Previous research on determinants of malaria in Burkina Faso has largely focused on individual risk factors. Malaria risk, however, is also shaped by community, health system, and climatic/environmental characteristics. The aims of this study were: i) to identify such individual, household, community, and climatic/environmental risk factors for malaria in children under five years of age, and ii) to produce a parasitaemia risk map of Burkina Faso.The 2010 Demographic and Health Survey (DHS) was the first in Burkina Faso that tested children for malaria parasitaemia. Multilevel and geo-statistical models were used to explore determinants of malaria using this nationally representative database.Plasmodium spp. Older children (>23 months) were more likely to be parasitaemic than younger ones, while children from wealthier households and whose mother had higher education were at a lower risk. At the community level, living in a district with a rate of attendance to health facilities lower than 2 visits per year was significantly associated with greater odds of being infected. Malaria prevalence was also associated with higher normalized difference vegetation index, lower average monthly rainfall, and lower population densities. Predicted malaria parasitaemia was spatially variable with locations falling within an 11%-92% prevalence range. The number of parasitaemic children was nonetheless concentrated in areas of high population density, albeit malaria risk was notably higher in the sparsely populated rural areas.Parasitaemia was collected from 6,102 children, of which 66.0% : 64.0-68.0%) were positive for Malaria prevalence in Burkina Faso is considerably higher than in neighbouring countries. Our spatially-explicit population-based estimates of malaria risk and infected number of children could be used by local decision-makers to identify priority areas where control efforts should be enhanced.The online version of this article (doi:10.1186/1756-3305-7-350) contains supplementary material, which is available to authorized users. In 2010, there were an estimated 216 million cases of malaria and an estimated 660,000 malaria deaths worldwide [Anopheles sp. bionomics, are associated with malaria transmission in endemic countries [According to the 2013 World Malaria Report, the entire population of Burkina Faso is at high risk of malaria . Althougountries –11.Although Burkina Faso is considered as one of the most highly malaria-endemic countries in Africa , geograp2 located in West Africa: it shares borders with Ivory Coast, Ghana, Togo and Benin in the South, Mali in the North and Niger in the North-West. The total population is estimated at 17 million. Most of central Burkina Faso is on a savanna plateau while the south is green and the north is arid.Burkina Faso is a landlocked country of 274,200 kmAn. gambiae s.s., An. arabiensis, and to a lesser extent, An. funestus[. The predominant malaria parasite is Plasmodium falciparum, which accounts for more than 95% of infections in children under five years of age [Burkina Faso has a tropical climate with two seasons: the country receives between 600 and 900 millimeters of rainfall during the rainy season and a hot dry wind from the Sahara blows during the dry season. The rainy season lasts approximately four months, July to October, and is shorter in the north of the country. Three climatic zones can be defined: the Sahel, the Sudan-Sahel, and the Sudan-Guinea. The Sahel in the north typically receives less than 600 millimeters  of rainf funestus. The pres of age .Children under five years of age were sampled from a total of 572 clusters in the 2010 Burkina Faso DHS. Clusters were defined as “Zones de dénombrement”: the smallest administrative unit with an average population of 1,000 and of 1,200 in rural and in urban areas, respectively. Our study sample included all 6,102 children. The DHS is a national household-based survey conducted by the National Center for Statistics and Demography (NCSD) and Macro International in order to assess disease and risk factors among the population. The survey had a stratified two-stage design. A total of 574 clusters were selected at the first stage with a proportional-to-population-size-probability sample. At the second-stage, a complete listing of all households was carried out and households were randomly selected for inclusion in the survey with equal probability. Data were collected between April 2010 and January 2011.Centre National de Recherche sur le Paludisme) and classified qualitatively as either negative or positive infection. The presence of Plasmodium sp. parasites in blood films was the primary outcome of this study. Detailed information on the survey’s design and laboratory procedures can be found in the Burkina Faso 2010 DHS report [Blood samples were drawn and interviews were conducted in the selected households . All children aged between 6 and 60 months living in these households were eligible for malaria testing. Parents or guardians provided consent for their children’s participation (non-consent was 1%). The parasite prevalence survey was based on microscopic examinations of stained blood films. Two blood slides, each composed of thick and thin films, were taken from each participant. Blood slides were read by two technicians at a reference laboratory in Ouagadougou , gender and a binary variable indicating if children slept under a bed net the night before the survey. At the household level, we considered: mother’s education; household size reporting the number of children aged under five years living in the household; the wealth index, based on ownership of household assets, used as a proxy for socio-economic status (SES) . The weaAt the community level, we considered a number of district-specific variables such as: rate of attendance to health facilities, the population per medical doctor ratio, and the population per health worker ratio. This information was extracted from the Ministry of Health Statistical Yearbook of 2010 [Environmental conditions can impact on malaria transmission and we examined the effect of a number of climatic and environmental variables: distance from rivers and water bodies, distance from major roads, normalized-difference vegetation index , altitude, minimum temperature, maximum temperature, mean diurnal range in temperature, rainfall before the survey date, average monthly rainfall, precipitation seasonality, vegetation cover, and population density . These climatic and environmental variables were assembled from a variety of sources summarized in Table Yij, indicating if child i living in community j was found to be parasitaemic. The logistic regression model had the following form:Descriptive statistics were used to examine the characteristics of the sample and, unless stated otherwise, sampling weights were not used in the statistical analyses. Given the hierarchical structure of the data, logistic random effect models were used in univariate and multivariate analyses to identify factors associated with malaria infection. Potential non-linear relationships between covariates and the malaria parasitaemia outcome were accounted for by either categorizing the covariate or including polynomial terms. The appropriate functional form was chosen by visually inspecting outcome-covariate scatter plots and through consideration of the Deviance Information Criterion (DIC) . Specifi0 + βXij) estimating the conditional vector of coefficients β = T for the matrix of explanatory covariates (Xij), a random intercept attributable to communities (ωj), and a random error for child i that doesn’t depend on community (ϵi). Note that preliminary analyses suggested that the inclusion of a household-level random effect was not required as its variance was extremely small. Statistical analyses were performed in a Bayesian framework and the marginal posterior distributions of the parameters were estimated using Integrated Nested Laplace Approximations (INLA) [This generalized linear model has a fixed part (βs (INLA) .In order to build parsimonious and well identifiable models, Bayesian variable selection was performed , 24, 25.The models described above do not consider the spatial relationships among community locations. The standard way of incorporating geographical dependence in the model is by introducing spatially correlated random effects at every sampled location, generally assuming that the spatial correlation decreases as distances between location increases. Predictions for un-sampled locations are also improved by explicitly modeling spatial dependence. Two models were fitted: model A that included individuals, household, community, and climatic/environmental variables, and model B that included the climatic and environmental variables. Producing a risk map of malaria requires that we have complete spatial information over the whole geographic area of Burkina Faso (at both sampled and unsampled locations) for all covariates included in the geo-statistical model. Because such information was unavailable in unsampled locations for SES, bednet usage, mother’s education, etc., only model B could be used for prediction. These geo-statistical models have the following general form:j ~ N with element where ωβ0, βXij, and ϵi are the same as described before but ωj is now the spatial random effect for community j. Σkl represents the Matérn covariance between location k and l. It was assumed that this random effect has a multivariate normal distribution with mean zero. The covariance is a function of the variance of the spatial process (σ2), a scaling parameter (κ), the Euclidean distance between location k and l (dkl), a smoothness parameter (ν) fixed to one in this particular application, and Κν and Γ(ν) are the modified Bessel function of second kind order and the Gamma function, respectively. Further, the range of the spatial process is defined to be Here, The relationships between climatic and environmental variables with malaria parasitaemia were visually examined to determine the appropriate functional form. Further, the Deviance Information Criterion (DIC) was used to choose among linear, categorical or polynomial form for each of the variables. Because of potential biologically determined lag between climatic variables and malaria transmission, the DIC was used to choose the appropriate lag time among a number of plausible lags ranging from 30 to 70 days for rainfall and 7 to 21 days for NDVI. Based on these preliminary analyses, the rainfall estimates were lagged by 60 days and NDVI by 7 days. The Gibbs variable selection algorithm described in the preceding section was used to select a parsimonious set of climatic/environmental variables for inclusion . These variables could be correlated with individual, household, or community characteristics