population that is estimated to be 75.5 million people. It is estimated that 97% of the population live in zones with stable transmission. In the WHO malaria report for 2014, 14 million of malaria cases were reported in DRC . Some st and PCR . Maketa d smears . The moslciparum . Plasmodium DNA [ Plasmodium [μL as the technique validated by Cnops et al. [Currently, Polymerase Chain Reaction (PCR) assays are the most sensitive manner to detectdium DNA , 17 and asmodium . Some PCs et al. . Plasmodium carriage. For example, Baliraine et al. reported 20.7% difference (12.6% by microscopy and 33.3% by PCR) [Discrepancies have been often found between microscopy and PCR results for the determination of asymptomatic by PCR) , and May by PCR) . P. falciparum in the DRC, based on a highly sensitive RT-PCR assay for malaria parasite identification.We conducted this study to assess the weight of asymptomatic carriage ofWe randomly identified health areas within 6 provinces of the DRC with different malaria transmission dynamics: Bolenge in Equateur, Luzizila in Kinshasa, Mweka in Kasai-Occidental, Butembo in Nord-Kivu, Punia in Maniema, and Kapolowe in Katanga Province. This household survey recruited one hundred individuals per province, except for Maniema where only 80 individuals could be included. Every age and sex was taken into account after being given an informed consent by adults and parents/guardians of children. People with fever or other malaria related symptoms were not included in this study. Samples were collected during the period from March to November 2014. Plasmodium DNA has been extracted using the QIAamp DNA Mini Kit®  according to manufacturer's recommendation. Briefly, 3 circles of approximately 3 mm diameter were punched out from a blood spot and placed into a 1.5 mL microcentrifuge tube in which 180 μL of buffer ATL was added. The final elution volume was 150 µL. Each dried blood spot was treated individually into a sterile petri dish to avoid contamination. One negative control (sterile water) was included for each dozen of blood spots treated. DNA was stored at −20°C till further analysis.Five hundred and eighty blood samples were obtained from finger prick and dropped on filter paper (Whatman 3MM®) that were dried and stored into individual plastic bags with desiccant.μL for human Plasmodium species identification was run, as previously described by Cnops et al. [A real-time PCR having a sensitivity varying from 0.02 to 0.006 parasites/s et al. . PCR tesThis study has received the ethical approbation of the Ministry of Public Health of the DRC and of the Institutional Committee of the Faculty of Medicine, University of Kinshasa.P. falciparum was correctly identified in 280 samples (48.2%) among which 6 infections were mixed . Prevalence of positive samples by age groups is represented in  Plasmodium species P. ovale and P. vivax were not found. The real-time PCR cycles thresholds (Ct) vary from 21.72 to 39.23. P. falciparum infection by province was 51% for Equateur, 62% for Kinshasa, 31% for Kasai-Occidental, 22% for Nord-Kivu, 63.7% for Maniema, and 63% for Katanga, as shown in Prevalence of Plasmodium carriers because elimination of parasites in only symptomatic patients will not be enough as long as the pool of asymptomatic carriers will continue to act as a parasite reservoir [Efforts to control or to eradicate malaria should take into account asymptomaticeservoir . Some reeservoir , 25. Plasmodium infections in asymptomatic carriers found in this study stressed the importance of including this item in malaria control programs by the DRC Ministry of Health.The high prevalence of


Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with protection from severe malaria, and potentially uncomplicated malaria phenotypes. It has been documented that G6PD deficiency in sub-Saharan Africa is due to the 202A/376G G6PD A-allele, and association studies have used genotyping as a convenient technique for epidemiological studies. However, recent studies have shown discrepancies in G6PD202/376 associations with severe malaria. There is evidence to suggest that other G6PD deficiency alleles may be common in some regions of West Africa, and that allelic heterogeneity could explain these discrepancies.Plasmodium falciparum malaria. Fifty-three G6PD polymorphisms, including 202/376, were genotyped across the 712 samples. Evidence of association of these G6PD polymorphisms and mild malaria was assessed in both ethnic groups using genotypic and haplotypic statistical tests.A cross-sectional epidemiological study of malaria susceptibility was conducted during 2006 and 2007 in the Sahel meso-endemic malaria zone of Mali. The study included Dogon (n = 375) and Fulani (n = 337) sympatric ethnic groups, where the latter group is characterized by lower susceptibility to vs Dogon 0.0%). There are differences in haplotype frequencies between Dogon and Fulani, and association analysis did not reveal strong evidence of protective G6PD genetic effects against uncomplicated malaria in both ethnic groups and gender. However, there was some evidence of increased risk of mild malaria in Dogon with the 202A mutation, attaining borderline statistical significance in females. The rs915942 polymorphism was found to be associated with asymptomatic malaria in Dogon females, and the rs61042368 polymorphism was associated with clinical malaria in Fulani males.It was confirmed that the Fulani are less susceptible to malaria, and the 202A mutation is rare in this group (< 1% versus Dogon 7.9%). The Betica-Selma 968C/376G (~11% enzymatic activity) was more common in Fulani (6.1% The results highlight the need to consider markers in addition to G6PD202 in studies of deficiency. Further, large genetic epidemiological studies of multi-ethnic groups in West Africa across a spectrum of malaria severity phenotypes are required to establish who receives protection from G6PD deficiency. The haplotype containing 968C (GAC[C]GGGGGCAC) in Fulani had non-significant protective effects across all phenotypes, including clinical malaria . For the immunological titres, there were few SNP associations , all in females. Haplotype analysis did not reinforce these effects.In light of potential associations amongst SNPs, haplotype association tests were performed using the blocks defined above  G6PD SNPs, including the established G6PD202, 376, and 968 markers, allele frequencies between the Dogon and Fulani ethnic groups were compared, and tests of association with mild malaria phenotypes performed. There were differences in haplotype frequencies between the ethnic groups, and association analysis did not reveal strong evidence of protective G6PD genetic effects against uncomplicated malaria in both ethnic groups and gender. However, the rs915942 polymorphism (and rs915941 in high LD) was found to be associated with asymptomatic malaria in Dogon females, not males. Similarly, the rs61042368 polymorphism  was associated with clinical outcomes in Fulani males, not females. Whilst one may expect a dose effect that is greater for males than in females, differential protective G6PD polymorphism effects between genders have been observed in studies of severe malaria, and the mechanisms are not fully understood. ThDifferences in malaria phenotypes and immunological titres between Dogon and Fulani ethnic groups have been observed, adding to the growing evidence that the Fulani in West Africa have reduced malaria susceptibility. It is thought that host genetics may play a part, and the role of polymorphism in the G6PD gene was investigated. G6PD A- deficiency is known to associate with reduced risk of severe malaria, and the 202A polymorphism has been used as a genotypable surrogate. Whilst there is strong evidence that G6PD A- deficiency is protective against severe malaria, the effect on mild forms of disease has not been demonstrated conclusively. The results indicate there is no strong evidence of protection to malaria from 202A or 968C mutations in either ethnic group, across gender. This could be explained by the limitations of the sample size, the absence of severe malaria cases in this study, or the lack of sensitivity of genotyping compared to enzymatic assays. HoweverObserved genetic differences in the G6PD gene between Fulani and Dogon ethnic groups reinforce the need to consider markers in addition to G6PD202 in studies of deficiency. The SNPs considered in this study provide a starting point for large-scale genetic epidemiological studies of deficiency across Africa. These studies should involve a broad range of ethnic groups, including Fulani, as well as both uncomplicated and severe malaria cases, so that it is possible to establish who receives protection from G6PD deficiency.LD: Linkage disequilibrium; SNP: Single nucleotide polymorphism; G6PD: Glucose-6-phosphate dehydrogenase; PQ: Primaquine.The authors declare that they have no competing interests.BM, AD; KAR, MT-B, OKD and TGC conceived and designed the study; BM and AD performed recruitment of study subjects; BM, SC, PC, KAR and MalariaGEN coordinated the laboratory experiments; BM, NS and TGC performed the statistical analysis: BM, NS, OKD and TGC wrote the manuscript. The final manuscript was read and approved by all authors.All haplotype-based association analyses for the clinical and immunological outcomes, as well as odds ratios for the clinical outcomes.Click here for fileAll SNP-based association analyses for the clinical and immunological outcomes.Click here for file


