Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention.P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7  but not in those without detectable parasitaemia .Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection.We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to  Plasmodium falciparum malaria is still a major cause of morbidity and mortality in sub-Saharan Africa where the greatest burden of disease is borne by young children P. falciparum such that clinical malaria tends to be less frequent in children over 5 years of age and adults. Despite this, older children and adults remain susceptible to asymptomatic, often chronic, P. falciparum infections to which immunity probably never occurs P. falciparum infection per se is further evident in the epidemiological pattern of clinical malaria and asymptomatic parasitaemia in areas endemic for P. falciparum transmission. Whilst the frequency of clinical malaria among individuals resident in these settings tends to be highly seasonal, that of asymptomatic P. falciparum infections remains relatively constant throughout the year P. falciparum infection must often develop clinical malaria perhaps following infection by parasites with different antigenic properties from those associated with the asymptomatic infection. The specific host factors underlying susceptibility to clinical malaria despite the ability to sustain asymptomatic P. falciparum infection are poorly understood.P. falciparum infection at the time of survey, among others P. falciparum infection at the time of survey have been observed to be higher and to exhibit stronger correlation with reduced risk of disease during follow-up when compared to responses in children without detectable parasitaemia Longitudinal studies in which individuals are recruited at cross-sectional surveys and their risk of developing disease assessed in a defined follow-up period have been a key tool in identifying correlates of immunity to clinical malaria P. falciparum infection at the time their full differential blood counts are measured, high ML ratio is associated with an increased risk of clinical malaria episodes during follow-up.Full differential cell counts from peripheral blood are routinely used as an aid to clinical diagnosis of a wide range of infectious diseases, including malaria, where they have been associated with prognosis in several studies The Kenya Medical Research Institute National Ethical Review committee granted approval for this study. Written informed consent was obtained from parents or guardians of all study participants.P. falciparum parasites by microscopy. Children that were febrile at the time of survey, whether carrying parasites in peripheral blood (“parasite positive”) or not (“parasite negative”), were treated appropriately by a clinician at the Kilifi District Hospital and excluded from this study. No treatment was administered to asymptomatic parasite positive children. A summary of the numbers of parasite positive and parasite negative children included in this study from each of the five surveys and their respective characteristics are shown in This study was conducted at Kilifi District Hospital on cohort data from children resident in Junju sub-location of Kilifi district, Kenya, and is part of the Kilifi Health and Demographic Surveillance System Weekly active surveillance was used to monitor episodes of clinical malaria by trained field-workers. In addition, passive surveillance was undertaken by trained field-workers based at villages across the study area and at local dispensaries. Monitoring for parasites was only done in children with a history of fever. ML ratios were not measured at the time of clinical malaria episodes. Detailed surveillance procedures have been published previously st December 2011. Observed associations were then confirmed using unadjusted Cox regression models to estimate the relationship between explanatory variables and time to first clinical malaria episode. Where data from all five cohorts were considered in the Cox regression modeling, ML ratio or the respective cell count data were included as time-varying covariates.All analyses were done in Stata™ version 11 and P value <0.05 used as the cut-off for statistical significance. Negative binomial regression models were used to estimate the associations between clinical malaria episodes and explanatory variables, offset by the period of observation during which children were at risk of clinical malaria. The risk period was defined as the period between date of recruitment to cross-sectional survey and either the date when an individual was lost to follow-up or 31st December 2011. Then, the effect of adjustment for age and antibodies to the parasite blood-stage antigens apical membrane antigen 1 (AMA1) and merozoite surface proteins 2 (MSP2) and 3 (MSP3), respectively, was examined. Finally, to assess the temporal stability of observed associations between ML ratio and risk of clinical malaria, full differential blood count data from children sampled in the May 2008 survey (N  =  319) or in any of four other cross-sectional surveys conducted in May 2007 (N  =  319), 2009 (N  =  332), 2010 (N  =  327) and 2011 (N  =  356), respectively, were used. The number of clinical malaria episodes in the respective inter-survey periods  were used as the dependent variable in the negative binomial regression modeling while accounting for: i) multiple sampling of individuals recruited into more than one survey using the robust sandwich estimator, ii) the year of survey as a fixed effect and, iii) age at each survey as a continuous variable.The May 2008 cross-sectional survey was used as the baseline survey for the primary analysis since children sampled at this time point had antibody data available, allowing adjustment for known antibody correlates of immunity to malaria. First, full differential blood count data from the 319 children sampled at the May 2008 baseline survey were used to estimate the association between ML ratio and total number of clinical malaria episodes experienced during the period between sampling at the May 2008 baseline survey and either the date when an individual was lost to follow-up or 31st December 2011. Parasite positive children accounted for fewer of these episodes (121 compared to 641 in parasite negative children). In a negative binomial model predicting frequency of clinical malaria episodes using parasite positive/negative status while accounting for age, year of survey and multiple sampling of individuals, carriage of asymptomatic P. falciparum infection at survey was associated with reduced risk of clinical malaria (Incidence rate ratio (IRR)  =  0.7, 95% confidence interval (CI) 0.56, 0.85, P  =  0.0004).A total of 762 episodes of clinical malaria  were experienced over the entire duration of the study, that is, between survey at May 2007 and 31To assess the relationship between ML ratio and risk of clinical malaria we first used the May 2008 survey as baseline since antibody data were available for children sampled at this time, allowing adjustment for known antibody correlates of immunity to malaria. Whereas all models were adjusted for age, associations were first estimated before adjustment for antibodies and the effect of this adjustment assessed thereafter.High ML ratios were associated with high frequency of clinical episodes among children parasite positive at baseline  but no association was observed among children parasite negative at baseline . Survival analysis using time to first episode as the primary endpoint confirmed the association between high ML ratio and risk of clinical malaria . FurtherP. falciparum blood-stage antigens There was no correlation between ML ratio and age at recruitment among parasite positive  or among parasite negative children . We considered the possibility that the association between ML ratio and disease might act through an association between ML ratio and protective antibodies to certain P. falciparum infection. Despite the statistical significance among parasite negative children the Spearman's rank correlation coefficients were relatively low  as a dependent variable in the negative binomial regression modeling while accounting for i) multiple sampling of individuals recruited into more than one survey using the robust sandwich estimator, ii) the year of survey as a fixed effect and, iii) age at each survey as a continuous variable. A positive association between ML ratio and risk of multiple episodes of malaria was evident among children that were parasite positive at the time of measuring their ML ratio . No such association was observed in parasite negative children . These findings were again confirmed by survival analysis using time to first episode as an endpoint . However, among parasite positive children, increased risk of clinical malaria episodes was associated with a high monocyte count  and a low lymphocyte count . This is in agreement with the high monocyte count and low lymphocyte count observed in previous studies of children presenting to hospital with clinical malaria Collectively our data suggests that in the context of an ongoing infection by malaria parasites, the relative frequency of monocytes to lymphocytes in peripheral circulation reflects an individual's capacity to mount an effective immune response. This is not surprising given that monocytes are an essential component of the innate immune response that acts as a link to the adaptive immune system through antigen presentation to lymphocytes. Thus any factors that perturb the function or relative frequency of either cell type could potentially affect an individual's response to infection. So why is the association between ML ratio and risk of clinical malaria only evident among parasite positive children?P. falciparum that allows greater power to detect associations between ML ratio and clinical malaria needs to be ruled out.Derangement in monocyte or lymphocyte function or frequency is likely to be most evident during an infection, and this may be supported by the observation that ML ratios were consistently higher among parasite positive children in all five cross-sectional surveys see . Thus, oP. falciparum, but to also correlate with development of clinical symptoms P. falciparum the adaptive immune response “learns” to produce anti-inflammatory cytokines that effectively temper the pro-inflammatory immune response, leading to a lower ML ratio and prevention of immunopathology. Assessment of the relationship between ML ratio and cellular immune responses is clearly needed if the mechanism underlying the association between elevated ML ratio and susceptibility to clinical episodes of malaria is to be determined.The innate immune response, through pro-inflammatory cytokines such as IFNγ, is thought to contribute to the initial control of parasitaemia following infection by P. falciparum infections hence there is no way of telling whether some children harbored asymptomatic infections in the previous week(s) during monitoring for clinical episodes or shortly before sampling of ML ratios at cross-sectional survey. This is further compounded by the possibility that our microscopy based parasite detection method, which is less sensitive than PCR P. falciparum infections and clinical malaria episodes. Finally, we did not assess the presence of co-infections, either at cross-sectional survey or during monitoring for clinical malaria episodes. Thus, the effect of potential helminth, bacterial or viral co-infections on the relationship between ML ratio and clinical malaria will need to be examined in future studies.Our study had several limitations. First, though we used data from children sampled at five annual cross-sectional surveys, both the ML ratios and parasite positive/negative status were measured at a single time-point within any year. Second, monitoring was not done for asymptomatic Despite these limitations our results support an association between high ML ratio at recruitment to cross-sectional surveys and increased risk of clinical malaria during follow-up. This observation now needs to be confirmed in other geographic settings.