and we wanted to give precedence to the covariates for which complete spatial coverage was available. Bayesian variable selection was therefore carried out in two stages. First, we considered only the set of 13 climatic/environmental variables for inclusion (Stage 1). Second, based on the reduced set of covariates from stage 1, we performed variable selection on an additional set of 9 individual-, household-, and community-level covariates while adjusting for the climatic/environmental variables selected during the first stage (Stage 2).The out-of-sample predictive ability of our model was assessed by fitting the model to a random sample of 80% of the locations (432 clusters) and predicting the remaining 20% (108 clusters). We calculated the posterior predictive distributions of the test locations and computed the proportion of the observed parasitaemia prevalence falling into different coverage distributions (e.g. 10% to 100%). The mean predictive error and the squared root of the mean square error (RMSE) between the observed and predicted cluster prevalence were also calculated as an index of accuracy. The Area Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) was used as an additional tool to assess model performance .The computational burden of the geo-statistical models was overcome by fitting the model using INLA , which pInformed consent was provided by all survey participants (or their guardian) before questionnaire administration and malaria testing. Further, all survey protocols have been approved by the Internal Review Board of ICF International in Calverton (USA) and by the “Comité National d'Éthique” in Burkina Faso.The total number of households selected for the survey was 4,029 in 572 communities, and microscopy data were available for 6,102 children. A total of 34 communities, totaling 361 children, were missing geographical coordinates and had to be dropped from our analyses. The mean age of children was 32.3 months (range: 6–59 months). Overall, 83.6% of the children lived in rural areas, 50.8% were boys, and most mothers had no education (84.4%). The average household size was 7.8 persons. Most of the data (62.3%) were collected between July and October 2010 but all data from Central Region (5.9%) were taken between May-June 2010. The ratio of population per medical doctor was higher than 20,000 for all communities, and only 13.5% of children had more than two contacts with health facilities in 2010. The weighted prevalence of malaria in Burkina Faso was estimated at 66.0% (95% CI: 63.1-66.7) ranging for 27.7% in the Central Region to 77.7% in the Sud Ouest Region , the climatic/environmental (model B) geo-statistical model’s predictions had a mean error of 0.005 and a RMSE of 0.19. This out-of-sample validation shows that predictions were fairly accurate, with 77% of tested locations falling within the 95% credible intervals (CrI) of the posterior predictive distribution. The proportions of test locations falling into specific CrI coverage of the predictive posterior distributions are presented in Figure 0%, the cTaking population density into account, the mean predicted malaria prevalence estimated from the geo-statistical model (model B) was 65%, a figure almost identical to that estimated from the DHS data alone. The map of the estimated number of infected children under 5 years showed that malaria burden was concentrated in major cities and in the districts around Ouagadougou Figure . Thus, aPlasmodium parasites in Burkina Faso. Prevalence of malaria was higher than the one reported from other sub-Saharan African countries where comparable nationally representative data are available . Although the geo-statistical model’s predictions proved to be relatively accurate, our prediction’s RMSE was of 0.19. Some of the models’ errors in the prevalence estimation could be due to important variables that were not accounted for in our risk maps such as coverage of indoor residual spraying (IRS) and ITN, and distribution and distances to mosquito breeding sites . UnfortuThe Burkinabé Ministry of Health set out the ambitious target to reduce the morbidity of malaria by 75% in 2015, using 2010 as a baseline, in its new ‘National Control Program on Malaria from 2011 to 2015’ [This study has some limitations. First, because the survey does not differentiate between symptomatic and asymptomatic cases, our maps indicate where parasitaemia detected through microscopy was highest but not necessarily where treatment is most needed. The nature of the data also imposed important limitations in that individual households were not geolocated and only the centroid of the community was mapped. Further, to insure data confidentiality, random spatial error was added to the geographic locations of each cluster: 0.90 km on average for urban clusters and 2.26 km for the rural ones . This raNational prevalence of malaria in children under five years of age in Burkina Faso was one of the highest ever recorded by DHS surveys. High spatial variation in malaria risk was exhibited and children living in densely populated areas had the lowest risk of infection. The number of parasitemic children was, however, highest in such urban areas. The determinants of malaria infection described in this study as well as the maps of parasitaemia risk and number of infected children could be used by managers of malaria control programs to define priority intervention areas. Even though the environment plays an important role in malaria transmission, increasing coverage of malaria interventions and disease knowledge but also, improving socio-economic conditions will be key to achieving sustainable malaria burden decreases in Burkina Faso.Text S1. Technical information related to the performed Bayesian Variable Selection and to the type of prior distributions used for fitting the multilevel and geo-statistical models. (DOCX 28 KB)Additional file 1: Additional file 2: Figure S1: Map of surveyed clustered and observed prevalence of malaria parasitaemia in 540 clusters in Burkina Faso (2010). (JPEG 1 MB)th and 97.5th percentiles of malaria parasitaemia risk in children under 5 years in Burkina Faso for August 2010. (JPEG 1 MB)Additional file 3: Figure S2: Maps of the posterior predictive distribution of the 2.5Additional file 4: Figure S3: Map of the thirteen administrative regions of Burkina Faso. (JPEG 1 MB)


Patients were evaluated for the presence of 21 symptoms before and after treatment and for reported side effects of these drugs after treatment was started. The most frequent symptoms before medication were headache, fever, chills, sweating, arthralgia, back pain, and weakness, which were present in between 40% and 76% of respondents. The treatment reduced the occurrence of these symptoms and reduced the lack of appetite, but gastrointestinal symptoms and choluria increased in frequency. There were no reports of pale stools before medication, but 12% reported the occurrence of this symptom after treatment started. Other symptoms such as blurred vision (54%), pruritus (22%), paresthesia (6%), insomnia (46%), and “stings” into the skin (22%) were reported after chloroquine was taken. The antimalarial drugs used to treat P. vivax malaria reduce much of the systemic and algic symptoms but cause mainly gastrointestinal side effects that may lead to lack of adherence to drug treatment. It is important to guide the patient for the appearance and the transience of such side effects in order to avoid abandoning treatment.Side effects of antimalarial drug can overlap with malaria symptoms. We evaluated 50 patients with  Plasmodium. There are five known species that parasitize man, the most prevalent being Plasmodium falciparum, P. vivax, and P. malariae in tropical areas such as Latin America, the Indian subcontinent, Southeast Asia, and parts of Africa [ P. ovale has been reported in Africa, Middle East, and the Indian subcontinent [ P. knowlesi has been recently described as responsible for large foci of infections in Southeast Asia [ Anopheles mosquito, Anopheles darlingi being the most prevalent species in the country [Malaria is one of the main scourges of the world, affecting half a million people a year . It is cf Africa . P. ovalontinent , while Past Asia . The nat country . P. vivax  is treated with chloroquine in Brazil at a dose of 30 mg/kg divided into three days and primaquine in the 30 mg dose/day for 7 days or 15 mg/day for 14 days. Also, low doses of chloroquine can be used for the prevention of vivax malaria. Pregnant women and children under 6 months of age with vivax malaria receive only chloroquine in different dosage schedules [The Ministry of Health directs therapy and provides free antimalarial medicines used throughout the national territory, in units of the Unified Health System (SUS), through a national policy on malaria treatment. Malaria caused by Plasmodium which cause malaria in humans, as well as against hypnozoites, latent forms of P. vivax and P. ovale responsible for relapses [Primaquine is the only antimalarial drug with effective activity against all the species gametocytes ofrelapses . Howeverrelapses , 9. Gastrelapses .With respect to chloroquine, there are relatively few adverse effects with use in usual doses, of which the most serious are retinopathy, cardiomyopathy, myopathy, and neuromyopathy. Prolonged treatment or use of high level doses may cause retinal toxicity, long subtle symptoms of decreased visual acuity, diplopia, and bilateral loss of vision . Adverse Plasmodium. This study evaluated the frequency of the symptoms that commonly occur in vivax malaria before and after treatment and the frequency of side effects reported during the term of antimalarial treatment with chloroquine and primaquine in standardized doses according to the protocol of the Brazilian Ministry of Health [The occurrence of symptoms specifically related to the intake of antimalarials and the overlap of symptoms of malaria with the side effects of primaquine and chloroquine may decrease adherence to treatment. Therefore, it becomes important to assess the main side effects of antimalarial drugs in patients from endemic areas, where treatment is aimed not only at improving the clinical status but also at reducing the transmission off Health .2, is bordered by the cities of Cruzeiro do Sul and Rodrigues Alves and the Republic of Peru. Mâncio Lima has 14,884 inhabitants living in urban areas (57.3%), rural or riparian areas (37.9%), and Indian villages (4.8%). The county is located 38 km from Cruzeiro do Sul and 650 km from Rio Branco.The study was conducted in the urban area of the municipality of Mâncio Lima, located in the Western part of the Brazilian Amazon, in the state of Acre. This municipality, with 5000 km vivax malaria were identified in microscopy stations