Plasmodium vivax is a human malaria parasite responsible for significant morbidity worldwide and potentially death. This parasite possesses formidable liver-stage biology that involves the formation of dormant parasites known as hypnozoites. Hypnozoites are capable of activating weeks, months, or years after a primary blood-stage infection causing relapsing bouts of illness. Elimination of this dormant parasitic reservoir will be critical for global malaria eradication. Although hypnozoites were first discovered in 1982, few advancements have been made to understand their composition and biology. Until recently, in vitro models did not exist to study these forms and studying them from human ex vivo samples was virtually impossible. Today, non-human primate (NHP) models and modern systems biology approaches are poised as tools to enable the in-depth study of P. vivax liver-stage biology, including hypnozoites and relapses. NHP liver-stage model systems for P. vivax and the related simian malaria species P. cynomolgi are discussed along with perspectives regarding metabolite biomarker discovery, putative roles of extracellular vesicles, and relapse immunobiology. Plasmodium, which have a complex life-cycle involving a vertebrate and invertebrate host. After infecting susceptible mammals, the parasite undergoes obligate, clinically silent development in the liver prior to entering the blood and causing the clinical symptoms and pathology associated with malaria. Neutralizing the parasites in the liver has been a goal to prevent blood-stage infection and, therefore, clinical disease and transmission. Indeed, targeting liver stage forms (LSFs) has been a strong theme in current anti-malarial drug and vaccine efforts  on these topics will undoubtedly include large-scale ‘omics,’ advanced immune profiling, mathematical modeling, computational biology, and the integration of clinical and ‘omics’ datasets .While research using NHPs will inevitably remain limited worldwide within a few capable biomedical research centers, such investigations with the public release of datasets will enable many more investigators to participate in associated areas of research and development. Moreover, collaborations with investigators at these centers can lead to new research directions including much needed translational studies to improve diagnostics and clinical care and to develop and test new anti-malarial interventions and vaccine candidates. From our perspective, these factors make NHP model systems critical to advancing the world toward malaria eradication.Plasmodium biology, malaria pathogenesis, or preclinical investigations pertinent to developing new interventions (Macaca mulatta) experimentally infected with P. cynomolgi . Different strains of P. cynomolgi and P. vivax that possess distinctive relapse patterns can be utilized to study the consequences of frequent versus infrequent relapses on the host immune system. A suitable mouse model is not currently available to study such phenomena. Indeed, humanized mice containing human hepatocytes have been demonstrated to support P. falciparum liver-stage growth . In contrast to relapses, recrudescences are the result of untreated or persistent blood-stage infections that become sub-patent, below the level of detection by microscopy, followed by an eventual return to patency; such recurring parasitemias are distinct from relapse parasitemias that are due to the activation of hypnozoites and release of a new brood of merozoites from the liver.To investigate hypnozoites and relapses, Table 1; . SimilarTable 1).Blood-stage parasitemias, which begin to develop within 8–10 days, can be curatively treated without destroying the hypnozoites. PCR testing can confirm the absence of blood-stage parasites, and thus, any subsequent blood-stage infections can be confirmed as relapses and not recrudescences. This experimental strategy is currently the only reliable means to study vivax relapses, with the caveats that these animals are small , parasitemia is typically low or moderate (1–2%), and only small blood volumes can be taken (6 ml/kg/month) based on Institutional Animal Care and Use Committee (IACUC) guidelines. One strain in particular (named the Brazil VII strain) is being developed at the Centers for Disease Control and Prevention (CDC) for studying relapses as it shows multiple relapse patterns over a period of several months similar to that observed previously in humans with “tropical strains” (M. mulatta) and long-tailed macaques (M. fascicularis), which possess similar genetic composition and physiology to humans . Two other simian malaria species that could be useful for studying hypnozoites or relapse mechanisms are P. fieldi and, especially, P. simiovale (Table 1).hrocytes . Tens ofgenerate . FurtherP. vivax, many P. cynomolgi isolates exist that have their own infection and relapse characteristics (Table 1). The number and frequency of relapses in rhesus infected with different strains of P. cynomolgi can be predicted with greater accuracy than typically possible with New World monkey-adapted P. vivax isolates although P. vivax Brazil VII has so far been uniquely dependable in this regard (unpublished data). In rhesus, variables such as the sporozoite inoculum can be altered to produce consistent infection patterns even though inter-individual variability between animals as well as inherent properties of the strain being used in the study can influence infection kinetics  gene  are a heterogeneous population of small vesicles found in virtually all bodily fluids including serum, plasma, urine, saliva, etc., and are categorized into subtypes based on their physical properties such as density, size, and shape as well as their biogenesis . These vPlasmodium liver-stage biology, but warrant attention. Indeed, EVs were recently shown to be released from Plasmodium infected erythrocytes and implicated in a “density-dependent sensing mechanism” that influences P. falciparum gametocytogenesis , EVs are currently being explored to develop sensitive methods to detect cancer in bodily fluids  during malaria is poorly understood although “immune exhaustion” during chronic infections has been investigated recently . Currently, the team is performing follow-up experiments to better understand this phenomenon and its immunological relevance using sampling strategies that are not possible in humans. Blood is being collected before, during, and after relapses to monitor the alterations in the B-cell compartment by flow cytometry, with a special interest in memory B cells. The identities of predominant B-cell clones based on immunoglobulin sequences are being examined using Ig-Seq technologies (The team of immunologists at the MaHPIC has been using NHP models to understand the effects of relapses on the host immune system. Indeed, the consortium has determined that relapses cause continuous expansion of the circulating memory B-cell pool using the nologies . The firPlasmodium life-cycle. Primaquine is the only FDA-approved drug against hypnozoites despite its contraindications. Additionally, excessive use of this drug can support the rise of primaquine resistant parasites (Non-human primate models of malaria have enabled major contributions toward understanding liver-stage biology, hypnozoites, and relapses, and will continue to provide the means to investigate this enigmatic part of the arasites . A biomaAll authors contributed to the writing, figure and table, and reviewed and approved the finalized article.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.