Congenital transmission by protozoan parasites is a worldwide important public health problem. Congenital infections affects the mother and the fetus or newborn. It is still surprising that despite the abundant immunoepidemiological knowledge of congenital transmission of protozoan parasite, no definite etiology or predictive diagnostic tests have been identified. Understanding the mechanisms by which host/parasites infection and interaction occurs is one of the most important topics that will help find specific biomarkers of infection, prevent congenital transmission, maintain the health of the newborn, and develop safe and efficient treatments. In addition, understanding of the biological mechanisms of host/parasite interactions will facilitate protection of mothers and their families and reduce costs in health services. Moreover, this will lead to gain insight into epidemiological aspects and association with other pathologies and preserve the wellbeing of the newborn. In this special issue we have invited a few papers that address such issues.Congenital malaria is underestimated and usually associated with low-density cord parasitemia. However, it is increasingly recognized as a potentially serious complication of maternal malaria during pregnancy in Sub-Saharian Africa. Earliest epidemiological studies have reported prevalence varying widely in malaria-endemic areas from 0% to 33%. At birth, infections are usually asymptomatic with low parasitemia and the diagnosis by microscopy is often missed. Infection may occur by transplacental passage of parasites during disruption of the placental barrier at the time of delivery, with subsequent clinical illness in the newborn baby. A paper of this issue assessed the prevalence of congenital malaria during the dry season  using peripheral and cord blood smears of new born babies in association with peripheral and placental blood smears of, respectively, near-term and term pregnant women. This study is interesting because the authors found that congenital malaria is not rare in their study area. Another paper is a literature review on the prevalence, burden, diagnosis, prevention, and control of congenital malaria in Sub-Saharian Africa. This review is of interest to individuals working in clinics and laboratory diagnostic of malaria but also to national governments and partners in Sub-Saharian Africa. The authors highlight the challenges in parasitological and clinical diagnosis, the challenges in prevention with the use of intermittent preventive treatment (IPT) and insecticide-treated nets (ITNs) and provides recommendation on how to strengthen the health system in Africa. Another paper reports the prevalence of transplacental malaria in Burkina Faso, a West African country, and determines the real burden of transplacental transmission. The authors show associations between levels of parasite in the maternal, placental, and umbilical cord blood. All papers about malaria published in this special issue show the interest of pursuing investigations for a better understanding of vertical transmission by malarial parasites.T. cruzi has increasingly become more important, and partly responsible for the “globalization of Chagas' disease,” constituting a public health problem of increasing relevance. The fact that only a percentage of the infected mothers transmit parasites to their fetuses raises the question of the ability of the placenta as well as the immunological status of mother and fetus/newborn to impair the parasite transmission. Therefore, it is thought that congenital Chagas disease is the product of a complex interaction between the parasite, the maternal, and fetus/newborn immune responses, and placental factors. Additionally, a paper of this special issue constitutes a morphological analysis by immunohistochemical and histochemical methods of placentas from women with chronic Chagas' disease. T. cruzi-infected placentas present destruction of the syncytiotrophoblast and villous stroma, selective disorganization of the basal lamina, and disorganization of collagen I in villous stroma. Changes in the extracellular matrix of placental tissues, together with the immunological status of mother and fetus, and parasite load may determine the probability of congenital transmission of T. cruzi. Another paper assayed the effect of T. cruzi on glucose transporter protein-1 (GLUT1), which is the main isoform involved in transplacental glucose transport. High glucose media as well as T. cruzi infection reduce GLUT1 expression. The effect of T. cruzi infection on GLUT1 expression may explain some of the clinical manifestation of congenital Chagas disease. Finally, a paper analyzing the congenital transmission of Chagas disease is a review about the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, in that review different available methods for studying the congenital transmission of T. cruzi are analyzed. In that context, the ex vivo infection of human placental chorionic villi by T. cruzi trypomastigotes constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms.During congenital Chagas transmission, the parasite reaches the fetus by crossing the placental barrier. In the past few years congenital transmission of Ricardo E. FretesRicardo E. FretesUlrike KemmerlingUlrike KemmerlingDemba SarrDemba Sarr


The inherited disorders of hemoglobin are by far the commonest monogenic diseases and there is considerable evidence that they have reached their very high frequencies due to heterozygote advantage against malaria. Recent studies have begun to clarify the effect of interactions between malaria and some of the more severe inherited hemoglobin disorders and demonstrated how complex epistatic interactions between different hemoglobin variants with respect to malaria resistance and modification of their phenotypic severity may explain the remarkable heterogeneity of distribution and the frequency of these conditions both between and within individual populations. It was estimated recently that over seven million babies are born each year with either a congenital abnormality or genetic disease, and that up to 90% of these births occur in low or middle-income countries.P. falciparum malaria. Other factors that maybe involved include the widespread practice of consanguineous marriage, increased maternal age in the poorer countries, and gene drift and founder effects.There is now extensive evidence that the extremely high frequency of the hemoglobin disorders compared with other monogenic diseases reflects natural selection mediated by the relative persistence of carriers against P. falciparum malaria, work that has been the subject of several extensive reviews.Over recent years there has been a major revival in interest in the study of interactions between the inherited hemoglobin disorders and Plasmodium) have humans as their natural hosts; P. falciparum, P. vivax, P. malariae, and P. ovale. More recently it has been found that many cases of malaria that were previously diagnosed as being due to P. malariae infection are in fact due to a fifth parasite, P. knowlesi, particularly in Malaysia and Borneo.4Despite progress towards its control malaria is still the most important parasitic disease. It is estimated that some three billion people reside in malarious areas and that the disease is responsible for between one and three million deaths each year. Until recently it was thought that only four species of malarial parasite  expression on α thalassemic red cells.+ thalassemia in a later section.These findings were later augmented by case control studies in Papua New Guinea where it was found that, compared with normal children, the risk of being admitted to hospital with severe malaria was significantly reduced, both for αP. falciparum or P. yoelli. Since there is clear evidence that the rate of decline of fetal hemoglobin production after birth is delayed in β thalassemia heterozygotesWith the exception of a small-scale case control study in Liberia, which suggested that the β thalassemia trait is protective against severe malaria, there have been no large-scale studies of this type.P. vivax and P. knowlesi, explaining the almost complete absence of P. vivax malaria in many parts of Africa.32Early studies in Africa,P. vivax malaria. In a cohort study of babies in Vanuatu it was found, surprisingly, that those with α thalassemia had a higher frequency of malaria during the very early years of life than normal infants and that this was most marked in the case of P. vivax malaria.P. vivax malaria, the later protection against P. falciparum reflecting cross-immunization between the two varieties of malarial parasite.P. vivax might occur.Given this remarkable example of a malaria protective polymorphism it is surprising that there have been, to date, very few studies of the possible interaction between the hemoglobin disorders and P. vivax and P. falciparum malaria was assessed.P. vivax. Interestingly, those who had been splenectomized appeared to be particularly susceptible, but even the malarial-antibody levels in children with intact spleens were significantly higher than those in age-matched controls. Evidence of previous vivax infection was also related to spleen size and hence to the severity of the thalassemic phenotype. Again, it is possible that the increased susceptibility to P. vivax reflects the rapidly-turning-over red cell population of children with HbE β thalassemia.Very recently a study has been reported from Sri Lanka in which the susceptibility of children with HbE β thalassemia to both P. vivax. From a clinical point of view, since P. vivax malaria is presenting an increasingly serious health problem in Asian populations where HbE β thalassemia is so common it will also be important to repeat these studies in other parts of Asia to determine whether P. vivax prophylaxis should become an integral part of thalassemia control programs in countries where this form of malaria is particularly common.Clearly therefore it will now be important to study the effects of the heterozygous states for β thalassemia and hemoglobin E with respect to susceptibility to The significance of interactions between different malaria-resistant polymorphismsP. falciparum.