of the city of Mâncio Lima before the medication and invited to participate in the study. Data collection took place between May and June 2013. After signing the informed consent or having it signed by parents or legal guardians, patients answered a questionnaire on malaria symptoms that occurred up to the time of diagnosis , to verify the occurrence of the following symptoms: nausea, vomiting, diarrhea, pale stools, loss of appetite, mesogastric and/or hypogastric pain, pain in hypochondrium, headache, myalgia, arthralgia, back pain, retroocular pain, fever, chills, sweating, malaise or weakness, itchy throat, jaundice, choluria, dyspnea, and bitter taste sensation, covering up thereby symptoms commonly occurring in malaria and overlapping with possible side effects of chloroquine and primaquine. Between 3 and 5 days after the start of medication, a second interview was conducted asking about the occurrence of the same symptoms plus some symptoms related to the use of chloroquine that are infrequent in malaria: blurred vision, itching, stinging sensation of the skin, convulsions, numbness, and insomnia.Patients with microscopic diagnosis ofThe data was entered into SPSS. The distributions of relative and absolute frequencies, median, mean, and standard deviations of the variables of interest were calculated. As primaquine and chloroquine may lead to the onset of symptoms and also intensify or reduce some of the symptoms of malaria, the occurrence of symptoms before and after drug treatment was analyzed with the McNemar's test for paired samples. Some of the symptoms considered to be side effects of chloroquine and primaquine and that are not usual in malaria were assessed only after drug treatment was started.The research protocol was submitted to the Ethics Committee for Experimentation with Human Beings of Universidade Federal do Acre and approved, CAE number 22876013.9.0000.5010 (2013). Informed consent was obtained from each adult participant or legal guardian in case of minors, before the start of the study. vivax malaria, 28 men and 22 women, aged between 7 and 68 years (average age 28.38 years). The average length of stay in an endemic area was 24.79 years, and about one-third of the study population (30%) lived in an endemic area for malaria between 11 and 20 years.The study included 50 people with3 in 66% of cases), 12% of them had between 301 and 500 parasites/mm3, and 12% were found to have 501–10000 parasites/mm3 , chills (54%), sweating (54%), and weakness (42%), as well as pain symptoms including headache (76%), arthralgia (48%), and lower back pain (52%). Less commonly reported symptoms were retroocular pain (36%) and myalgia (30%). Gastrointestinal symptoms occurred in low frequency, such as bitter taste (30%), decreased appetite (32%), nausea (22%), vomiting (16%), and pain in hypogastric or mesogastric region (20%). Before treatment, only 20% of patients reported dark urine, and no patient reported having had pale stools.P < 0.05, P < 0.05, P = 0.01, After initiation of treatment, a significant reduction in the prevalence of fever (38%), arthralgia (26%), back pain (32%), and retroocular pain (18%) , insomnia (46%), pruritus (22%), the feeling of “stings” into the skin (22%), and paresthesias (6%). No patient reported occurrence of seizures or unconsciousness after the start of antimalarial treatment. Most gastrointestinal symptoms appeared after the patient started the antimalarial treatment . vivax or falciparum malaria, the most frequent symptoms were also fever (97.9%), chills (71.7%), and headache (62.4%) [The symptoms most commonly seen in patients with malaria reported in the literature are fever, chills, sweating, headache, myalgia, and arthralgia. Da Silva-Nunes and Ferreira  evaluate (62.4%) . vivax malaria with chloroquine, a blood schizonticide, and primaquine  is very effective in reducing parasitemia and some of the symptoms caused by the Plasmodium infection, such as fever and algic symptoms. Fever tends to disappear between 24 and 48 hours after the beginning of treatment; parasitemia decreases significantly at about 48 to 72 hours, and pain symptoms decrease rapidly [The treatment of rapidly . Pinto e rapidly  reportedHowever, chloroquine can cause side effects or intensify symptoms already present, such as abdominal discomfort, nausea, vomiting, and diarrhea. These side effects can also occur during the use of primaquine  being suChloroquine may block the entrance of potassium into the cells and primaquine also has an effect on blocking sodium channels. Both may also have an effect on the chloride channels present in cardiac myocytes, which may explain the occurrence of cardiac and gastrointestinal side effects . In studThe itching and stinging sensation are explained by formation of haptens during the metabolism of chloroquine, which bind to degraded products erythrocytes or phospholipids involved in allergic reactions, stimulating the production of IgE-like antibodies and degranulation of mast cells and basophils. Also, the binding of chloroquine at the dermoepidermal junction may possibly result in stimulation of nerve fibers , causingRegarding primaquine use, the most prevalent side effect is hemolytic anemia, resulting in jaundice and choluria. Weakness, malaise, and abdominal pain are also reported as primaquine side effects. Data on primaquine use worldwide indicates that, among 200 million people, there were 14 deaths in six decades related to its use, from which 12 were caused by severe hemolysis . HemolytThe gene encoding the G6PD is located on the X chromosome (locus Xq28), with a recessive pattern of inheritance linked to sex. It can manifest as homozygous or heterozygous inheritance in females and hemizigous in males . In heteThe prevalence of G6PD deficiency is between 3 and 30% in endemic countries for malaria, corresponding to 350 million people . In the  falciparum malaria transmission (0.25 mg of base per kilogram) are usually considered to have low toxicity, because it is a single-dose treatment [ vivax malaria, aimed at preventing relapsing events, is a longer course (between 7 and 14 days), with varying dosage among malaria control policies, and therefore hemolytic events are more prone to happen [The consequences of severe hemolysis are severe anemia and acute renal insufficiency. These events are dependent on the extent of hemolysis, which is related to the severity of G6PD deficiency and the dose of primaquine . Recommereatment . Howevero happen .In a systematic revision, Monteiro et al.  detectedIn our study, the frequency of choluria (“dark urine”) increased significantly from 6% to 56% in malaria patients after primaquine was started. Since it is an event referred by the patient, we cannot be sure how many of the patients really presented with choluria, or how severe was the choluria, but it is known that G6PD deficiency occurs among Amazonian residents. Malaria and weakness also increased significantly from 42% to 64% after primaquine use. It is possible that the hemolytic anemia is the cause of weakness and malaise, reported by these patients. However, none of the patients studied suffered from severe hemolysis or severe anemia (data not shown) or required hospitalization during or after malaria treatment. Although severe events related to the use of primaquine were infrequent in our and other studies in the Amazon, it is important to bear in mind the possible adverse side effects of primaquine and also mild anemia occurring in patients with the less severe forms of G6PD deficiency, which can be associated or not with weakness. Kondrashin et al.  reported falciparum malaria patients from Myanmar, occurring in 16% of patients receiving a single dose. There is no clear evidence why primaquine can cause abdominal pain; it can be related to the direct effect of the drug on an empty stomach [Smithuis et al. reported abdominal pain as a side effect for primaquine in stomach  or relat vivax malaria with a combination of chloroquine and primaquine leads to decrease in fever and pain symptoms, it can lead to the onset of gastrointestinal symptoms and hemolysis or enhances these symptoms when already present in the patient with malaria, contributing to reducing treatment adherence, especially during the use of primaquine, which has a longer course of treatment. Moreover, the sensation of itching and stinging, although they do not occur in all patients, can be quite intense, hindering treatment with chloroquine. It is important to warn the patient of these possible side effects and seek measures to minimize them, either with concomitant use of antihistamines or antiemetics, according to the individual needs of each patient. The occurrence of severe hemolysis after primaquine use, although not common in Brazil, can still occur, and since Brazilian control programs currently do not have a policy for G6PD deficiency screening before malaria treatment is offered, health care personnel must be aware of it to interrupt primaquine use and manage its side effects properly.Although the present study has a limitation in the number of subjects studied, it was possible to detect significant association of symptoms and malaria treatment. Thus, while the treatment of