Malaria among school children is increasingly receiving attention, yet the burden of malaria in this age group is poorly defined. This study presents data on malaria morbidity among school children in Bungoma county, western Kenya.Plasmodium falciparum infection, clinical malaria, and anaemia among 2346 school children aged 5–15 years, who were enrolled in an individually randomized trial evaluating the effect of anthelmintic treatment on the risks of malaria. At baseline, children were assessed for anaemia and nutritional status and information on household characteristics was collected. Children were followed-up for 13 months to assess the incidence of clinical malaria by active detection, and P. falciparum infection and density evaluated using repeated cross-sectional surveys over 15 months.This study investigated the burden and risk factors of P. falciparum infection was 42 % and ranged between 32 and 48 % during the five cross-sectional surveys. Plasmodium falciparum prevalence was significantly higher among boys than girls. The overall incidence of clinical malaria was 0.26 episodes per person year  and was significantly higher among girls . Both infection prevalence and clinical disease varied by season. In multivariable analysis, P. falciparum infection was associated with being male, lower socioeconomic status and stunting. The risk of clinical malaria was associated with being female.On average prevalence of P. falciparum parasitaemia, clinical malaria and anaemia among school children is not insignificant, and suggest that malaria control programmes should be expanded to include this age group.These findings show that the burden of The online version of this article (doi:10.1186/s12936-016-1176-y) contains supplementary material, which is available to authorized users. Significant progress in malaria control has been realized in the last decade with a number of countries, including Kenya, reporting decline in malaria transmission and hospital admission , 2. The Malaria prevention typically targets the highest risk groups; pregnant women and children under five who are most affected by the severe forms of the disease. However, there is increasing evidence suggesting that school-aged children 5–15 years) bear the highest burden of asymptomatic malaria irrespective of the transmission setting with a prevalence range between 14 and 64 % –14, and  years beBoth asymptomatic and clinical malaria have been shown to have a negative impact on the health and cognitive development of school children –22. AlthPlasmodium falciparum infection and clinical malaria among school children living in an area of high malaria transmission in western Kenya. Specifically, this study investigated the burden of P. falciparum infection, clinical malaria, and anaemia, as well as associated risk factors.In order to adapt malaria control strategies to changes in transmission patterns, there is urgent need for data on asymptomatic and clinical malaria among different age groups. The present study aimed to describe patterns of P. falciparum. A school survey conducted in 2009 reported that the prevalence of P. falciparum among school children in western Kenya was 21.6 % and with only 19.0 % of school children sleeping under a bed net the previous night [The study presents secondary analysis of longitudinal data arising from an individually randomized clinical trial evaluating the impact of repeated (every 4 months) anthelmintic treatment with albendazole on clinical malaria and malaria parasitaemia among school children . The trius night . The mosus night . There wus night .P. falciparum infection, while parasite density was not significantly different between the two groups  in accordance with national guidelines. No cases of complicated malaria were encountered.Prior to the trial, a baseline household survey was conducted to collect information on socioeconomic characteristics, and children’s use of malaria prevention measures. During the baseline health survey, haemoglobin concentration was assessed from a finger prick blood sample using a HemoCue haemoglobin photometer , weight and height were recorded. Follow-up cross-sectional surveys at schools were conducted at 3, 7, 11, and 15 months . Estimates of land surface temperature (LST), enhanced vegetation index (EVI), elevation and normalized difference vegetation index (NDVI) were extracted for each school after averaging the values of covariates within a 1-km catchment area around each school and household. Permanent water bodies were identified through intersecting gridded surfaces of the normalized difference water index (NDWI) for rainy and dry season. Those water areas which remain throughout the year were considered potential permanent breeding sites for mosquitoes. Straight-line distance (Euclidean distance) from schools to the nearest permanent body was calculated. A detailed description on how the data was obtained and extracted is provided in an additional file enclosed , the London School of Hygiene and Tropical Medicine (LSHTM) Ethics Committee (6210), and the Makerere School of Public Health, Institutional Review Board (IRB00005876). The study was registered with the Clinical trial.gov NCT01658774.P. falciparum infection at any density irrespective of presence fever as diagnosed by expert microscopy, (2) P. falciparum parasite density categorized in two groups: light (1–999 parasites/μL), and heavy (≥1000 parasites/μL), and (3) clinical malaria, defined as the presence of asexual P. falciparum parasitaemia as determined by microscopy plus either an axillary temperature >37.5 °C or a reported history of fever during the preceding 24 h. Children were considered at risk from their date of entry into the study until completing follow-up at 13 months. Children with documented or reported clinical malaria or known to have received medical attention from any source other than the survey team were censored for 28 days and those children who were absent from school for ≥10 days were censored for the time of absence [P. falciparum infection and clinical malaria were investigated by season: wet season (May–August and November–December) and dry season (January–April and September–October).Three malariometric outcomes were considered; (1) malaria parasitaemia defined as  absence . Anaemia absence  with a mnever (reported none use), sometimes (reported use at least once), and always (reported use at each survey) based on bed net use information collected during the five cross-sectional surveys.Principal component analysis was used to construct a household wealth index based on information on wall, floor, and roof construction materials, source of fuel, and education level of household head. The wealth index was then divided into two groups (termed poor and less poor) based on the median. Z-scores of height-for-age (HAZ), weight-for-age (WAZ) and body mass index for age (BMIZ) were calculated using the AnthroPlus software that uses the new 2006 WHO growth Ref. . ChildreP. falciparum infection and anaemia together with their 95 % confidence interval (CI) was calculated using binomial regression analysis, adjusted for clustering by school. To allow for over dispersion in the distribution of parasites, arithmetic mean parasite counts with their 95 % CIs were estimated using a negative binomial regression model taking into account school clustering. Estimates of prevalence and density of P. falciparum infection were calculated by sex and survey. Incidence rates were calculated as number of events divided by person years at risk using survival analysis, and were estimated for each calendar month, stratified by sex and age group. Survival analysis for clinical malaria was presented using Kaplan–Meier curves. Prevalence rate ratios  and incidence rate ratios were calculated adjusting for clustering in schools. Most of the infections were of low parasite density and preliminary