+ thalassemia. This may reflect the intracellular level of Hb S. Those who inherit the α thalassemia gene together with the sickle cell trait have significantly lower levels of Hb S compared with those who have the sickle cell trait alone.Since different hemoglobin variants or forms of thalassemia that offer protection against malaria frequently occur together in the same population it is important to determine how they interact with one another with respect to the level of malaria protection. The term epistasis is used to describe potential interaction between two or more loci of this kind. Interestingly, the first study of this kind, in east Africa, has shown that there is negative epistasis between the sickle cell and α thalassemia genes with respect to protection against Recently, there has been a particularly interesting twist to the story of these interactions with respect to the distribution of the sickle cell and thalassemia genes in Africa and the Mediterranean populations. Although the sickle cell gene occurs to a modest degree in some though not all Mediterranean populations the predominant hemoglobin variants in these regions are the α and β thalassemias. On the other hand, β thalassemia, with the exception of some localized parts of Liberia, is relatively rare across the African subcontinent, where the sickle-cell and α thalassemia mutations predominate. It has been shown recently that this state of affairs can be explained by two epistatic interactions, one that we have just described in the case of the sickle cell trait and α thalassemia, together with the ameliorating effect of α thalassemia on the phenotype of the severe forms of β thalassemia.In this short review I have tried to highlight some recent information obtained from the studies of the relationship between the hemoglobin disorders and malaria, set against the background of the considerable amount of progress that has been made in this field over recent years. Several aspects of this work have interesting and potentially important implications for the hemoglobin field.Currently, we have only an extremely approximate estimate of the global burden of disease that is being produced by the hemoglobin disorders. This is largely due to lack of accurate information about gene frequencies in individual populations P. vivax malaria of patients with Hb E β thalassemia in those parts of Asia where P. vivax malaria is presenting a major health problem and where Hb E β thalassemia is particularly common. Similarly, the observations of the apparent lack of susceptibility of patients with sickle cell disease to P. falciparum malaria provides important information about future design of programs for the better control and management of sickle cell anemia in African populations.The early data that has come from studies of the interaction of malaria with the more severe forms of thalassemia, notably sickle cell anemia and Hb E β thalassemia, are also of potential importance. It will be extremely important, for example, to repeat the susceptibility studies to P. vivax on red cells, which led to the discovery of the parasite antigen which interacts with this receptor, has led to the development of at least one promising target for vaccine production.Finally, although some progress has been made the precise mechanisms of malaria protection by the hemoglobin variants and different forms of thalassemia is still uncertain. This is an important question because it is possible that if these mechanisms were better understood they might offer valuable clues to the development of completely different approaches to the control and management of malaria. It is interesting that the discovery that DARC is the receptor for


Plasmodium falciparum malaria remains endemic in sub-Saharan Africa including Ghana. The epidemiology of malaria in special areas, such as mining areas needs to be monitored and controlled. Newmont Ghana Gold Limited is conducting mining activities in the Brong Ahafo Region of Ghana that may have an impact on the diseases such as malaria in the mining area.Prior to the start of mining activities, a cross-sectional survey was conducted in 2006/2007 to determine malaria epidemiology, including malaria parasitaemia and anaemia among children < 5 years and monthly malaria transmission in a mining area of Ghana.Plasmodium falciparum represented 98.1% (95% CI 96.2 - 99.2) of parasitaemia. The geometric mean P. falciparum asexual parasite count was 1,602  and 1,195  among children < 24 months and ≥ 24 months respectively. Health insurance membership  and the least poor  were protected against malaria parasitaemia. The prevalence of anaemia was high among children < 24 months compared to children ≥ 24 months (44.1% (95% CI 40.0 - 48.3) and 23.8% (95% CI 21.2 - 26.5) respectively. About 69% (95% CI 66.3 - 70.9) of households own at least one ITN. The highest EIRs were record in May 2007 (669 ib/p/m) and June 2007 (826 ib/p/m). The EIR of Anopheles gambiae were generally higher than Anopheles funestus.A total of 1,671 households with a child less than five years were selected. About 50% of the household heads were males. The prevalence of any malaria parasitaemia was 22.8% (95% CI 20.8 - 24.9). The baseline malaria epidemiology suggests a high malaria transmission in the mining area prior to the start of mining activities. Efforts at controlling malaria in this mining area have been intensified but could be enhanced with increased resources and partnerships between the government and the private sector. Plasmodium falciparum malaria remains endemic in sub-Saharan Africa [n Africa . In Ghann Africa . Malarian Africa . The burn Africa , Kenya [n Africa ,6 and Tan Africa , which cNewmont Ghana Gold Limited, a mining company in Ghana recognizWith this realization, the Ghana Health Service in partnership with Newmont Ghana Gold Limited conducted a household and malaria epidemiology study as a baseline prior to the start of mining activities of Newmont Ghana Gold Limited in 2006/2007 to plan and evaluate malaria control programme within the area. This effort to control malaria in a mining area builds on a similar partnership in another mining area in Obuasi, Ghana that has led to a successful malaria control programme in the area . This baThe study was carried out in four districts in the Brong Ahafo Region of Ghana; the Asutifi District (between latitudes 6°40' and 7°15' North and Longitudes 2°15' and 2°45' West), the Tano North and South (Tano N/S) Districts (between latitudes 7°00'N and 7°25' N and between longitudes 1°45 W and 2°30 W), and the Techiman municipality (between latitudes 7°31'N and 8°0' N and between longitudes 1°60 W and 2°00 W). The study areas are in the wet semi-equatorial forest zone where the mean annual rainfall is about 1,200 mm per annum. Farming of cash crops, such as Cocoa, Oil Palm, Coffee are the major economic activity in the study communities. All health facilities in the study area implement both clinical and public health services under the supervision of the Brong Ahafo Regional Health Directorate. Malaria is the leading cause of morbidity and mortality in the region. About eighty percent of residents in the study area belong to the Akan ethnicity.A household cross-sectional study was carried out between July 2006 and March 2007 to determine baseline household morbidity and malaria transmission prior to the start of mining activities in the area. The districts in the mining area were categorized into two; the Impact area involving communities within the Asutifi, and Tano N/S districts whose health and/or socio-economic indices are likely to be affected by mining activities being undertaken and located within approximately 25 Km radius from the mine sites. The Non-impact area representing communities within the Asutifi, Tano North or Tano South districts that are not likely to be affected by the mining activities and located more than 25 Km from the mines site Figure . The TecAll households in the selected communities were enumerated and encoded into a database. Each household member in the community including children less than five (5) years was given a unique identification number as part of the household morbidity survey. All households who were respondents for the household morbidity survey and with children less than five (5) years in the database formed the sampling frame. One child less than five (5) years was randomly selected per household using a computer software for anthropometric assessment, hematological and malaria parasitaemia.Household ownership of ITNs and other household characteristics were assessed using a pre-tested structured questionnaire which were administered by trained fieldworkers. The quality of the ITNs was assessed by direct observation for holes.Malaria parasitaemia was determined by collecting finger prick blood samples from participants to prepare thick and thin blood smears. The smears were stained with 10% Giemsa and air-dried. They were examined under the microscope using ×10 eyepieces and ×100 objective. Malaria