P = 0.055). A HRP–2/pLDH positive RDT was associated with an increased risk of severe malaria–related anemia compared to a negative RDT (adjusted odds ratio (AOR) 18.8, 95% CI 4.32 to 82.0, P < 0.001) and to a HRP–2 only RDT . There was no significant association between RDT result and the administration of parenteral quinine. These results offer preliminary evidence that specific patterns of antigen positivity on RDTs could be utilized to identify patients at an increased risk for complications of severe malaria.The detection of severe malaria in resource–constrained settings is often difficult because of requirements for laboratory infrastructure and/or clinical expertise. The aim of this study, therefore, was to explore the utility of a multiple antigen (HRP–2/pLDH) rapid diagnostic test (RDT) as a low–cost, surrogate marker of patients at high risk for complications of severe malaria. We reviewed programmatic data at a peripheral health center in Western Uganda. Available demographic and clinical data on all individuals presenting to the center who underwent an RDT for suspected malaria infection were reviewed. We fit logistic regression models to identify correlates of two outcomes of interest: 1) severe malaria–related anemia, defined here as hemoglobin ≤7g/dL and 2) receipt of parenteral quinine. 1509 patients underwent malaria testing with an SD FK60 RDT during the observation period. A total of 637 (42%) RDTs were positive for at least one species of malaria, of which 326 (51%) exhibited a single HRP–2 band and 307 (48%) exhibited both HRP–2 and pLDH bands, while 4 exhibited only a single pLDH band. There was a trend towards more severe anemia in patients with a HRP–2/pLDH positive RDT compared to a HRP–2 only RDT (β = –0.99 g/dl, 95% CI –1.99 to 0.02,  P. falciparum malaria, and approximately 550 000 die from manifestations of severe disease [Each year millions of African children are infected with  disease . In area disease . While d disease -5, effec disease ,7. Morta disease -11, and  disease ,13.A major challenge to the identification of severe malaria stems from the rigorous laboratory–based diagnostic criteria ,14,15. HPlasmodium species infections. Additionally, these RDTs have shown some ability to provide a quantitative estimate of parasite density due to the differing sensitivities of the respective antigens [Rapid diagnostic tests (RDTs) are low–cost, simple tools for the diagnosis of malaria and are particularly valuable in settings where microscopy is not readily available -21. Neweantigens -24. BecaWe evaluated programmatic data during the period January–March 2013, collected from the Bugoye Level III Health Center (BHC), in the Kasese District of Western Uganda . The health center functions as a referral center for the Bugoye sub–county, and serves a rural population of approximately 50 000 residents. The center houses a small laboratory with staff trained to perform light microscopy and RDTs, as well other basic tests, such as hemoglobin estimation.The objective of this retrospective, observational pilot study was to assess the diagnostic utility of a three–band (HRP–2/pLDH) RDT at a resource–limited, rural health center in malaria–endemic Western Uganda.We reviewed data on all individuals with fever who underwent an RDT for suspected malaria infection. We abstracted data on patient age, sex, village of residence, patient disposition, laboratory results, and medication prescriptions from clinic registers.P. falciparum/Pan RDT . The RDT is a validated antigen detection test with three individual bands signifying the control, the HRP–2, and the pLDH antigens [P. falciparum, whereas a unique pLDH line indicates infection with one or more of the other Plasmodium species. The presence of a positive HRP–2 line together with a pLDH line indicates an infection with either P. falciparum or a mixed–species infection.Laboratory diagnosis of malaria was made using the Standard Diagnostics FK60 Malaria Ag antigens ,25,26. TRDTs (Lot #090192) for the study were obtained from Kampala Pharmaceutical Industries , and stored in their original packaging at room temperature. RDTs were performed and interpreted by two members of the BHC laboratory staff in accordance with the manufacturer’s instructions. In cases in which the control line did not appear, the result was considered invalid and the test was repeated. Faint HRP–2 and pLDH test lines were considered positive.When available, we reviewed matched thick and thin smears for specimens with three–band positive tests. Smears were prepared using a modified Field’s Stain and examined by light microscopy . AsexualData were entered into Microsoft Excel  and analyzed with Stata 12.1 . We summarized patient characteristics and compared those with two– and three–band positive RDTs using Pearson chi–squared testing. We fit univariable logistic regression models to identify correlates for two outcomes of interest: 1) severe anemia, defined as a hemoglobin <7 g/deciliter (g/dL) and 2) receipt of parenteral quinine. While the WHO traditionally define severe malaria-related anemia a normocytic anemia of Hb <5 g/dL in children and <7 g/dL in adults, we used hemoglobin values of <7 g/dL as the clinically relevant outcome as it represents the transfusion threshold for severe malaria in adults and approaches the threshold at which transfusion should be considered in children when present with other clinical features .P–value of <0.25 [P–value of <0.05 was considered statistically significant in the final models.Our primary explanatory variable of interest was RDT test result, defined as HRP–2 vs HRP–2/pLDH positive. Secondary explanatory variables included sex, age, village distance from the health center, village elevation, and transmission season, defined as high (January) or low . We fit multivariable models including all variables that were significant in univariable models with a pre–specified of <0.25 . A resulEthical approval for study procedures and data collection was provided by the institutional review boards of Partners Healthcare and the Mbarara University of Science and Technology. Informed consent was not required by the ethical review committees due to the programmatic nature of the project.During the observation period, 1509 patients underwent malaria testing with an RDT. A total of 637 RDTs (42%) were positive for malaria. Of the positive RDTs, 326 (51.2%) exhibited a single HRP–2 band, 307 RDTs (48.2%) exhibited both HRP–2 and pLDH bands, while only 4 RDTs (0.6%) exhibited a single pLDH band. Characteristics of the study sample, stratified by RDT result, are summarized in Notably, the prevalence rate of HRP–2/pLDH positive results was twice as high among patients <15 years compared to those ≥15 years. The absolute number and proportion of HRP–2/pLDH positive RDTs declined during the transition from high to low transmission seasons, while that of HRP–2 only positive RDTs remained relatively stable.P. falciparum mono–infections, although there were 4 mixed–species infections . In addition, there were two non–P. falciparum mono–infections , which may have represented sub–patent P. falciparum co–infection not seen on microscopy. All four of the pLDH–only positive RDTs were identified as P. ovale mono–infections. Parasite density was reported for 89 of 90 of the three–band positive individuals. The mean density was 73 105 parasites/μL with a median density of 40 200 parasites/μL (IQR = 12 100–103 238). Parasite densities were only available for three patients with HRP–2 positive RDTs.Of the 307 patients with HRP–2/pLDH positive RDTs, 90 (29.3%) had corresponding blood smears available for interpretation. The majority  of smears from patients with HRP–2/pLDH positive RDT results demonstrated P. falciparum infections (including mixed–species infections) was 99.1% (95% CI 94.5% to 99.9%). The specificity was 75% (95% CI 53.0% to 89.4%), although only a small number of smears were prepared in patients with paired HRP–2 positive (n = 19) and negative (n = 19) RDTs. If, however, we assume that the two RDTs that were three–band positive but negative on microscopy represented true P. falciparum infections, as seen when confirmed by PCR in other studies [The sensitivity for  studies ,32, the P = 0.055, During the observation period, a total of 85 paired hemoglobin (Hb) levels were assessed in patients also undergoing an SD FK60 RDT. Children <5 years of age were more likely to have a Hb performed and had a higher proportion of values <7 g/dL (56.4% vs 26.1%) compared to those individuals ≥5 years of age. The mean Hb was 7.3g/dL (95% CI 6.6 to 8.1) in patients with a HRP–2 only positive RDT, and 6.3g/dL (95% CI 5.6 to 7.1) in patients with a three–band positive RDT  with a trend toward statistical significance.In multivariate logistic regression models, a HRP–2/pLDH positive RDT was the strongest predictor of severe anemia  of these patients. There were no differences in rates of inpatient admission between those with HRP–2 only and HRP–2/pLDH positive RDTs. Patients with HRP–2/pLDH positive results were more likely to receive treatment with parenteral quinine and anti–pyretics. In the multivariate analysis for correlates of receipt of parenteral quinine, the strongest associations were seen with three–band positive RDT and infection during high transmission season, although neither of these reached statistical significance (Our results show a trend towards patients with HRP–2/pLDH positive RDTs having more severe malaria–related anemia that those patients with an HRP–2 only positive RDT. While previous work with the SD FK60 in a reference laboratory found a correlation between parasite density and the presence of the pLDH band , our finWe postulate that the positive pLDH band, which is less sensitive than HRP–2, is a marker of higher parasite density -35. FindAn alternate explanation for the relationship could be HRP–2 antibody persistence leading to false–positive HRP–2 positive RDTs. Prior studies in Uganda have reported HRP–2 band specificities as low as 62% . These fP. falciparum malaria exceeding 95% [However, our clinical experience in Bugoye suggests that very few patients seek treatment for malaria outside of the government health center, given difficult terrain and high costs associated with private facilities. In settings where there is a low probability of prior treatment, such as our site, the specificity of the HRP–2 antigen is typically higher than that associated with studies conducted at urban referral centers ,41,42. Ading 95% .Our study, which was hypothesis generating in nature and primarily involved a retrospective review of routinely collected data, has a number of limitations. Foremost among these is the lack of available paired slides for all specimens. The available slides and Hb results were prepared in a non–random manner at the direction of the supervising clinical officer. We did, however, review smears for nearly 30% of those patients with three–band positive RDTs and Hb values for nearly 15% of patients with positive RDTs. The second major limitation of our study was that the use of the Sahlis method for Hb estimation, which while simple and inexpensive, can be prone to dilution and reading errors. We believe there should be minimal bias stemming from this method because errors would be stochastic and a single staff member interpreted all results. Finally, we performed this study at a single site and with a relatively small sample size, which reinforces the need for corroboration of these findings in larger, and more diverse patient populations. A follow up, prospective study is now under way.In summary, our findings suggest that patients with HRP–2/pLDH positive RDTs have more severe malaria–related anemia than patients with HRP–2 only positive RDTs. We postulate that the positive pLDH band, which is less sensitive than HRP–2, is a marker of higher parasite density and correspondingly, more severe anemia. These results offer preliminary evidence that multiple antigen RDTs could be utilized to identify patients at an increased risk for complications of severe malaria. If confirmed, multiple antigen RDTs may play a role in case management algorithms at peripheral health facilities. Further studies with more comprehensive data collection methods, larger sample sizes, and longer timeframes should be pursued before this approach is adopted.