analysis indicated little variation and therefore no further analysis was undertaken of this outcome.Data were analysed using Stata version 13 . Summary statistics were calculated for all baseline data. Proportion of children with P. falciparum infection were identified using mixed effects logistic regression modelling, where P. falciparum infection status was treated as repeated measures (child random intercept), with additional random intercepts to adjust for clustering of children within schools and households. As most households had a single child, the study area was divided into 191 hexagons of 1 km diameter to which households were assigned based on their geographic location (1–66 children per hexagon). Hexagon defined clusters were favored over actual household clusters due to model convergence. All univariable and multivariable models for P. falciparum infection included a fixed term for survey round; this adjusted for the number of times a child contributed to the analysis, even if there was missing data at a particular follow-up survey. Univariable analysis was first performed including one covariate at a time and significant variables  were included in multivariable analysis. Parsimonious regression models were developed using a backwards variable selection approach, eliminating one variable at a time based on the highest P value and retaining only variables with P ≤ 0.05.Risk factors of P. falciparum infection and anaemia was however subsequently tested adjusting for variables included in the minimum model. To investigate any potential effect of previous P. falciparum infections on incidence of clinical malaria, the final model was further tested including the number of P. falciparum infections experienced by a child as a fixed term.Risk factors for incidence of clinical malaria were investigated using mixed effects Poisson regression. The models included a random intercept for children, however they failed to converge when clustering at both school and household level was considered. Hence models were adjusted only for clustering at household, as this is where infection is more likely to occur. Anaemia was not included in the variable selection procedure, because of potential reverse causality. The association of Factors associated with baseline anaemia prevalence were identified using mixed effects logistic regression modelling with a random intercept to adjust for clustering within schools. Model selection procedures were followed as described above.In total, 2346 children aged 5–15 years from 1712 households were included in the study. Baseline characteristics of children are presented in Table P. falciparum infection was 42 % and was significantly higher among boys  and among younger (5–10 years) compared to older children (11–15 years) irrespective of sex  . The majority (68 %) of the infections were light, having parasite densities < 1000 parasites/µl of blood. Densities did not vary by sex, but varied significantly by season (P < 0.001).The overall prevalence of P. falciparum infection reduced with increasing age of children (P < 0.001). Additionally, P. falciparum infection was more common among boys, in children from households with lower socioeconomic status and who were anaemic  episodes per person-year. Of the 2346 children enrolled into the study, 1815 (77.4 %) did not experience any episode of clinical malaria during the follow-up period, 405 (17.3 %) children had only one episode and 93 (4 %) had two or more episodes of malaria. Having any episode and repeated episodes of clinical malaria was significantly higher among girls than boys   episodes per person year with the peak corresponding with the wet season. When stratified by season and sex, the incidence rate varied among boys and girls between the seasons with broadly similar trends, Table ; Fig. 2aIn univariable analysis with season as a fixed term, clinical malaria was associated with being female ,  were anaemic and anaemia was most common among 11–15 year-olds and among boys , rupturing of RBCs, hypersplenism, and reduced RBC production in the bone marrow [P. falciparum infection were associated with poor nutritional status: thinness and stunting, respectively. Malnourished children have previously been shown to have increased risk of malaria [P. falciparum infection further added to the strain of anaemia [Furthermore, in this study anaemia was prevalent (two in five children), and was shown to be associated with n Africa , 43–46. e marrow , 48. Bot anaemia . In addi anaemia  and may P. falciparum with low socioeconomic status is in agreement with previous studies that show malaria is more common among people of lower socioeconomic status who often live in poorly constructed houses increasing their exposure to infection [The observed association of nfection , 30, 51.nfection . In thisnfection , 54 whilnfection . Modern nfection , 57. Impnfection .P. falciparum infection or clinical malaria as would be expected, however the finding is agreement with the last malaria indicator survey that did not show an association of bed net use and Plasmodium infection in Lake Victoria region [Anopheles spp. mosquitoes as previously has been reported in western Kenya [In contrast to cross sectional studies conducted in various parts of Africa, where bed net use has been shown to be lowest among school children , 89 % ofa region . It is pa region , 30. An rn Kenya .P. falciparum infection prevalence was important also during the dry season suggesting that transmission control interventions should not only be targeted to the wet season. Mapping of P. falciparum infection and clinical malaria by survey at school and household level showed pronounced spatial clustering in the western part of the study area. This may be explained by an insufficient spatial resolution of the environmental data used according to the size of the study area. It is well known that the major malaria vectors in the area (Anopheles gambiae s.s.) has greater affinity for small breeding sites such as animal footprints and small ponds formed during rainy season [As expected, the prevalence parasitaemia and incidence of clinical malaria fluctuated depending on the rainfall patterns. Western Kenya is a high malaria transmission area with a annual precipitation ranging 1200–1800 mm . Higher y season  which isy season . It is my season .The major strength of this study was the temporal dimension afforded by examination of a cohort over 1½ years, with 85 % follow-up rate. The study had a number of limitations. First, the children included in the main trial were from schools that were purposively selected based on accessibility, and therefore may not necessarily be representative of all school-aged children in the study area. However, these findings are similar to a study based on a representative sample of schools conducted in western Kenya . Second,P. falciparum infection and anaemia was high among school children in Bumula district, western Kenya. Although reported bed net ownership was high, consistent use was low. Therefore, promoting bed net use among this age group may help to achieve the desired protective effect of this intervention. The study demonstrated the need to include school children in standard malaria interventions, which may alleviate the observed high anaemia burden.The results show that the burden of clinical malaria,