parasites were counted against 200 white blood slides on the thick smear. A slide was declared negative only after 200 oil immersion fields of the thick smear had been examined. Parasites were expressed per microlitre, assuming a total white cell count of 8,000 per microlitre. Species identification was done using the thin smear. A sub-sample (10%) of the blood slides were randomly selected and sent to an independent expert microscopists for examination and the results compared with > 90% concordance.Part of the blood from the finger prick was collected into Hemocue microcuvettes and haemoglobin levels were determined using a calibrated hemocue . All children found to be unwell, parasitaemic or anaemia during the survey were referred to a study clinician and treated.Anopheline morphological identification keys [et al [Anopheles positive by ELISA also termed as sporozoite rate (SR) and the Human Biting Rate which is estimated as the geometric mean of Anopheles caught per CDC light traps set.Entomological surveys were carried out in the Asutifi and the Tano N/S districts from November 2006 to August 2007. The dry season period (Nov. 2006 - Feb. 2007) and wet season period (May 2007 - Aug. 2007) were purposefully planned to compare transmission with or without the influence of rainfall in a mining area. No entomology work was carried out in the Techiman district as similar studies were carried out in the Kintampo district, an adjacent district with similar rainfall pattern in 2004 . Centre ion keys . They wes [et al . An avers [et al -13. The Anopheles gambiae s.l. specimens were further analysed by polymerase chain reaction (PCR) in order to establish members of the species complex and molecular forms composition [A total of 200 morphologically identified position .® Visual FoxPro 6.0. All data management processes were done at the computer laboratory of the Kintampo Health Research Centre. Data analysis was done using StataCorp Stata 10, TX USA. Averages and proportions were used to provide descriptions of age- and parasite prevalence, density, infection rates and the proportions of parasite clones that persisted during the survey. Socio-economic status was calculated for each household using World Bank asset scores for Ghana, based on ownership of a television, radio, refrigerator, car, motorcycle, bicycle, farm, electricity in house, ceiling, floor cover, and number of individuals/sleeping room. The anthropometric indices height-for-age (HA), weight-for-age (WA), weight-for-height (WH), were expressed as Z-scores using the WHO Anthro for personal computers, Version 3.1, 2010: Software for assessing growth and development of the world's children. A child was identified as being malnourished if s/he scored < -2 in one of the HA, WA, or WH indices.All data collected in the field or the laboratory were logged for traceability, and then batched for double data entry and processing using MicrosoftMalaria parasitaemia, anaemia (haemoglobin < 10 g/dL) and ITN usage were the main outcome variables. Odds ratios were calculated with their 95% confidence interval to assess the relationships between exposure and outcome variables using univariate and multivariate logistic regression models. The Wald's test and likelihood ratio test were used to estimate P values for exposure variables with two (2) levels and more than two (2) levels respectively in both the univariate and multivariate logistic regression models. P values less than 0.05 were considered statistically significant. Missing values were not included in the logistic regression models.Ethical approval to conduct the study was granted by the Kintampo Health Research Centre Institutional Ethics Committee. Community entry involved explaining the study to key community opinion leaders followed by community durbars/meetings. At these meetings, the study's aims, objectives, risk and benefits were explained to all participants. A signed/thumb printed written consent was obtained from each respondent. All data collected are kept confidential.A total of 1,671 households with a child less than five years were selected. Response rate was approximately 100%. About 50% of the household heads were males (Table Majority of household heads were between ages 30-39 years (27.8%) and 40-49 years (27.3). Majority of children  in the households were ≥ 24 months. Health insurance coverage was 42.3%, (95% CI 40.5 - 45.3).Plasmodium falciparum constituted 98.1% (95% CI 96.2 - 99.2) of parasitaemia. Plasmodium malariae was uncommon -1.9% (95% CI 0.81 - 3.8); no Plasmodium ovale or Plasmodium vivax was identified; there were no mixed infections. The geometric mean P. falciparum asexual parasite count was 1602  and 1,195  among children < 24 months and ≥ 24 months respectively. The proportion of children with parasite counts at various cut-off points was similar among the two age groups . The prevalence of anaemia (haemoglobin < 10 g/dL) among all children < 5 years was 30.5% (95% CI 28.2 - 32.8). The prevalence of anaemia was high among children < 24 months compared to children ≥ 24 months 44.1% (95% CI 40.0 - 48.3) and 23.8% (95% CI 21.2 - 26.5) respectively. About 32.7% (95% CI 28.9 - 36.7) of the children < 24 months and 19.0% (95% CI 16.6 - 21.5) of children > = 24 months had haemoglobin levels between 8.0 - 9.9 g/dL ; childhood anaemia was less likely among household heads with characteristics, such as educational level > middle/Junior high school , the least poor socioeconomic status  and household ITN ownership . There was however no significant association between childhood anaemia and household health insurance membership or household size (Table About 68.7% (95% CI 66.3 - 70.9) of households own at least one ITN. The median ITN per household was 2 (range 1 - 7). About 18.1% (95% CI 15.9 - 20.5) of ITNs observed for quality had holes in them. About 86.4% (95% CI 84.3 - 88.4) of household that owned ITNs had at least one person sleeping under an ITN the night before the interview. Majority of ITNs  were obtained free of charge as part of a national campaign while 26.4% (95% CI 23.8 - 29.0) bought their ITNs on the open market at commercial prices. There was no significant association between household ITN ownership and household head characteristics such as sex and educational level in the univariate logistic regression model  of the mosquitoes caught, Anopheles funestus 4.3% (n = 227) and non-Anopheles species 31.6%  which were predominantly Culex and Aedes species of mosquitoes. A few Mansonia species were recorded in Asutifi (4) and Tano N/S districts (2).A total of 5,257 mosquitoes were caught from 818 light traps set; Anopheles gambiae and Anopheles funestus were generally high. In the impact and non-impact areas of the Asutifi district, the median EIR was 46 ib/p/m (range 0-826 ib/p/m) and 51 ib/p/m (0 - 216) in the impact and non impact areas respectively. The highest was recorded in June 2007 at both impact and non impact areas respectively  and 102 (range 14 - 669 ib/p/m) in the impact and non-impact area respectively with the highest in May 2007  compared to Anopheles funestus median 0 ib/p/m (range 0 - 5 ib/p/m) alone  mutations. One hundred and ninety three out of the 200 selected mosquitoes (97%) possessed the kdr mutation (kdr+), whilst seven (3%) did not (kdr-).Two hundred mosquitoes were randomly selected to determine knockdown resistance  compared to reports from other rural parts of Ghana; 58% in a forest savanna transitional area of Kintampo in 2004 [The prevalence of  in 2004 ; between in 2004 ,16. The  in 2004 .et al [The relationship between malnutrition and malaria are inconsistent in the literature. Studies conducted in Ethiopia  and soutet al  that sugIn 2003, the government of Ghana introduced the National Health Insurance Scheme with the aim of reducing financial constraints as a gap in accessing health. Household membership of the insurance scheme provides children to seek health in health facilities closest to them. Common illnesses, such as malaria, are treated without direct cash payment at the point of service. In this study, health insurance membership was protective against malaria parasitaemia but had no significant relationship with anaemia. Participants who subscribe to the health insurance may regularly seek health care and may have been treated for malaria thus, the observed protection of malaria parasitaemia by health insurance membership.ITNs are known to prevent malaria infection by reducing human contact with the malaria vector and thus able to reduce all-cause mortality in children less than five years old by 17% in northern Ghana  and abouDespite the benefits of ITNs in controlling malaria, its patronage has not been quite encouraging and its usage is for some reasons other than for malaria control. For instance, in Savalou, Benin mosquito nets were often seen as a means of protection against mosquitoes and other biting insects in order to sleep better rather than as a means of preventing malaria . SeveralOne of the commonest causes of anaemia in sub-Saharan Africa is malaria. In this study, the prevalence of anaemia among all children < 5 years was moderately high 30.5% 95% CI 28.2 - 32.8) with children less than 24 months having a higher risk of anaemia. Although high, it is still lower than reported in other parts of Ghana and follows the same trend as malaria parasitaemia ,16,28. T.2 - 32.8There are few longitudinal malaria entomology studies carried out in mining communities in Ghana and other parts of the world. The abundance of mosquitoes in the Asutifi and Tano N/S districts differed slightly due to several contributing factors. Unlike