Malaria was estimated to result in 214 million clinical cases and 438,000 deaths in 2015, primarily in children under 5 years of age. In Africa, malaria causes approximately 10% of all deaths in children under 5 years of age. The RTS,S/AS01 malaria vaccine has been tested in young children in phase III clinical trials and shown to be 18 to 36% efficacious against clinical malaria (P = 0.0006) but not in boys  in both age groups in which the vaccine was tested  . The sex-differential effect is highly significant (P = 0.001). There also was a tendency for RTS,S to be associated with a higher risk of fatal malaria in girls  but not in boys . It is counterintuitive that there was no reduction in fatal malaria associated with RTS,S; however, RTS,S was associated with a twofold-higher case fatality ratio in children who got severe malaria  recently published a position paper on malaria vaccines , with emP value of 0.0006 for girls and a mortality rate after RTS,S of 2.4% in girls compared to 1.8% in boys   . AlthougThere is precedent for the observation that infant girls experience increased mortality following receipt of vaccines. For example, in the 1980s, when the high-titer measles vaccine (HTMV) was introduced to prevent measles in children under 9 months of age, there was a twofold increase in all-cause mortality in girls, but no increase in boys, which led to withdrawal of the vaccine . It was Preclinical studies in animal models can help provide insights into the biological basis of these observations, but here too, analysis of potential sex effects has been lacking. Published studies of RTS,S or recombinant circumsporizoite protein in mice and nonhuman primates have only reported using adult females or have not reported the sex of the animals –9. Gener–a priori hypotheses in vaccine trials to identify and address potential risks early in the vaccine development process.The RTS,S vaccine is modestly effective at reducing clinical malaria in children, but the sex differences in all-cause mortality should be rigorously studied in both clinical trials and experimental animal models, particularly in light of prior experience with the HTMV. We seek to raise awareness about the need for additional research into how the RTS,S vaccine and, possibly, other vaccines are associated with greater mortality in girls but not boys. This will only be achieved if age and sex are considered in


Background. Malaria's prevalence during pregnancy varies widely in parts of sub-Saharan Africa, including Burkina Faso. The objective of this study was to evaluate the incidence of mother-to-child malaria transmission during childbirth at St. Camille Medical Centre in the city of Ouagadougou. Methods. Two hundred and thirty-eight (238) women and their newborns were included in the study. Women consenting to participate in this study responded to a questionnaire that identified their demographic characteristics. Asymptomatic malaria infection was assessed by rapid detection test Acon  and by microscopic examination of Giemsa-stained thick and thin smears from peripheral, placental, and umbilical cord blood. Birth weights were recorded and the biological analyses of mothers and newborns' blood were also performed. Results. The utilization of long-lasting insecticidal nets (LLINs) and intermittent preventive treatment with sulfadoxine-pyrimethamine (SP) were 86.6% and 84.4%, respectively. The parasitic infection rates of 9.5%, 8.9%, and 2.8% were recorded, respectively, for the peripheral, placental, and umbilical cord blood. Placental infection was strongly associated with the presence of parasites in the maternal peripheral blood and a parasite density of >1000 parasites/µL. Conclusion. The prevalence of congenital malaria was reduced but was associated with a high rate of mother-to-child malaria transmission. Plasmodium falciparum malaria transmission [ Plasmodium falciparum caused malaria is responsible for anemia in pregnant women [ Plasmodium falciparum in utero [In sub-Saharan Africa, about thirty-two million pregnant women are living in areas at risk forsmission . The presmission , Uganda smission , and Nigsmission . Two presmission , 6. Malasmission . Even asnt women . The sevnt women , 10. Thint women  and causnt women , 13. Gesnt women . Malariant women , 16 and nt women , 18. Thiin utero –21. Placin utero . HoweverThis cross-sectional study was conducted from September 2013 to February 2014 at Saint Camille Medical Center (CMSC) of Ouagadougou. Ouagadougou is located in the center of Burkina Faso with over a million and a half inhabitants. The climate in the area is characteristic of the Sudanese savannah, with a rainy season from June to October, the cold and dry season from November to January, and the hot and dry season from February to May. The transmission of malaria is stable with a high seasonal transmission from June to November. Pregnant women without clinical signs of malaria and who gave birth were enrolled in this study. Symptoms of clinical suspicion of malaria include fever, headache, vomiting, nausea, abdominal pain, diarrhea, and myalgia. Since malaria symptoms can be confused with clinical manifestations occurring in pregnant women during labor and delivery, only nonfebrile pregnant women were considered asymptomatic for malaria. Only 238 women who consented to participate in the study were recruited. These women completed a questionnaire to determine their demographic characteristics and the malaria prophylaxis used.From the women who consented to participate in the study, venous, placental, and umbilical cord blood was collected in an EDTA impregnated tube for hematological tests or dry tube for biochemical analysis.μL. Parasite densities were calculated according to the following formula: number of parasites counted × 8000/number of leucocytes counted [Asymptomatic malaria infection was assessed by rapid detection test (RDT) Acon  and by microscopic examination of Giemsa-stained thick and thin smears from peripheral, placental, and umbilical cord blood. All slides were read twice by experienced microscopists and the discrepancies resolved by a third reader . We used the standard method to determine parasitaemia as in most studies conducted in Africa. For thick films, parasites and leukocytes were counted in the same fields until 200 or 500 leukocytes were counted. Parasite densities were estimated using an assumed leukocyte count of 8,000 leukocytes/ counted . HaemoglP values below 0.05 were considered statistically significant.Data were analyzed using Statistical Package for Social Sciences  and EPI-Info version 6.04 dfr . A total of 238 parturients aged from 17 to 42 years were included in the study. The mean age of women was 26.46 ± 4.96 years. The age group of 21 to 25 years old was the most represented. The majority of women (86.6%) used long-lasting insecticidal net (LLIN) for vector control. Most women took at least one dose of sulfadoxine-pyrimethamine (SP) as an intermittent preventive treatment against malaria. Anemia was diagnosed in 18.4% of pregnant women, and 3.4% (8/238) had severe anemia .Two hundred and thirty-eight (238) mother-infant pairs were included in the study. Newborns had a mean weight of 3157 ± 394 grams (2400–4430 g). A small number of children (1.7%) were less than 2500 g birth weight. The mean of hemoglobin level in newborns rate was 14.9 g/dL. Anemia was present in 10.1% of neonates. Over 90% of the infants included in our study had an elevated creatinine. Transaminases were also measured in newborns and over 84% of them had normal aminotransferase levels .No association was found between the malaria infection in parturients and placental and umbilical cord blood and the clinical parameters of newborns.The parasites were present in the peripheral blood of 8.8% (21/238) of the pregnant women.P. falciparum was present in all cases of infections. The plasmodial mixed infections were found in 6 of the 21 mothers diagnosed positive by RDT. Thick smears revealed the presence of P. malariae in 2 mixed infections and P. ovale in one case. The geometric mean of parasites in the parturients was 14,477 parasites/μL (118–115073 parasites/μL) (ites/μL) . The inf P. malariae. The geometric mean of the parasitaemia observed was 21,630 parasites/μL   of the specimens tested. RDT also showed the presence of 6 mixed plasmodial infections in placental blood, including 2 cases ofites/μL) . The pla Plasmodium falciparum was the only parasite species present in the umbilical cord blood. The parasite density was low in the cord blood compared to those observed in placental and peripheral blood with a mean of 1444 parasites/μL (231–5102 parasites/μL)  .μL  for pregnant women in order to prevent malaria and its effects during pregnancy. IPT with at least two doses of sulfadoxine-pyrimethamine administered during the second and third trimester of pregnancy is an alternative prevention strategy whose effectiveness has been demonstrated by several studies in Burkina Faso and elsewhere in sub-Saharan Africa [ Plasmodium falciparum infestation. Most women of our study received two doses of SP in the second and third trimester of their pregnancies. Those who received one dose came in their third trimester.In areas with moderate or highn Africa , 24 by rIn this study, the use of malaria preventive measures such as LLINs and IPT-SP was high: 86.6% and 84.5%, respectively. We observed an increase rate in the use of LLINs and IPT compared to the rates of 42% and 27% reported in 2012 in the same health center . HoweverThe mean birth weight in the present study is comparable to that reported in a previous study in Burkina Faso . We foun Plasmodium malariae was identified in 11.1% of asymptomatic malaria cases in pregnant women. Our results are consistent with those of a previous study among children which had reported the prevalence of P. malariae ranging from 0.9 to 13.2% in Burkina Faso [ P. malariae reported in this study is higher than the prevalence of 3.6% found in Nigeria in pregnant women [ P. ovale in Burkina Faso, which varies from 0.5 to 1.8 [ P. ovale asymptomatic cases in parturients in this study. This is consistent with a recent study which has shown that P. ovale and P. malariae represent 5.9% of malaria infections in Senegal [In this study,ina Faso . The prent women . Despite5 to 1.8 , we iden Senegal . The pre Senegal , respect Senegal . The mal Senegal .There is a strong association between maternal parasite density, placental malaria, and cord blood parasitaemia. This association was also shown in Burkina Faso , 36. HowOur study has some limitations such as the sample size but also the threshold of parasites detection related to standard microscopy. In fact, the latter may underestimate the number of asymptomatic malaria cases among parturients and placental and cord blood malaria.The high use of preventive measures such as LLINs and IPT-SP among parturients has resulted in the reduction of asymptomatic malaria and severe anemia. These efforts should be pursued because there is a high risk of mother-to-child asymptomatic malaria transmission.Asymptomatic malaria in women is underestimated because placental malaria can be observed in parturients in the absence of parasites in their peripheral blood.