Malaria remains a major cause of febrile illness in Nigeria and interventions to reduce malaria burden in Nigeria focus on the use of insecticide-treated nets. This study determined the prevalence of malaria parasitaemia and the use of insecticide-treated nets (ITNs) for the control of malaria amongst under-five year old children in Calabar, Nigeria.A total of 270 under-5 year old children were recruited and structured questionnaires were used to obtain information on the background characteristics of the respondents from their caregivers. Capillary blood samples were collected from each of the patients through finger-pricking and tested for malaria parasites by Rapid Diagnostic Test and microscopy.An overall parasitaemia prevalence of 32.2 % (by Rapid diagnostic test kit [RDT]) and 40.1 % (by microscopy) were obtained in this study. Forty-six (45.5 %) of the febrile patients had malaria parasitaemia (by RDT) or 41 (59.4 %) by microscopy. One hundred and fifty (55.6 %) of the caregivers acknowledged the use of nets on doors and windows for malaria prevention and control. One hundred and thirty-nine (51.5 %) mentioned sleeping under mosquito net while 138 (51.1 %) acknowledged the use of insecticide sprays. Although 191 (71.5 %) of the households possessed at least one mosquito net, only 25.4 % of the under-5 children slept under any net the night before the survey. No statistically significant reduction in malaria parasitaemia was observed with the use of mosquito nets among the under-5 children. Almost all the respondents (97.8 %) identified mosquito bite as the cause of malaria. Fever was identified by the majority of the respondents (92.2 %) as the most common symptom of malaria.The findings of the study showed high prevalence of parasitaemia and that fever was significantly associated with malaria parasitaemia. Mosquito net utilization among the under-fives was low despite high net ownership rate by households. Therefore, for effective control of malaria, public health education should focus on enlightening the caregivers on signs/symptoms of both uncomplicated and complicated malaria as well as encourage the use of ITNs especially among the under-fives.The online version of this article (doi:10.1186/s12879-016-1459-5) contains supplementary material, which is available to authorized users. Plasmodium parasites and transmitted by female Anopheles mosquito /Insecticide-Treated Nets [ITNs] and Artemisinin Combination Therapy (ACT). The distribution of LLINs was integrated with Ante Natal Care, immunization as well as stand-alone campaigns . Also, oThe role caregivers, especially mothers, play in attending to their febrile child is very important in reducing morbidity and mortality due to malaria. This is most important where the place is considered high risk for malaria, i.e., if > 5 % of fevers among children is caused by malaria. For example, a child with fever in a high-risk area who does not appear to have any other underlying reason for the fever, e.g., measles on physical evaluation should be considered as having malaria. Such a child should receive anti-malarial drugs. This is the WHO programme guidelines for Integrated Management of Childhood Illness [IMCI], used in resource limited settings to evaluate and treat children .Despite the evidence-based benefits of sleeping under ITNs, and the efforts made by the Federal Government and Non-governmental Organizations to tame the public health scourge of malaria in Nigeria, some geopolitical zones of the country still record low average number of ITNs ownership/usage per household . DiffereThe study is descriptive and cross-sectional in design. It was carried out from November, 2012 to December, 2013 to determine malaria parasitaemia and the perception and practices of care-givers of under-five children on the use of ITNs amongst the under-five children. It was carried out in the University of Calabar Teaching Hospital, Calabar which is a second generation Teaching hospital in the country, Nigeria. The hospital currently has over 600 beds distributed between the three annexes and renders services in specialized areas in medicine such as paediatric surgery, haemodialysis, neuro-surgery, ophthalmologic surgery and maternal health.The study target population consisted of women/men aged 15 – 50 years who had the responsibility of taking care of at least one Under-five year old child. A total of 270 under-5 children who came to the hospital as outpatients with their care-givers were recruited in the study.Approval was obtained from the Research and Ethics Committee of the University of Calabar Teaching Hospital, Calabar, Nigeria. Informed consent was also obtained from the patients’ relatives. Those who declined consent were excluded from the study.2 pq/d2) was used to determine the sample size assuming the ITN usage rate among under five children in the South-South zone of Nigeria to be 20.0 % [Single population proportion formula  was interviewed by trained interviewers using structured questionnaires  software . Continuous variables were presented as the mean ± standard deviation. Categorical variables were presented as actual numbers and percentages in table forms, or figures. All categorical variables were compared using Pearson’s Chi-square test or Fisher’s exact test. 0 + b1X1 + b2X2 + … + bpXp, where Ý is the predicted probability of the event which is coded with ‶0″  rather than “1” . “1-Ý” is the predicted probability of the other decision and X1 through Xpare distinct independent (predictor) variables and b0 through bpare the regression coefficients. The “-2 Log Likelihood” statistics and Hosmer-Lemeshow test were used to show how well the model predicts the decisions. Two tailed P-values was reported, odds ratios and 95 % Confidence interval was used to estimate the association between dependent (outcome) variables and independent variables.ln (Odds) = ln (Ý/1-Ý) = bA summary of the baseline characteristics of the respondents is given in Table Table Majority of the households  that owned at least one net got them from the Primary health centre closest to them, twenty-nine (15.2 %) of them got theirs from Non-Governmental Organizations. Only 2 (1.0 %) got theirs from the Patent medicine store. About half the population of the respondents obtained their nets within 2–12 Months prior to the study, only 14 (7.5 %) got their nets more than 2 years prior to the study. Almost all the nets (93.7 %) were already-treated nets by the time they were procured. Twenty-one (11.0 %) care-givers admitted a secondary treatment of their nets by themselves after procurement.Sixty-eight (25.4 %) of the children studied were reported to have slept under any net the night before the survey. Amongst thosewho had nets 191), only 68 (35.6 %) had at least one under-5 year old child who slept under the net the night before the study. Those care-givers’ household in which no child slept under a net the night before this study gave different reasons for not sleeping under the net. Some of the reasons included: ‘weather was too hot’ (77.2 %), ‘difficulty at hanging the net’ (7.3 %), ‘there were no mosquitoes’ (7.3 %) , ‘don’t like to use nets’ (13.9 %), and ‘there is no mosquito’ (12.5 %).Figure Two hundred and seventy (270) patients were recruited, RDT tests were carried out on 258 patients and 32.2 % (83/258) had positive parasitaemia. On the hand, one hundred and sixty seven (167) were tested using slide microscopic method, 40.1 % (67/167) had positive slide (parasitaemia).Two hundred and sixty-four (97.8 %) care-givers correctly identified mosquito bite as the cause of malaria. One care-giver (0.4 %) admitted that it was due to ‘too much intake of oily food’ while 5 (1.8 %) did not know the cause of malaria. Majority of the respondents, 249 (92.2 %) correctly identified ‘hotness of the body or fever’ as the most common sign and symptom of malaria. Other signs and symptoms identified included: ‘generalized body weakness’ (59.3 %), ‘loss of appetite’ (47.4 %), and headache (43.0 %). On malaria prevention, one-hundred and fifty (55.6 %) admitted that ‘using mosquito nets at doors/windows’ can be a means of preventing mosquito bite and of course malaria, one hundred and thirty nine (51.5 %) of them accepted that ‘sleeping under ITN prevents malaria, while 79 (29.3 %) opted for ‘spraying insecticide every night’.One hundred and nine respondents (41.6 %) admitted that at least one under-5 