Asutifi where the mosquito abundance was highest in the impact area, Tano experienced the reverse within the six month surveyed period. Though reasons for these differences in vector densities were not investigated, they may be attributed to environmental factors, such as changes in climate or vegetation, rainfall pattern, temperature; human factors such as human created breeding sites from urbanization; and intrinsic vector characteristics as previously reported ,32.ib/p/y) two years prior to this survey and used as an estimate for the control. The high EIRs recorded occurred in months that experienced moderate rainfall; June-July in Asutifi and February-May in Tano N/S, which indicates the impact of rainfall in vector populations and their breeding potential. Moderate rainfall creates pockets of water which favours breeding of larvae unlike torrential rains which washes away larval breeding sites of the major malaria vectors in Africa [An. funestus in this survey may probably be because a large proportion of young vectors with a low potential to transmit malaria may have been caught in the light traps; however this could not be confirmed since samples were not subjected to physiological age grading technique (parity detection) in this study. All An. gambiae s. l. analysed by PCR were of the S-molecular forms which is widespread in West Africa and other parts of Africa including the Kintampo area estimated for the control area [The EIRs calculated in this survey were as high as the EIRs found in the Kintampo area  and community members (two ITNs per household); refurbishment of a community health centre and training of health staff for accurate malaria diagnosis . The impThe malaria epidemiology suggests a high malaria transmission in the mining area prior to the start of mining activities. Efforts at controlling malaria in this mining area have been intensified but could be enhanced with increased resources and partnerships between government and the private sector. Periodic evaluations will be key to ascertain the impact of the malaria control activities currently ongoing in the mining area.The authors declare that they have no competing interests.KPA led in the proposal writing with contribution from CM, CZ, GK, KT, DD and SOA. KPA, CZ, DD, and KT coordinated the data collection during the study with support from YAD and CA on behalf of Newmont Ghana Gold Limited. Laboratory analysis was performed by DBD, CB, KT and DD. Data management and analysis was done by KPA, GD, MA, SAE and SOA. KPA wrote the manuscript with contribution from all authors. All authors read the final version of the manuscript.


Plasmodium vivax has been reported in Brazil, mortality is rarely observed. This is a report of a gestational malaria case that occurred in the city of Manaus  and resulted in foetal loss. The patient presented placental mixed-infection by Plasmodium vivax and Plasmodium falciparum after diagnosis by nested-PCR, however microscopic analysis failed to detect P. falciparum in the peripheral blood. Furthermore, as the patient did not receive proper treatment for P. falciparum and hospitalization occurred soon after drug treatment, it seems that P. falciparum pathology was modulated by the concurrent presence of P. vivax. Collectively, this case confirms the tropism towards the placenta by both of these species of parasites, reinforces the notion that co-existence of distinct malaria parasites interferes on diseases' outcomes, and opens discussions regarding diagnostic methods, malaria treatment during pregnancy and prenatal care for women living in unstable transmission areas of malaria, such as the Brazilian Amazon.Gestational malaria is a multi-factorial syndrome leading to poor outcomes for both the mother and foetus. Although an unusual increasing in the number of hospitalizations caused by  Plasmodium falciparum infection, clinical complications are associated with the sequestration of P. falciparum-infected erythrocytes (Pf-iE) within microvasculature and placental syncytiotrophoblasts [Plasmodium vivax-infected women [P. vivax experience maternal anaemia, and some of their babies present a low birth weight [P. vivax infection [In severe cases of hoblasts -5. Vivaxed women . Furtherh weight ,7, whichh weight ,2. Despinfection , informaP. vivax was responsible for the majority (83.7%) of registered cases in 2008. Plasmodium falciparum infections accounted for 16.3% of cases, and Plasmodium malariae infection was rarely observed [P. vivax have emerged in the Brazilian Amazon [In Brazil, where malaria incidence is almost exclusively restricted to the Amazon (99.8% of the cases), P. vivax infection at the nearest Health Center and showed approximately 90,000 parasites/mm3. In Brazil, the microscopic examination of Giemsa-stained thick blood smear is the official method for malaria diagnosis. This was her fourth pregnancy, and she had no medical history of previous abortion, stillbirth or pre-term delivery. The patient had three previous malaria episodes, the last occurring two years ago. Additionally, she reported a plasmodial infection during her third pregnancy. At the time, the patient was treated and no further complications were observed.A 19-year-old pregnant woman, estimated to be 35 weeks of gestation (WG), living on the boundary of the city of Manaus - Amazon State , surrounded by the Amazon rainforest, was diagnosed for 3), with 67% lymphocytes. Serological tests for syphilis, toxoplasmosis, measles and HIV were negative.She was given a three-day regimen (25 mg/kg) of oral chloroquine, with four pills (150 mg each) administered in the first day, followed by three pills on the two subsequent days. However, after the second dose, the patient presented with vomiting, which led to cessation of the drug treatment. The patient was subsequently transferred to a tertiary-care maternity hospital in Manaus, where she was hospitalized until delivery. At the maternity hospital, the patient presented symptoms of fever, headache, jaundice, anorexia, chills and hypertension. Urine sediment analysis revealed that bilirubin and biliary pigments were three-fold above the standard levels. Furthermore, blood analysis revealed slight anaemia  and leukocyte count were normal , abnormal foetal symmetry and abnormal placental texture. Although pregnancy was estimated to be 35 WG, with a foetal weight of 2,500 g, according to the patient's last menstruation date, foetal growth was approximately 38-39 WG. Thus, the estimate of 35 WG after ultrasound analysis might represent impairment of intra-uterine growth. Two days later in a routine follow-up, an abnormal foetal heart rate was observed. Another ultrasound analysis was performed, and no foetal heartbeat was detected, and oligohydramnios was observed. Next, labour was induced by administration of oxytocin, and foetal loss of a male weighing 2,670 g was confirmed. No foetal autopsy was performed due to the lack of authorization by relatives. Macroscopic examination of the placenta revealed an abnormal dark colour; following patient consent, a sample of the placental tissue was collected for further microscopic and molecular analysis. Molecular analysis of the placenta revealed a mixed infection with P. falciparum-specific PCR product was detected, despite the maintenance of negative thick blood smears. At the time, the patient was treated with artemether/lumefantrine for three days. Figure Because of the absence of parasite forms in the thick blood smear performed at the maternity, the patient did not receive any anti-malarial treatment during her stay and immediately after being released from the hospital. In the second month after foetus loss, nested-PCR analysis was conducted on the asymptomatic mother's peripheral blood and a Plasmodium spp. gDNA in the blood sample, sensitive nested-PCR was performed using species-specific oligonucleotides based on human malaria parasite genes for the 18S small subunit ribosomal RNA (ssrRNA) as described previously [A small fraction (1 × 1 × 1 cm) of maternal placenta was collected, frozen in liquid nitrogen and crushed in 2 mL of digestion buffer  before incubation overnight at 37°C in the presence of proteinase K . The DNA used for PCR amplification was purified by two phenol/chloroform extractions, followed by ethanol precipitation; DNA samples were resuspended in water. Two samples of the patient's peripheral blood were collected, the first one at few hours after the foetus loss and other approximated two months later. Genomic DNA (gDNA) was purified using the Charge Switch gDNA Blood Kit (Invitrogen) according to the manufacturer's protocol. To determine and discriminate the presence of Nested-PCR results conducted with patient's placental tissue were also confirmed by semi-nested multiplex malaria PCR (SnM-PCR)  and in the Amazonian Annual Parasitological Index  over the last few years [ommended .P. vivax infection and pregnancy outcomes such as stillbirth and miscarriage remains unclear in the literature. However, studies conducted in Southeastern Asia indicate a correlation between P. vivax infection and adverse pregnancy outcomes, eventually leading to maternal anemia and low birth weight, and with the presence of malaria pigment in the placenta [P. falciparum infections in pregnant women in a cohort of 1,699 childbearing women in the Brazilian Amazon [The relationship between placenta ,7. Furthn Amazon .P. vivax and P. falciparum were amplified from infected placental tissue. In contrast, only P. vivax-infected erythrocytes (Pv-iE) were found in patient peripheral blood following diagnosis by Giemsa-stained thick blood smear. Although