Plasmodium falciparum are the main confirmatory means for malaria diagnosis in most health facilities without microscopy services. As a consequence of this P. falciparum species diagnostic approach, non-falciparum malaria is not only under-diagnosed but entirely missed, thereby making the exact disease burden unknown. We thus investigated the prevalence of various Plasmodium spp. and associated burden of infection in selected communities in Zambia.Although malaria is preventable and treatable, it still claims 660,000 lives every year globally with children under five years of age having the highest burden. In Zambia, malaria rapid diagnostic tests (RDTs) that only detect Plasmodium spp. Multivariate logistic regression was employed to examine the association between background factors and malaria.Data from two malaria hyper-endemic provinces (Eastern and Luapula) of the 2012 National Malaria Indicator Survey (MIS), conducted between April and May 2012, were used. The MIS is a nationally representative, two-stage cluster survey conducted to coincide with the end of the malaria transmission season. Social, behavioural and background information were collected from households as part of the survey. Thick blood smears, RDTs and dried blood spots (DBS) were collected from children below six years of age. Slides were stained using Giemsa and examined by microscopy while polymerase chain reaction (PCR) was used to analyse the DBS for malaria Plasmodium spp. by PCR was 54.3% (95% CI 51–57.6%). Of the total Plasmodium isolates, 88% were P. falciparum, 10.6% were mixed infections and 1.4% were non-falciparum mono infections. Among the mixed infections, the majority were a combination of P. falciparum and P. malariae . Children two years and older (2–5 years) had three-fold higher risk of mixed malaria infections (aOR 2.8 CI 1.31–5.69) than children younger than two years of age.Overall, 873 children younger than six years of age were surveyed. The overall prevalence of Plasmodium spp. infections in this population stresses review of the current malaria RDT diagnostic approaches. The observed less incidence of mixed infections in children under two years of age compared to their older two-to-five-year-old counterparts is probably due to the protective maternal passive immunity, among other factors, in that age group.The high prevalence of mixed  Plasmodium spp. is not clearly known, although estimates of the global distribution of Plasmodium falciparum and P. vivax, the most predominant malaria Plasmodium spp., are widely cited [et al [P. malariae and P. ovale are unknown and the burdens of P. ovale and P. malariae are even more underrepresented in surveys where microscopy is employed since there are morphological resemblances between P. ovale and P. vivax.Malaria remains a significant public health problem in many countries throughout the world, especially in sub-Saharan Africa, despite being preventable and treatable . In 2010ly cited -6. MuellP. malariae cases are routinely reported and the anecdotal prevalence is estimated to be approximately 2%. Furthermore, Blossom et al. [P. vivax. Low prevalence of P. vivax on the African continent is attributed to high prevalence of the Duffy negative factor, a gene inherent among the majority of indigenous Africans that is otherwise responsible for refractoriness to P. vivax [Non-falciparum malaria may be present in Zambia given that m et al.  describeP. vivax -10.Plasmodium spp. are possible and are called mixed infection. Mixed malaria species infections are often not recognized or are underestimated by microscopists [P. vivax or P. falciparum malaria have demonstrated a high prevalence of up to 30% of mixed infections. Other malaria species have also been reported during convalescence, suggesting covert co-infections [P. malariae and P. ovale occurred almost exclusively in mixed infections [Infections with more than one scopists ,12. In Afections . In Manhfections . This siP. falciparum is the predominant cause of malaria (98%), Zambia has relied on the exclusive use of histidine-rich protein 2 (HRP2) or P. falciparum-based antigen detection RDTs. [The Zambia National Malaria Control Programme (NMCP) has been at the forefront of scaling up malaria interventions over the past decade, including the early adoption of artemisinin-based combination therapy and use of rapid diagnostics tests (RDTs) for clinical management of malaria. The 2011-2015 National Malaria Strategic Plan calls for the creation of malaria free zones during its current five-year span. Therefore, understanding the extent to which current tools are able to achieve these goals is important. It follows that further understanding of the distribution of local malaria parasite species is important for developing appropriate preventive as well as diagnostic and treatment option. Unfortunately, many malaria-endemic countries, especially in sub-Saharan Africa, are just beginning to understand the relative levels of prevalent malaria parasite species as malaria control programmes begin to expand in the pursuit of malaria elimination. With the anecdotal evidence that Plasmodium spp. causing malaria in Eastern and Luapula provinces of Zambia using stored samples from the Malaria Indicator Survey of 2012, conducted to monitor the progress of malaria control implementation efforts by the National Malaria Control Program (NMCP). This information is necessary to guide and inform malaria management and control strategies as well as provide country-specific evidence for strategic malaria diagnostic service choices and treatment options.This study sought to determine the proportion of mixed Data and samples from MIS conducted between April and May 2012 for two provinces in Zambia were utilized. MIS’s have been conducted biannually since 2006 and are used by the NMCP to evaluate progress in scaling up malaria control interventions and to monitor levels of malaria parasite prevalence among children aged five years and younger . The 201Study samples from children under six years of age were used from two provinces, Eastern and Luapula. These two provinces were noted to be hyper-endemic for malaria, with the highest levels of malaria parasite prevalence among sampled children during the 2012 MIS study . For thiPlasmodium spp. was performed using nested PCR, performed at the National Malaria Control Centre molecular laboratory with further validation on random samples at Tulane University. DNA was extracted using the chelex method [et al [Identification of x method  and the d [et al .P. falciparum, P. vivax, P. ovale and P. malariae). In the second reaction, different primers were run for the four species separately. The band sizes of the products were as follows: P. falciparum 205 bp, P. malariae 140 bp, P. ovale 800 bp and P. vivax 120 bp.The protocol was designed such that in the first reaction a conserved region for the four species was amplified (2), 200 μM of each dNTP 2.5 units of Taq DNA polymerase, PCR primers (10 μM of rPLUf and rPLUr) and 5 μL of DNA template and water in a final volume of 25 μL. In the second reaction, the same master mix was used but with different primer concentration (10 μM of each of the species-specific primers in separate tubes), 2 μL DNA template from the first reaction in a 25 μL reaction volume. The PCR was run in a gene Amp PCR system 9700  under the following conditions: the first reaction had denaturation at 94°C for one minute followed by 35 cycles at 94°C for one minute, 58°C for two minutes and 72°C for five minutes; the second reaction (with four tubes for each primer) had denaturation at 94°C for one minute, followed by 30 cycles at 94°C for one minute, 58°C for two minutes and 72°C for five minutes. The final cycle was followed by an extension time of five minutes at 72°C. A 2% agarose gel stained with ethidium bromide was run for visualisation of the PCR product [The PCR mixture for the first reaction contained 12.5 μL of a 2X master mix containing Taq buffer , and in some cases Fisher’s exact tests for proportions, were used to test for independence. Multivariate logistic regression was used to examine the association between background factors with malaria between comparable categories. The distribution of age as a continuous variable conformed to normality as assessed by probability plots.Demographic and laboratory data of the participants’ records was analysed with Stata version 11  with the cluster effect accounted for in the analyses. The Mantel-Haenszel chi square test  children from Eastern Province and 369 (42.3%) from Luapula Province. Larger proportions (95.7%) of these children were classified according to the national census as living in rural clusters, with only 4.4% living in an urban cluster  with 11.6% due to non-falciparum species and 10.3% were mixed infections ; P. falciparum, P. malariae and P. ovale (6%); and P. falciparum and P. ovale (2.1%). The combinations of P. falciparum, P. malariae and P. vivax (0.2%), and P. falciparum and P. vivax (0.2%) were rare  (Figure –57.6% (Fns Table . The obsP. ovale .1%. The Plasmodium infections (Table Assessment of factors associated with mixed ns Table  showed aPlasmodium species in the studied population. Although the malaria prevalence is predominantly due to P. falciparum, the prevalence of non-falciparum mixed infections was significantly higher than previously reported. This high prevalence partly explains why malaria still remains one of the leading causes of morbidity and mortality in Zambia despite the current control and management measures. The high prevalence of malaria due to all four species and in both mono and mixed infections patterns clearly reveals not just the presence of non-falciparum species but also the dynamic and evolving nature of the malaria epidemic. The 11.6% is actually close to the anecdotal level of non-falciparum-only infections known, but mixed falciparum infections account for a more significant contribution of the total infection prevalence than previously considered. These findings suggest that there is need for continued monitoring of non-falciparum infection prevalence in this population so as to decide when species-specific RDTs should be introduced for diagnostic purposes. The species-specific diagnosis will be important when the prevalence of malaria in this population reduces and as the country moves towards malaria elimination. In addition, Coartem, the first-line treatment drug in Zambia, does not clear Plasmodium hypnozoites and radical treatment with primaquine, effective against hypnozoites may play an important role in the control and eventually elimination of vivax and ovale malaria, and malaria consequently [The study showed a significantly high prevalence of malaria in two provinces of Zambia, demonstrating for the first time to our knowledge the prevalence of the four major equently .falciparum malaria cases were present in the study population necessitates the need to have diagnostic tools or approaches at the national level that should facilitate the detection of such non-falciparum malaria cases. While it should not necessarily be the primary diagnostic tool, diagnostic approaches that have the capacity to further investigate and detect non-falciparum malaria may become important in future. For health facilities where microscopy services are present, there is a need to conduct refresher courses for laboratory staff and microscopists with an emphasis on Plasmodium species-wide