year child under their care had fever 2 weeks prior to the index study; one hundred and fifty respondents (57.3 %) did not have any child with fever for the same period as in the above. For the question to determine incidence of under-five mortality from among the care-givers, a total of 35 under-5 deaths were recorded. Out of the 35 deaths, fourteen (40.0 %) were fever-associated, twelve (34.3 %) were non-fever associated, while for 9 (25.7 %), it could not be ascertained whether the deaths were associated with febrile illness or not.p < 0.001). There was no significant difference between tribe, care-givers’ age and ownership of ITNs.There was significant association between those with fever and presence of parasitaemia. Those with parasitaemia either by RDT or microscopy testing were more likely to develop fever than those with no parasitaemia, (The proportion of febrile patients that had parasitaemia by RDT testing was 55.4 % (46/83), while that by microscopy was 61.2 % (41/67).p > 0.05). Malaria parasitaemia increased with the age of the child; it was 15.5 % for age group 1–11 months, 38.0 % for age group 12–35 months and 46.5 % for age group 36–59 months (p > 0.05).The proportion of children under the age of five years that had positive parasitaemia was less among care-givers who had tertiary education (46.3 %), than among those who did not (53.7 %) (Table p < 0.05) (Table p > 0.05)  Table . No sign5) Table .Table 4Dp < 0.05; OR = 0.52). A similar model was fitted for possible predictors for ‘sleeping under the net’. Only care-givers age gave a statistically significant result, with younger care-givers not likely going to have under-five children that will sleep under a net (p > 0.05)  Table .p < 0.05). This malaria prevalence of 40.1 % was higher than 12 % reported in Tanzania [In this study, the prevalence of parasitaemia (by RDT) was 32.2 % while that by microscopy was 40.1 %. The proportion of febrile patients that had parasitaemia by RDT was 55.4 % while that by microscopy was 61.2 %  of the caregivers mentioned using nets on doors and windows, 51.5 % mentioned sleeping under a mosquito net, while other responses were: destroying mosquito breeding places (51.1 %) and spraying insecticides every night (29.3 %). The Nigerian Malaria indicator survey of 2010  reportedet al. [Net ownership, as determined by possession of at least one mosquito net in a household was found to be 71.5 %. The proportion of households who had at least one mosquito net has risen far above the reported proportion of 42 % obtained in 2010 , 8 % in et al.  in theiret al.  in whichet al. [Some variables are known to influence ownership of mosquito nets. Some of these factors have been reported to include level of education, wealth index, family size and residence among others . In thiset al.  observedAbout two-third of the respondents who do not own at least a net stated that ‘nets were not available’ as a reason for not having one. For most of them, the non-availability of the nets meant they did not know where/how to get one. Other reasons given for not having mosquito net included: ‘does not like to use net’ and ‘there is no mosquito in my residence’. Misconceptions about causes of malaria and prevention modalities are also valid reasons for non-ownership and utilization of nets .et al. [Twenty five-point four percent  of children under age 5 among those interviewed slept under a net the night before the survey. Compared to previous NDHS and NMIS surveys in Nigeria, the sustained rise in net utilization was lost. The percentage of children under age five who slept under ITNs has increased steadily and substantially from 6 % in 2003 , to 12 %et al. .This current study showed that whereas 71.5 % caregivers with under- 5 children owned at least one ITN, only 25.4 % of the children used a net a night before the survey. The high discrepancy (46 %) between ownership and utilization of ITN in this study could not easily be explained out and as such calls for great concern. It probably shows that there is need for adequate motivation before ownership will translate to utilization. It does appear that in the last 2 years before this index study, large scale net campaigns and distribution was carried out in Calabar and since ITNs were given free of charge, caregivers were poised to have them; however, they lacked the motivation to use them. Tobin-West and Alex-Hart  also repThe most common reason given among the caregivers; that had at least one ITN for not using it was that the weather was too hot (77.2 %). This same reason has also been reported in other similar studies and has been attributed to the hot tropical climate of the sub-Saharan African region , 30, 31.p > 0.05). Demographic characteristics like child age, care-givers’ age, education, and ethnicity have been known as possible predictors of net use in other studies [p < 0.05). Our study showed that older care-givers are more likely to have their children sleep under net than younger care-givers. This finding could have been as a result of past experience these older care-givers have had with caring and parenthood.Higher educational levels in previous studies , 26 haveAssessment of the impact of ITN coverage and/or utilization on health outcomes is usually difficult. This is usually due to poor routine health information and vital registration systems; making determination of malaria-specific mortality and morbidity almost impossible . Few stuet al. [Malaria prevention and control measures aim at preventing mortality and reducing morbidity and also malaria-associated economic losses. Lack of knowledge about malaria and its mode of transmission will hamper appropriate preventive measures. In our study, care-givers were asked questions to ascertain their knowledge of causes, signs and symptoms, and means of prevention of malaria. Almost all the caregivers (97.8 %) identified mosquito bite as the cause of malaria. This finding was consistent with those of previous studies , 36 but et al. . This awet al. , 39. Theet al. . A worriet al. . The asset al. .Treatment seeking behaviour among caregivers has been shown to be related to the cost, availability and cultural beliefs about the causes and effective cures for malaria-like symptoms . Among tOur study has some limitations. First, we tried to replicate a Nigerian Malaria Indicator Survey (NMIS) of 2010 , howeverA parasitaemia prevalence of 40.1 % obtained in this study can still be seen to be high considering recent scale up in malaria prevention campaigns in the area. Fever was significantly associated with malaria parasitaemia. This means a lot of febrile illnesses among the under-five children in this area might still be due to malaria infection. Respondents identified various methods that are used to prevent/control malaria infection, with majority acknowledging putting net at windows and doors, followed by sleeping under mosquito nets and the use of insecticide sprays. Household ownership of nets was very high compared to many recent studies, however, the net ownership did not translate to use as there was much discrepancy between ownership and usage of the net. There was no statistically significant reduction in malaria parasitaemia with the use of mosquito nets over non-use among the under five children studied, an effect that could have arisen due to smallness of sample size. The respondents demonstrated good knowledge of the cause and symptoms of uncomplicated malaria, however, only few knew the signs and symptoms of severe malaria. Majority of the respondents also demonstrated the deadliness of malaria among the under five children via their treatment seeking behaviours. Most of them would prefer to take their children to government hospital first, possibly hoping to obtain best care delivery there. Fairly good number of care-givers would rather prefer to ‘try their luck’ by giving self-medication at home first.