the patient's chloroquine treatment was incomplete, the utilized diagnostic method failed to detect P. vivax in the peripheral blood after treatment; thus showing some efficacy of the drug treatment against this species of parasite. Moreover, as the nested-PCR analysis conducted by us detects only viable parasites, we assume that both of these species of parasites were accumulated or sequestered in the placenta and played a role in pathology.This is a case report of gestational malaria with foetal loss, in which DNA of both P. falciparum  amplified products in the presence of species-specific human malaria parasite oligonucleotides specific for P. ovale, P. malariae, P. vivax or P. falciparum ssrRNA gene. Both P. vivax and P. falciparum gDNA were found in the placenta. Abbreviations are as follows: MW, molecular weight; bp, base pair; S, patient placental sample; C-, negative control, absence of nuclear material; Po+, Pm+, Pv+ and Pf+, positive controls representing amplification product of P. ovale (436 bp), P. malariae (269 bp), P. vivax (499 bp) and P. falciparum (395 bp) ssrRNA gene.Click here for filePlasmodium falciparum infection performed two months after deliveryMolecular diagnosis of . Agarose gel electrophoresis representing nested-PCR performed in the presence of patient's peripheral blood collected two months after delivery and species-specific human malaria parasite oligonucleotides . Only P. falciparum gDNA was detected in peripheral blood. Abbreviations are as follows: MW, molecular weight; bp, base pair; B, patient peripheral blood sample; niB, non-infected peripheral blood sample; C-, negative control, absence of nuclear material; Pv+ and Pf+, positive controls representing amplification product of P. vivax (120 bp) and P. falciparum (200 bp) ssrRNA gene.Click here for file


Plasmodium vivax is scarcely reported in comparison to Plasmodium falciparum. In complicated malaria, thrombocytopaenia and haemostatic alterations lead to increased activation of coagulation cascade and fibrinolytic system. Thromboelastography (TEG) is a haemostasis system which measures the viscoelastic strength of blood clot in the coagulation pathway. It detects the initial derangement in clotting cascade involving in platelet interaction and fibrinolysis. Hence, it can document the early changes in coagulation in vitro, and thereby guide the management. The current study was aimed at detection of DIC in patients with P. vivax malaria based on TEG.Disseminated intravascular coagulation (DIC) due to Ethylene diamine tetraacetic acid (EDTA) blood samples from acute febrile patients were tested by microscopy and immunochromatographic test for malaria. A total of 31 confirmed cases of vivax malaria were enrolled for this study. All the samples were tested by thromboelastography and conventional tests parameters for detection of any coagulation derangement.Of 31, 17 (55%) were classified as complicated and 14/31 (45%) were uncomplicated. Among 23 cases with thrombocytopaenia, non-overt (early stage) DIC was detected in 18 cases by TEG and 17 cases by the conventional methods.It seems that the burden of DIC in vivax malaria is much higher than the world literature reported. TEG can be utilized as an important tool for early detection of DIC and guiding the management in malaria patients. Plasmodium, Plasmodium falciparum and Plasmodium vivax account for majority of malaria infection worldwide. Complicated malaria is observed to be a consequence of P. falciparum infection mainly [P. vivax is found to be relatively benign. However, many recent reports from Africa, Southeast Asia and India highlighted its association in causation of complicated malaria [P. vivax infection. DIC is diagnosed as a combination of clinical features such as haemorrhagic manifestations and laboratory tests , activated partial thromboplastin time (aPTT), D-dimers, fibrinogen degradation product, etc.). However, absence of bleeding is often considered as negative for DIC and misdiagnosed many non-overt (early) cases of DIC. Apart from routine laboratory tests, thromboelastograpy (TEG) is a popular method for early and accurate detection of DIC in trauma, organ transplant, cardiac surgery, and obstetric procedures [Among the different species of n mainly . In cont malaria -5. The c malaria . It is a malaria ,8. But, ocedures . TEG meaP. vivax was confirmed in 31 blood samples by the above methods. Speciation was characterized and parasitaemia was calculated in all the positive slides as per WHO guidelines. PCR could not be performed in these samples. However, considering the high sensitivity and specificity of QBC and ICT, the possibility of mixed infection with P. falciparum was ruled out. All patients were categorized as complicated or uncomplicated as per WHO criteria [A prospective study was carried out over a period of four months in which patients with diagnosis of malaria were enrolled after receiving clearance from the institute ethics committee . Blood sample from acute febrile patient with clinical diagnosis of malaria was tested by microscopy , and quantitative buffy coat assay (QBC)) and immunochromatographic test(ICT) . Infection of criteria . PatientP. vivax and tested by TEG. TEG consists of a cup which holds the blood. Citrated blood was reconstituted by adding 20 μl of CaCl2 to 340 μl of blood in the cup for thromboelastographic test. The cup oscillates through an angle of 4˚45 with a pin suspended freely in the blood by torsion wire. The torque of the rotating cup is transmitted to the pin, thereby monitoring the motion. The clot formation, clot strength, lysis, and rate of fibrin-platelet bond formation affects the magnitude of pin motion. The signals are monitored and converted into graphical form by the computer. Thus TEG documents the initial fibrin formation, clot strengthening, and fibrin-platelet bonding, and clot lysis detecting hypocoagulation, normal coagulation, hypercoagulation, and fibrinolysis. Abnormal values were calculated in comparison to the normal values given by the standard reference and analysed for different coagulation dysfunction.Three ml blood was collected in citrate (0.129 M trisodium citrate) vacutainer tubes from all patients positive for Calculation of coagulation index (CI) by TEGAll samples were also analysed for the conventional coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen degradation product, D-dimer, etc. The International society for haemostasis and thrombosis (ISTH) scoring algorithm was used to determine overt and non-overt DIC conditions . Fully aP. vivax. Among them, 17 (55%) were classified as complicated and 14 (45%) as uncomplicated (as per WHO criteria). The parasitaemia in complicated and uncomplicated groups ranged from 200/μl to 36,000/μl and 200/μl to 56,000/μl, respectively. In the complicated group, severe anaemia  was the commonest complication followed by acute renal failure . The other complications observed were delirium and altered sensorium , pulmory oedema , jaundice . Nine out of 17 (53%) patients in the complicated group required intensive care unit for the management of complications. The number of cases in complicated and uncomplicated groups were found to be 13 (76%) and nine (64%), respectively, with alteration of TEG; in comparison to 10(59%) and 7 (50%), respectively, as detected by the conventional techniques. However, only one patient out of 31 complained of haemorrhagic manifestation and was clinically diagnosed to have DIC. None of the patient progressed to overt DIC during this period. A total 23/31 (74%) patients  showed thrombocytopaenia (platelet <1.5 lakh/dL). Eighteen out of 23 (78%) patients with thrombocytopaenia were detected to have non-overt (early) DIC by thromboelastography and 17 out 23 (74%) by the conventional test parameters (Table In the present study, 31 febrile patiens were diagnosed positive for P. vivax. In this study, more than 50% of vivax cases were categorized as complicated malaria and required hospitalization. The available literature was searched which revealed isolated case reports of DIC as a consequence of vivax malaria [The present study highlights a surge of complicated malaria cases caused by  malaria ,13. In t malaria . Alterat malaria . In uncoRecently, many reports published show an increasing rate of severity, with vivax malaria presenting with a variety of complications such as renal failure, pulmonary oedema, severe anaemia, thrombocytopaenia, etc. This is the first study to emphasize coagulation dysfunction in vivax malaria. Despite the small number, the majority of patients in this study with thrombocytopaenia showed a clear coagulation derangement in TEG as well as the conventional tests. Thrombocytopaenia can be taken as a valid marker of development of coagulation dysfunction, irrespective of the severity of the disease. Moreover, TEG is an important tool in the early diagnosis and monitoring of the development of DIC in these patients.DIC: Disseminated intravascular coagulation; ICT: Immunochromatographic test; TEG: Thromboelastogram; PT: Prothrombin time; aPTT: Activated partial thromboplastin time; TT: Thromboplastine time; AO: Acridine orange; QBC: Quantitative buffy coat assay; CI: Coagulation index.The authors have declared that they have no competing interests.SM participated in the conception, design, interpretation of data and drafting of manuscript. JCS reviewed critically the manuscript for final approval. SA participated in interpretation, and analysis of data, and drafting of manuscript. AG helped in acquisition, interpretation and analysis of data. All authors read and approved the final manuscript.