microscopy so as to strengthen non-falciparum malaria diagnosis as has been reported and advocated elsewhere [Differential species burden patterns observed were not surprising as similar observations have been reported elsewhere ,23. Howelsewhere ,10,24.Plasmodium malaria compared to individuals older than five years of age. Children younger than two years in our study were found to be less likely to have mixed malaria infections compared to those aged from two to five years. This could be partly explained by the maternally derived antibodies believed to offer protection from infections. Riley et al reported that neonates and infants are relatively protected from clinical malaria, although the mechanism of the protection is not clearly understood [Among the factors associated with mixed malaria infections, age was found to be a predictor. This is in agreement with a similar study by Guerra-Neira et al  who repoderstood . Indeed,derstood ,28. Notederstood , our stufalciparum infections might vary with transmission patterns, demographic trends and geographical contrasts. There is the need for these to be monitored through a functional surveillance system so as to understand the epidemiological profile. Given that most of the non-falciparum malaria cases occur as mixed infections, the use of HRP2-based RDTs can still continue in health facilities but need to be strengthened with improved and enhanced microscopy to facilitate detection of the non-falciparum malaria cases. Finally, there is a need for further studies to have a complete, detailed profile and epidemiology of non-falciparum species of malaria in the country, especially in the era of malaria elimination activities in the country and region.This study reveals the presence of mixed infections in two provinces of Zambia with an overall prevalence of 10.3%. The major factor associated with mixed infection was the age of the child. This might call for repackaging of control and management measures at national level. The presence of non-


Red blood cell (RBC) polymorphisms are common in malaria endemic regions and are known to protect against severe forms of the disease. Therefore, it is important to screen for these polymorphisms in drugs or vaccines efficacy trials. This study was undertaken to evaluate associations between clinical malaria and RBC polymorphisms to assess biological interactions that may be necessary for consideration when designing clinical trials.In a cross-sectional study of 341 febrile children less than five years of age, associations between clinical malaria and common RBC polymorphisms including the sickle cell gene and G6PD deficiency was evaluated between November 2008 and June 2009 in the middle belt of Ghana, Kintampo. G6PD deficiency was determined by quantitative methods whiles haemoglobin variants were determined by haemoglobin titan gel electrophoresis. Blood smears were stained with Giemsa and parasite densities were determined microscopically.The prevalence of clinical malarial among the enrolled children was 31.9%. The frequency of G6PD deficiency was 19.0% and that for the haemoglobin variants were 74.7%, 14.7%, 9.1%, 0.9% respectively for HbAA, HbAC, HbAS and HbSS. In Multivariate regression analysis, children with the HbAS genotype had 79% lower risk of malaria infection compared to those with the HbAA genotypes . HbAC genotype was not significantly associated with malaria infection relative to the HbAA genotype . G6PD deficient subgroup had a marginally increased risk of malaria infection compared to the G6PD normal subgroup .These results confirm previous findings showing a protective effect of sickle cell trait on clinical malaria infection. However, G6PD deficiency was associated with a marginal increase in susceptibility to clinical malaria compared to children without G6PD deficiency. It is estimated that there are about 216 million cases of malaria each year and about 655, 000 deaths worldwide Glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemoglobins S (HbS) are common genetic disorders in sub-Sahara Africa Sickle cell trait and other red blood cell−related genetic factors, such as alpha-thalassaemia, as well as metabolic abnormalities including glucose-6-phosphate dehydrogenase (G6PD) deficiency have also been associated with protection against clinical malaria The prevalence of G6PD deficiency in Ghana is estimated to be about 12% among pregnant women The Kintampo North Municipality lies within the forest-savannah transition belt of Ghana and has high levels of malaria transmission Ethical approval of the study was given by the Kintampo Health Research Centre Institutional Ethics Committee. At enrolment, the study objectives, risks, benefits and procedures of the study were explained to all caregivers whose children participated in the study to seek their written informed consent. Caregivers gave written informed consent on behalf of participating children. All data collected are kept in locked cabinets to ensure confidentiality and participants’ identity and records were anonymized prior to analysis.This was a cross-sectional study, carried out at the Kintampo North Municipal Hospital in the Brong Ahafo Region of Ghana from November 2008 to June 2009. Children less than five years of age who reported of fever  and without symptoms of severe illness were enrolled. All enrolled children were residents of the Kintampo North Municipality as per study inclusion criteria where malaria transmission intensity is as high as 269 infective bites/person/year and malaria parasite prevalence is >50% Individual written informed consent was obtained from all caregivers prior to enrolment. A study clinician initially took clinical history and carried out a physical examination on all children irrespective of their disease condition, after which blood samples were taken to test for malaria and host genotypes including sickle cell. Recruitment into the study covered both the dry (November) and rainy (June) seasons as the causes of fevers are not the same throughout the year and each child was seen once at the outpatient department (OPD) without any follow up visit. Any child diagnosed as having clinical malaria, defined as fever  plus a positive malaria blood smear, was treated with artesunate/amodiaquine; other diseases diagnosed were treated according to the Ghana Health Service (GHS) standard treatment guidelines. Information on socio-demographic background was obtained through the use of a short questionnaire. About 0.5 ml of finger-prick capillary blood was collected into EDTAK2 anticoagulant tubes and used for the laboratory analysis at the Kintampo Health Research Centre (KHRC) Clinical Laboratory. All blood samples were obtained prior to treatment with artesunate/amodiaquine.G6PD status was determined by the Randox quantitative method The Randox quantitative method classifies the G6PD deficiency status of the participants using a cut-off of 4.1 U/g Hb. All participants who measured enzyme activity <4.1 U/g Hb were classified as “Deficient” while those with enzyme activity at 4.1 U/g Hb and above were “Normal”. There was no classification for partial deficiency. Two levels of quality controls, deficient  and normal levels  were analyzed before the analysis of test samples.Haemoglobin levels were determined using an ABX Micros 60 Haematology analyzer . Scheduled external quality assessment programme and daily internal quality controls were followed as quality measures The Helena electrophoresis system  was used to electrophorese haemolysates on Titan III cellulose acetate plates. The haemolysate was prepared by mixing 1 part of whole blood to 3 parts Helena haemolysate reagent (0.005 M EDTA with 0.01% potassium cyanide). Electrophoresis was performed at 350 V for 25 minutes in a tris-EDTA/boric acid buffer . The electrophoretic procedure was performed as recommended by the manufacturer. Helena Hemo quality controls provided and performed daily were used as a maker for band identification.Thin and thick blood smears were prepared; the thick film was fixed in methanol after drying and both thin/thick films stained with 10% Giemsa. The parasites were counted against 200 leukocytes and then extrapolated to parasites per microlitre of blood using the absolute leucocytes counts 2-test and means of continuous and normally distributed variables such as haemoglobin level were compared using t-test (two-group comparison) and one way ANOVA (three-group comparison). Statistical significance was set at p<0.05. Using a forward selection method, a logistic regression model controlling for potential confounding variables such as antimalarial use and bed net use were used to investigate the associations of G6PD and sickling status on clinical malaria. High density malaria parasitaemia (HDP) was defined as malaria parasites ≧10,000/µL of blood and severe anaemia was defined as haemoglobin concentration level less than 6.0 g/dL.All the data were double-entered and verified for consistency using Microsoft Access software version 2007. Final dataset was analyzed using STATA statistical software . Proportions of categorical variables such as bednet use or antimalarial use were compared using the χA total of 364 children were approached to be consented. Ninety-four percent (341/364) were enrolled; 75% of the children enrolled into the study were above 11 months of age with remaining 25% either aged 11 months or below . About 5About one-fifth (19%) of the children were G6PD deficient ; 55.4% . HbSC (1Children with HbAC genotype had the highest mean haemoglobin (mHb) of 10.1 g/dL (95% CI: 9.0–10.5) and HbAS genotype had the lowest of 9.5 g/dL (95% CI: 8.4–9.7) . G6PD noThe overall prevalence of clinical malaria in the entire cohort of children was 31.9% (107/336). Univariate and multivariate models for clinical malaria infection defined as the presence of fever and parasites have been summarized in The risk of malaria in children who used bed nets was not significantly different from those who did not use bed nets . Children who did not use antimalarial had higher odds of contracting clinical malaria as compared to children who used antimalarial . Children aged 12–59 months were more likely to have malaria as compared to children aged 0–11 months . Among male children, the G6PD deficient children had higher odds of contracting malaria but this was not statistically significant . Similarly among females, children who were G6PD deficient were more likely to contract malaria as compared to G6PD normal children .2 = 1.232, p = 0.54).The geometric mean parasite density (GMPD) observed among children with the red cell polymorphism variants were: HbAC [65451/µL (95% CI: 22424–91039)], HbAS , HbAA [43954/µL (95% CI: 30154–64071)] . There w2 = 0.162, p = 0.69) (GMPD among G6PD deficient [29776/µL (95% CI: 14858–59672)] and G6PD normal  children were similar (one way ANOVA Bartlett’s test Chi = 0.69) .High-density parasitaemia–HDP  was observed in 16.8% (22/131) of HbAC and 3.1% (4/131) among the HbAS when compared to 80.2% (105/131) of the HbAA subgroup (P = 0.007) . G6PD noOur results have demonstrated a reduction in malaria risk in children with the sickle cell trait, an association that remained statistically significant in multivariate regression analysis after controlling for bed net and antimalarial drug use. Protection associated with HbAC was less significant as compared to HbAS genotype. G6PD deficiency was however associated with