P. falciparum eba-175, ama-1, and msp-3 polymorphism in the pathogenicity of malaria disease was investigated. We therefore compared the prevalence of different alleles between symptomatic and asymptomatic malarial children under five years of age living in Burkina Faso. Blood filter papers were collected during the 2008 malaria transmission season from 228 symptomatic and 199 asymptomatic children under five years of age. All patients were living in the rural area of Saponé at about 50 km from Ouagadougou, the capital city of Burkina Faso. P. falciparum parasite DNA was extracted using QIAGEN kits and the alleles diversity was assessed by a nested PCR. PCR products were then digested by restriction enzymes based on already described polymorphic regions of the eba-175, ama-1, and msp-3 genes. The individual alleles eba-175_FCR3 and msp-3_K1 frequencies were statistically higher (p < 0.0001) in the asymptomatic group compared to the symptomatic ones. No statistically significant difference was noted in the prevalence of ama-1-3D7, ama-1-K1, and ama-1-HB3 genotypes between the two groups (p > 0.05). The comparative analysis of P. falciparum genotypes indicated that the polymorphism in eba-175 and msp-3 genotypes varied between asymptomatic and symptomatic clinical groups and may contribute to the pathogenesis of malaria.The association between  Based on the above clinical associations and the polymorphism in eba-175 [ msp-3 [ ama-1 [ eba-175, msp-3, and ama-1 haplotypes in symptomatic and asymptomatic children living in a malaria endemic and specifically in seasonal areas is of scientific interest. Indeed, the results from this current study could help to improve our understanding of the role, if any, of P. falciparum genotype diversity in malaria pathogenicity. Moreover, data generated from this study might be relevant to vaccine studies in the context of EBA-175, AMA-1, and MSP-3 based malaria vaccine development.According to the latest WHO malaria report , there w studies –11. In a malaria . In 2001 malaria  revealed eba-175 , msp-3 [ [ msp-3 , and ama [ ama-1 , 16, we The study was conducted in two community clinics, Saponé Marché and Kounda located in the Saponé Health District, approximately 50 km southwest of Ouagadougou, the capital of Burkina Faso. The Saponé Health District is located at 12°13′N, 1°48′W and has approximately 80,000 inhabitants, most of them (>95%) belonging to the Mossi ethnic group. Malaria transmission is perennial and seasonal in this district and is characterized by a peak during the rainy season (May–October). The entomologic inoculation rate was estimated at 200 infective bites/person/year in the study area . The incAbout 500 children under the age of five living in the two communities were targeted for this study.During the follow-up period, all malaria cases were managed by the study clinical staff with no cost. Clinical management was based on the National Policy guidelines, which consisted of artemisinin-based combination therapy  for uncomplicated malaria cases and quinine salt for severe malaria cases.The study was part of a large epidemiological study aimed at assessing malaria morbidity and mortality in children under the age of five years (DMID protocol 06-0020). The study protocol was reviewed and approved by Office of Clinical Research Affairs (OCRA) within the Division of Microbiology and Infectious Disease  of the USA). It was also approved by the Health Research Ethics Review Committee in Burkina Faso.A clear description of the study and all anticipated risks and benefits was explained to the participants before a written informed consent was voluntarily obtained from the participants or their legal guardians.Blood samples were obtained by finger prick from children aged 3–59 months. Thick and thin smears and blood spotted on filter paper (Whatman No. 2) were collected during two cross-sectional surveys conducted in February and September 2007, respectively, dry and rainy season time points, for asymptomatic cases detection. Children were then followed up during one year at their relevant community clinics for malaria symptomatic cases detection.Thick and thin smears were stained with Giemsa and examined under light microscope for malaria parasites identification and counting.The spotted filter paper was kept at room temperature with silica gel desiccant prior to genotyping analysis. P. falciparum parasite irrespective of parasite density; asymptomatic malaria case was defined as the presence of asexual P. falciparum parasite irrespective of parasite density, in the absence of clinical symptoms.For the purpose of this study, the following definitions were adopted: symptomatic malaria case was defined as children with axillary temperature ≥37.5°C bearing asexual P. falciparum. Asexual P. falciparum parasites were estimated against 200 leucocytes and the parasite density was calculated based on a mean of 8000 leucocytes per microliter of blood.Thick and thin blood smears were stained with Giemsa and microscopically examined to identify monoinfections withParasite DNA was extracted from blood spots using QiAmp DNA blood mini kit according to the manufacturer's protocol . DNA samples were aliquoted in sterile tubes and stored at −20°C until polymerase chain reaction (PCR) amplification.Eba-175 genotyping was performed by nested PCR as described elsewhere [ msp-3 gene , 2.5 µL of 10x PCR buffer (Invitrogen), 2.0 mM MgCl2, and 200 µM dNTP (Invitrogen). The PCR cycling conditions for the first and the second reaction are described in lsewhere . The prigene see . This re msp-3 first amplification reaction using a set of primers (VM785/3 and VM990) indicated in  ama-1 were performed in a final volume of 50 µL that contained 5 µL of 10x PCR buffer (Invitrogen) and 2 units of Taq DNA polymerase with 1 µM each of the ama-1 VM815 and VM990 primers. The PCR products from the ama-1 second reaction were digested by using three restriction enzymes Mse I, Ssp I, and BfCU I, which generated fragments whose size corresponded to the 3D7, K1, and HB3/7G8 ama-1 allele classes, respectively.The first PCR reaction was similar to the above P. falciparum 3D7 (MRA-102G), HB3-B2 (MRA-149G), and K1 (MRA-159) was provided by the Malaria Research and Reference Reagent Resources Center, American Type Culture Collection , and used as positive controls during the PCR amplification. The positive controls were used to score and interpret fragment sizes.Purified DNA from Eba-175 alleles (CAMP and FCR3) were identified as single fragments, approximately 714 and 795 base pairs in length, respectively. The msp-3 K1 and 3D7 alleles were identified as single fragments, approximately 500 and 400 base pairs in length, respectively. All other msp-3 fragments of different sizes were also reported. Finally, the ama-1-K1, ama-1-3D7, and ama-1-HB3/7G8 alleles were identified as single fragments of 285, 400, and 335 base pairs in length, respectively, when subjected to digestion with Mse I, Ssp I, and BfCU I, respectively.The eba-175 fragments or the presence of two or three of the ama-1 fragments within the same sample.Mixed infections were defined as the simultaneous presence of K1 and 3D7 msp-3 alleles or the F and Chttp://www.cdc.gov/epiinfo/Epi6/EI6dnjp.htm). This software was used to obtain the carrier frequencies of the different alleles. Individual allele frequencies were estimated using GenAlEx 6.5 [The allelic data input was double entries in Excel format and was analyzed using the statistical software Epi Info v6.04a  based on the difference in the development of acquired immunity.In order to assess if the prevalence ofp < 0.05 value used as statistical significance threshold end point.The chi-square and Fisher's exact tests were used for data analysis with h) = 1 − ∑pi2 ranged from 0 to 1, where pi is the frequency of the ith allele. The t-test was used to compare mean heterozygosity between the different groups.Genetic diversity was estimated by determining the heterozygosity based on the three antigens combined, where heterozygosity , 2.1–2.4) were included in this study. The genetic analyses included 427 samples representing 228 and 199 samples collected, respectively, from asymptomatic and symptomatic malaria children. This genetic analysis included only samples which were microscopically known positive for eba-175 alleles. Genotyping analysis revealed that the frequency of the FCR3 allele was significantly higher in asymptomatic infections compared to the symptomatic infections (p = 0.0001). The ama-1-HB3 allele was also overrepresented in the asymptomatic infections, but without reaching a statistically significant difference   . ama-1_HB3 allele and the eba-175_FCR3 allele were the most prevalent ones in both groups. The msp-3_3D7 was the most common msp-3 alleles in the asymptomatic group.A comparison of the allele's prevalence within each group (asymptomatic or symptomatic) revealed that the eba-175 FCR3 and the msp-3 K1 alleles were more common in asymptomatic versus symptomatic group and that the difference was significant  based on the difference in the development of acquired immunity. Statistical analysis of the distribution of the different alleles by age demonstrated some structure in the allele frequencies. The distribution of msp-3 alleles showed a higher and statistically significant prevalence of msp-3 3D7 compared to msp-3 K1 alleles in all age groups irrespective of the clinical features, except in the “1–3 years” category where a statistically significant lower frequency (p = 0.005) of msp-3 3D7 alleles was observed .A higher prevalence of>3 years . The dif msp-3 gene within and flanking the heptad-repeat selected for this genetic analysis was identified in previous study as the site of antigen diversity among MSP-3 polypeptides [ eba-175 subject to the present genetic analysis was the exon I located in region II which is known to code for a repeated sequence of two Duffy binding-like domains (F1 and F2). The two domains F1 and F2 are being separated by highly divergent dimorphic region III, through the insertion of either a 342-base pair segment in FCR-3 strains (F-loop) or a 423-base pair segment in CAMP strains (C-loop).The present study was designed to analyze the allelic diversity of the three main malaria vaccine candidates in symptomatic and asymptomatic malaria infected children living in the southern region of Burkina Faso. The study was focused on domain I of AMA-1 representing the most polymorphic region of the gene , 16, 21 peptides . Finally eba1-175 dimorphism and malaria clinical status was demonstrated in children living in central Africa and specifically from Gabon [The genetic diversity influence of such known surface antigens in malaria pathogenesis was discussed in various previous studies generating conclusions with a need for further investigations in different malaria setting. Indeed, the importance of the relationship betweenom Gabon , 23. ama-1 genetic diversity between symptomatic and asymptomatic infections was highlighted in a previous study [The importance of AMA-1 as a malaria vaccine candidate provides further justification for exploring the role of genetic diversity in this antigen. A strong imbalance inus study  suggesti eba-175 gene was dimorphic with F and C alleles present in general prevalence as shown in previous studies [The results from this present study confirmed that the studies –27. Whil studies  and in A studies . ama-1 alleles in our study showed lack of association in genotype prevalence with malaria infection contrasting the finding from previous studies. Strong imbalance between symptomatic and asymptomatic infections was found in the distribution of residues at several clustered polymorphic positions of ama-1 genotypes [ ama-1_HB3/7G8 genotypes were overrepresented in the study area in both symptomatic and asymptomatic groups compared to the other genotypes (K1 and 3D7). The common PCR method used in this study does not provide enough resolution to discern individual point mutations in the domain I predicted to arise from diversifying selection due to a protective immune response [ ama-1 gene product would probably better address the role of such polymorphism in the pathogenesis of malaria.The analysis ofenotypes . Howeverresponse , 30–32.  msp-3_K1 alleles were associated (p < 0.0001) with higher risk of clinical malaria in  msp-3_3D7 alleles was associated with asymptomatic malaria only in the age group of 1–3 years. These findings highlight the relevance of analyzing the polymorphism of the P. falciparum parasite based on subclasses with closer interval of age instead of large range of age. Previous studies analyzing the age-allele specific immunity constitute a perfect illustration of that complexity of malaria antigen polymorphism [Themorphism  in whichmorphism .The PCR-RFLP is use as a technical approach to analyze the allelic diversity in this study. Even though this is a useful approach based analysis, inexpensive and ease to determine the number of different parasite genotypes infecting an individual patient or in assessing the multiplicity of infection (MOI), the approach is of limited utility for interpreting some patterns as the size itself (principle of polymorphism) may be under selection , 36 or f eba-175, msp-3, and ama-1 gene in P. falciparum isolates from asymptomatic and symptomatic malaria cases indicated that the polymorphism was comparable between asymptomatic and symptomatic malaria groups in our study area. Except for few differences observed in the eba-175 and msp-3 alleles frequencies, the overall polymorphism of the three genes together did not show any alleles association with symptomatic or asymptomatic malaria. Nevertheless, these findings give broad insights about the general distribution of the P. falciparum polymorphism for the development of malaria vaccine trial sites. Our results highlighted the need of using more advanced techniques such as DNA sequencing in order to shed light on this relevant debate.This comparative analysis of the role of allelic polymorphism of the