marginal susceptibility with clinical malaria.Many studies globally Studies conducted in Nigeria Nineteen percent (19%) of our study participants had G6PD deficiency, as determined phenotypically. This is consistent with the prevalence of G6PD deficiency assessed as part of a Phase III antimalarial drug trial in six African countries including Ghana et al. found that G6PD deficiency was associated with a 46–58% reduction in risk of malaria in African children Plasmodium falciparum were less ill than non–G6PD-deficient individuals Plasmodium falciparum infection is usually not lethal in G6PD deficient individuals Plasmodium parasites can proliferate less efficiently in their erythrocytes The protective role of G6PD deficiency in clinical malaria has also produced conflicting results among various studies et al. (2007) found out that partially deficient females suffer the same morbidity and mortality of Plasmodium falciparum infection as non-deficient patients et al. (2003) also found fewer Plasmodium falciparum infections in pregnant women with partial G6PD deficiency Plasmodium falciparum infection was 60% among the G6PD deficient subgroup and 42% among G6PD normal subgroup with a weak evidence to support a statistical difference (p = 0.03).However it is still uncertain whether this advantage applies only to individuals who are totally deficient of the G6PD enzyme. Guindo falciparum malaria is that of Bienzle et al. in Nigeria where 702 children aged 9 months to 6 years presenting at hospital with acute febrile illness were assessed P. falciparum parasitaemia which is quite similar to what has been observed in our study population One of the most frequently cited field studies of the relationship between G6PD deficiency and Plasmodium parasitisation in G6PD deficient children.Nevertheless variations in our findings with others reported elsewhere could have resulted from differences in methods and populations involved. We were unable to screen for additional co-infections, such as HIV-1 and bacteraemia, believed to influence the course of malaria infection Unlike insecticide treated bed net usage, children who had not been treated with antimalarial drug were more likely to have clinical malaria. Therefore anti-malaria use both as a preventive or curative treatment is likely to reduce prevalence of the clinical malaria especially in children less than 5 years.We could not examine the effects of three genotypes  due to their small proportion and the statistical limitations associated. This suggests the need for a bigger study in the area and under different levels of malaria transmission to better understand the patterns of protection or risk of malaria that these other phenotypes may confer in this population.We acknowledge some limitations of this study. First, we did not investigate the role of thalassaemia in clinical malaria which could impact on the risk estimation in this study. The prevalence of thalassaemia particularly α-thalassaemia is more than 80% in parts of Africa, Southeast Asia and Melanesia From the perspective of protecting individuals from clinical malaria, HbAS genotype has showed a clear advantage and superiority as compared with HbAC and G6PD deficient genotypes in our study population. The biological interactions between HbAS and clinical malaria has suggested the need to critically look at HbAS as an innate host factor in randomized trials or observational studies for detecting the effect of interventions, especially in areas of high HbAS prevalence.As millions of people are exposed to malaria annually, there is the need to continually explore all possible associations that red cell polymorphisms may have with disease susceptibility or protection when designing malaria intervention programs.Table S1Final dataset for public repository (in STATA file).(DTA)Click here for additional data file.


Microscopy of peripheral blood thin and thick films remains the reference for malaria diagnosis. Although Giemsa staining is most commonly used, the Leishman staining method provides better visualization of the nuclear chromatin pattern of cells. It is less well known whether accuracy of parasitaemia assessment is equally accurate with the latter method.Peripheral blood thin and thick smears from consecutive febrile patients admitted to Ispat General hospital, Rourkela, Odhisa, India, were stained with Giemsa and Leishman stain. Methods were compared for species identification, parasite quantification, and ability for identification of alternative diagnoses.10parasitaemia between methods were strongly correlated (r2 = 0.9981). In parasite negative patients, thin smear assessment contributed to making a diagnosis in 276/1,180 (23%) of cases. These assessments were better made in Leishman-stained preparations, especially for the assessment of morphological changes in red and white cells.Blood films from 1,180 fever patients were compared according to staining method, of which 111 were identified as parasitaemic using Giemsa and 110 with Leishman staining. The Kappa value as a measure of agreement between methods was 0.995 (p < 0.001), and the logPlasmodium parasites. The Leishman method is superior for visualization of red and white blood cell morphology.Leishman’s staining method for thin and thick smears is a good alternative to Giemsa’s stain for identifying  Early parasitological diagnosis of malaria is the cornerstone of malaria treatment and control. Although rapid diagnostic tests for malaria are increasingly used, microscopy remains the reference standard for malaria diagnosis . MicroscPlasmodium parasites in Leishman compared to Giemsa-stained thin and thick peripheral blood films was assessed in patients presenting with fever at the time of admission in the hospital. The staining methods were also compared, regarding their contribution to suggesting alternative diagnoses than malaria.In malaria-endemic regions, thin and thick smear preparation are used to diagnose malaria, but examination of the blood elements can also reveal other causes of fever including leukaemia, or clues for viral infection or bacterial sepsis. For this purpose, Leishman stain could be preferable to Giemsa, since visualization of the nuclear chromatin pattern and cytoplasmic colour contrast are known to be clearer with the Leishman method. The Leishman stain also takes less time for preparation than Giemsa staining. However, these advantages should not be at the expense of accuracy in parasitaemia assessment, which is the primary purpose of the malaria blood slide examination. In the current study the qualitative and quantitative assessment of Plasmodium falciparum[The study was performed at Ispat General Hospital (IGH), Rourkela situated in the Sundergarh district of Odisha, India. With a population of 36.7 million , Odisha has the highest population at risk for malaria in India. In 2010 Odisha contributed nearly 24% of total cases and 17% of total malaria mortality in India [In the current study, EDTA anti-coagulated blood samples were collected from consecutive febrile patients admitted to the hospital. Samples were processed without delay to avoid morphological alteration of parasites related to storage . A samplSlides were independently read by two of the authors (SS and AKM). Both readers have more than 15 years experience in slide assessments. Assessments of presence of malaria parasites, quantification of parasites, and non-quantitative assessment of alternative diagnoses were compared between readers. In case of non-congruent results in the qualitative assessment of malaria species or in suggestions of alternative diagnoses, a consensus was reached through joint review of the case. In both Giemsa- and Leishman-stained slides, the thick smear was used for diagnosis and quantitative assessment, whereas the thin smear for species identification. Parasitaemia in all malaria- positive cases was quantified per 200 white blood cells (WBC), independently by two microscopists. Parasitaemia (PRST) per μL was calculated as: PRST/μL = 40 × PRST/200 WBC, assuming a WBC count of 8,000/μL. In case of high parasitaemia (>50-70 parasites/field in thick smear) a pinhole eyepiece was used to avoid over-estimation of parasitaemia.The microscopists were blinded to the status of the patient and the reading results of the alternatively stained slide from the same patient.10of the parasitaemia per μL was used to account for the natural increase in variability with increasing parasite density [10 parasitaemia between methods was assessed by the method of Pearson. A Bland-Altman plot was produced comparing the differences between methods according to parasitaemia. Agreement of methods was assessed by calculation of the Kappa value and the intraclass correlation coefficient was calculated using SPSS statistical software package For comparison of the quantitative parasite counts, the log density . The corA peripheral blood film was taken from 1,180 patients with fever, and stained according to both Giemsa and Leishman. A total of 111 patients were parasitaemic by light microscopic assessment of the Giemsa-stained slide, compared to 110 patients with Leishman-stained slides. The Kappa value as a measure of agreement between methods was 0.995 (p < 0.001). A total of 276 patients showed other abnormalities  of cases  PRST/μL in the Giemsa-stained slides and 281  PRST/μL in the Leishman-stained slides. LogPlsmodium falciparum and Plasmodium vivax parasitaemia, and has the advantage that abnormalities in other blood elements, in particular WBC, are better identified. Parasite counts compared between the two separate methods were highly correlated and the methods showed a high level of agreement. As expected there was an increase in the absolute difference in parasite counts at a higher parasitaemia [This study shows that the Leishman-staining method is a good alternative to the Giemsa method for quantification of sitaemia , 9. Specsitaemia . AssessmExamination of the thin film enables additional diagnoses. In the series one case of acute lymphoblastic-leukaemia, five cases suggestive of infectious mononucleosis, 68 cases suggestive of viral infections based on the presence of atypical lymphocytes, and 202 cases of probable invasive bacterial infections (left shift and toxic granulation in neutrophils) were identified. In all these cases Leishman stain was superior to Giemsa stain for the morphologic assessment of leukocytes. The fineness of chromatin pattern and colour of the nucleus was clearer with Leishman stain compared to Giemsa. Also red blood cell morphology was more easily assessed with Leishman stain. In addition to this, the time taken for staining is shorter with the Leishman method, which can be an important advantage in countries such as India where diagnostic laboratories are sometimes understaffed.Since light microscopy of the Giemsa-stained slides were used as the reference method, the study did not evaluate the sensitivity of the two staining methods for detection of low parasitaemias. Also, the comparison of preservation of the staining quality could be subject to future study.Plasmodium parasites. The Leishman method is superior for visualization of red and white blood cell morphology, which can be an advantage for the diagnosis of diseases other than malaria identifiable by changes in these blood elements.This study shows that the Leishman’s staining method for thin and thick smears is a good alternative to Giemsa for identifying