Rapid diagnostic tests for malaria (RDTs) allow accurate diagnosis and prompt treatment. Validation of their usefulness in travellers with fever was needed. The safety of a strategy to diagnose falciparum malaria based on RDT followed by immediate or delayed microscopy reading at first attendance was evaluated in one referral hospital in Switzerland.A retrospective study was conducted in the outpatient clinic and emergency ward of University Hospital, covering a period of eight years (1999–2007). The study was conducted in the outpatient clinic and emergency ward of University Hospital. All adults suspected of malaria with a diagnostic test performed were included. RDT and microscopy as immediate tests were performed during working hours, and RDT as immediate test and delayed microscopy reading out of laboratory working hours. The main outcome measure was occurrence of specific complications in RDT negative and RDT positive adults.2,139 patients were recruited. 1987 had both initial RDT and blood smear (BS) result negative. Among those, 2/1987 (0.1%) developed uncomplicated malaria with both RDT and BS positive on day 1 and day 6 respectively. Among the 152 patients initially malaria positive, 137 had both RDT and BS positive, four only BS positive and five only RDT positive (PCR confirmed) (six had only one test performed). None of the four initially RDT negative/BS positive and none of the five initially BS negative/RDT positive developed severe malaria while 6/137 of both RDT and BS positive did so. The use of RDT allowed a reduction of a median of 2.1 hours to get a first malaria test result.A malaria diagnostic strategy based on RDTs and a delayed BS is safe in non-immune populations, and shortens the time to first malaria test result. Plasmodium parasitaemia in the blood is the mainstay to initiate appropriate treatment. However, accurate diagnosis remains a challenge. Diagnosis by microscopy is still considered as the gold standard, but turnaround time often exceeds 2–3 hours and accuracy is acceptable only when performed by experienced microscopists . Of note the median time between the blood sample and RDT result was 15 minutes .Overall non-compliance to timelines defined in the diagnostic strategy was 31.5% (40/127). It was 20% (10/50) for patients attending during working hours and 39% (30/77) for those attending out of hours (p = 0.04). Non-compliance to timelines was 29% (20/69) for patients with danger signs and 34.5% (20/58) for those without danger signs (p = 0.64). Non-compliance to timeline recommendations (for those with or without danger signs) did not affect heath outcome.Median time before getting a BS result was 2.1 hours  for the cases without any complication and 3.2 hours  for the six cases who developed severe malaria after admission (p = 0.14). Among the four patients with negative RDT but positive microscopy, the delay between the RDT and the BS result was 14, 18.3, 37.7 and 70.8 hours. One of the four had a danger signs at first attendance and none developed severe malaria (as mentioned above).Among the six cases that developed severe malaria after admission, all six had a positive RDT and BS at first testing. Three had microscopy reading within three hours after RDT result; for the other three, the delay to get microscopy results was respectively of 4.3, 17 and 27 hours. One had no danger sign, three had at least one danger sign at first attendance and another two had thrombocytopaenia <100 G/L. Among those with danger signs or thrombocytopaenia, all except one received intravenous quinine at first place. All six cases who developed severe malaria after admission recovered uneventfully.The present study provides, for the first time, evidence that a malaria diagnostic strategy based on a RDT followed by immediate or delayed microscopy reading at first attendance is safe and does not expose travellers or migrants to an increased risk of severe malaria or death. These findings can probably be generalized to most setting in non-endemic countries since they derive from data collected in predominantly non-immune patient population under routine clinical and laboratory conditions of an ordinary outpatient clinical and emergency hospital ward. No patient with a negative RDT developed severe malaria, despite a planned delay before getting the blood smear results out of hours. This was true even when established timelines between RDT and microscopy were not complied to. The use of RDTs was not associated with the development of complications since all 6 cases who developed severe malaria after admission had a positive RDT at first testing. The availability nowadays of artemisinin-based combination therapy (ACT) for the treatment of uncomplicated malaria renders the strategy with RDT even safer since ACT is very effective and well-tolerated ,22. ACT et al.[et al.[Even if the study was not designed to validate the accuracy and performance of RDTs, which has already been extensively demonstrated, RDTs were as good as microscopy to diagnose malaria. Indeed 5 malaria diagnoses were based on positive RDT results only (negative blood slide) and 4 on blood smear results only (negative RDT result). Recently, Gillet et al.,24 and Ll.[et al. demonstrl.[et al.,24. NegaDiagnostic strategies based on RDT have been adopted in other centres managing non-immune patients ,26 but, The less rigorous follow-up of the malaria negative patients is a limitation in the overall assessment. However, it is highly unlikely that secondary malaria cases were missed after having attended the outpatient clinic since feverish patients are advised to come back daily to repeat malaria tests, especially so if symptoms persist or worsen. In addition, the University Hospital in Lausanne has a long tradition of reference centre for travel related diseases in the area and is easily accessible. At least, secondary malaria deaths that would have occurred outside the hospital have been virtually excluded by our investigation of malaria death records in the region. This study was undertaken in one hospital only and should be repeated in different settings to accumulate more evidence. Since non-falciparum malaria patients and children were excluded, the safety of this strategy should be confirmed for malaria due to other species  and in a paediatric population .In conclusion, this study - conducted in a routine clinical and laboratory non-endemic setting without 24-hour expert microscopy available – provides some evidence that a malaria diagnostic strategy based on RDTs followed by immediate or delayed microscopy reading is safe. Indeed no patients with a negative RDT developed severe malaria or died. This study adds information about the safety of a malaria diagnostic strategy based on RDTs, of which accuracy and performance have been extensively demonstrated. There was also a clear benefit of using RDT, as it allowed decreasing significantly the delay before getting a test result (and thus onset of appropriate treatment), even during laboratory working hours and increasing overall sensitivity when combined with microscopy. The results of this analysis provide evidence and lessons for considering large-scale implementation of malaria diagnostic strategies that include RDTs in non-endemic settings.Authors declare that they have no competing interests.IAR was responsible for the design of the retrospective investigation, data extraction, entry, management and analysis, interpretation of results and writing the manuscript. VDA contributed to the development and design of the strategy, clinical supervision, interpretation of the results and writing the manuscript. GP’H contributed to the development and design of the strategy, and data extraction. BG contributed to the development and design of the strategy, clinical supervision, interpretation of the results and writing the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum is a major cause of severe malaria in Southeast Asia, however, there is limited information regarding clinical factors associated with the severity of falciparum malaria from this region. We performed a retrospective case-control study to compare clinical factors and outcomes between patients with severe and non-severe malaria, and to identify clinical factors associated with the requirement for intensive care unit (ICU) admission of patients with severe falciparum malaria among hospitalized adults in Southeast Asia. A total of 255 patients with falciparum malaria in the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012 were included. We identified 104 patients with severe malaria (cases) and 151 patients with non-severe malaria (controls). Patients with falciparum malaria with following clinical and laboratory characteristics on admission (1) referrals, (2) no prior history of malaria, (3) body temperature of >38.5°C, (4) white blood cell counts >10×109/µL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL, were more likely to develop severe malaria (P<0.05). Among patients with severe malaria, patients who met ≥3 of the 2010 WHO criteria had sensitivity of 79.2% and specificity of 81.8% for requiring ICU admission. Multivariate analysis identified the following as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai  or Myanmar ; (2) referrals ; (3) no prior history of malaria ; and (4) albumin concentrations of <3.5 g/dL . Our findings are important for the clinical management of patients with malaria because it can help early identification of patients that could develop severe malaria and require ICU admission. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of these patients. Plasmodium, namely P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Of these, P. falciparum and P. vivax are responsible for the majority of infections P. falciparum malaria P. falciparum is a major cause of severe malaria and approximately 10–20% of patients with falciparum malaria require urgent and intensive medical care Malaria is caused by various species of the protozoan parasite per seThe World Health Organization (WHO) provides case definitions for malaria infection as well as guidance on the management of malaria and for severe malaria The Standards for the Reporting of Observational Studies in Epidemiology (STROBE) procedure were followed in this study.P. falciparum forms in thin or thick peripheral blood smear were included in our study. Patients who had mixed infections with other forms of Plasmodium species, community acquired infection, dengue, rickettsiosis and leptospirosis; pregnancy; and underlying medical illness were excluded. In the Hospital for Tropical Diseases, blood culture for microbiology, urinalysis, and chest X-ray were routinely performed in hospitalized patients with malaria in order to exclude other infections. In addition, serological testing for diagnosis of dengue, rickettsiosis and leptospirosis were performed when clinical findings were suspected.The study protocol was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University in Bangkok, Thailand. The written consent was not obtained and it was specifically waived by the approving ethic committee of the Faculty of Tropical Medicine. This was a retrospective case-control study conducted among adults admitted at the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012. Patients who met the criteria for having severe falciparum malaria defined by the 2010 WHO criteria (cases) and patients who did not meet these criteria (controls) were identified from a prospective cohort of 300 patients with falciparum malaria. Patients aged ≥15 years and diagnosed with falciparum malaria by presence of asexual The demographic information, clinical and laboratory findings, and patient outcomes were transferred into a pre-defined case record-form. Medical records and discharge summaries of the 300 patients with a diagnosis of falciparum malaria at the time of death or discharge from hospital were summarized and classified as cases and controls. Of the 300 patients with falciparum malaria, 45 patients were excluded because 14 patients had a lack of proof of malarial infection, 9 patients did not have available laboratory data, 7 patients had evidence of mixed infections, 12 patients had underlying medical diseases, and 3 were pregnant .The remaining 255 patients with falciparum malaria were included. Using the 2010 WHO criteria for diagnosing severe malaria, 104 patients met the criteria for severe malaria defined as cases and the remainder (n  = 151) were classified as controls. Among 104 patients with severe falciparum malaria, 49 patients were admitted at the ICU and 55 patients were admitted at the wards.In our study, patients with falciparum malaria were considered to have had severe malaria if they presented with ≥1 of the following clinical or laboratory findings: (1) impaired consciousness, defined by a Glasgow Coma Score (GCS) of <10; (2) multiple convulsions, defined as >2 episodes of convulsions in 24 hours; (3) acidotic breathing; (4) shock, defined by a systolic blood pressure of <80 mmHg or the need for inotropic drugs; (5) clinical jaundice with evidence of other vital organ dysfunction including acute kidney injury defined by increase of serum creatinine ≥0.3 mg/dl within 48 hours, shock, alteration of consciousness defined by GCS <15, and hypoxemia defined by oxygen saturation <93% in room air; (6) dark colored urine; (7) spontaneous bleeding, and 8) pulmonary edema detected by chest X-ray. The laboratory findings that indicated severe malaria included: (1) hypoglycemia, defined as blood sugar levels of <40 mg/dL; (2) severe metabolic acidosis, defined as serum bicarbonate levels of <15 mmol/L; (3) severe anemia, defined as hemoglobin (Hb) levels of <5 g/dL; (4) hemoglobinuria, defined as positive hemoglobin with the presence of ≤2 red blood cells per high power field in urine; (5) hyperparasitemia, defined as >2%/100,000/µL parasites in a blood smear in areas with low intensity transmission, or >5%/250,000/µL parasites in a blood smear in high, stable malaria transmission areas; and (6) renal impairment, defined as serum creatinine levels of >3 mg/dL. Time to febrile clearance was defined as the time (in hours) from the initial treatment dose of an anti-malarial drug to the start of the first sustained decrease in oral temperature of ≤37.5°C for a period of ≥48 h. Time to parasite clearance was defined as the time (in hours) from the initial treatment dose of an anti-malarial drug to the first of two consecutive peripheral blood smears with no evidence of parasitemia. Community acquired infection is defined as any infection diagnosed within 48 hours of hospitalization and hospital acquired infection is defined as any infection diagnosed after 48 hours of hospitalization.The study sample size was estimated with the use of the Power and Sample Size Program, version 3.0, 2009 . We planned a study with a ratio of 1∶1.5 for independent cases of severe malaria to control patients. The probability that control patients could have had severe malaria was estimated at 0.2. If the true odds ratio was 2.25 for severe malaria relative to non-severe malaria, then we had to include 101 cases of severe malaria and 152 control patients to be able to reject a null hypothesis if the odds ratio equaled 1. The power of the study was set at 80% and the Type I error probability associated with a null hypothesis were 0.05.t-tests and the data for these variables are reported as mean ± standard deviation (SD). Variables that did not pass the normality test were compared with Mann–Whitney U tests for comparison of 2 groups and data for these variables are reported as median and inter-quartile range (IQR). Univariate analysis was performed to determine possible factors that could be associated with severe malaria. All variables with P≤0.2 in the univariate logistic regression analysis was included in the stepwise multiple logistic regression analysis by using the backward selection method for determining independent associated factors for severe malaria. All tests of significance were 2-sided, and P<0.05 was considered statistically significant. Diagnostic sensitivity and specificity of the 2010 WHO criteria was defined against those requiring ICU admission, and was expressed as a proportion with a 95% confidence interval (CI).All data analyses were performed with SPSS for Windows 18.0 . Categorical variables were compared with Chi-square analyses or Fisher’s exact test where appropriate, and data for these variables are summarized as number and percentage. We used the Kolgomorov Smirnov test to ascertain whether numerical variables were normally distributed. All variables with normal distributions were compared with Student Of the 255 patients diagnosed with falciparum malaria, 104 met the 2010 WHO criteria for severe malaria defined as cases and the remainder of the patients (n  = 151) were included in the control group.There were no significant differences in the age, gender and ethnicity among the patients with and without severe malaria. There was also no significant difference in the number of patients with and without severe malaria who visited to the Thai-Myanmar border or other areas. Patients with severe malaria were significantly more likely to have been referred for treatment to the Hospital for Tropical Diseases in Bangkok and had no a prior history of malaria. All statistical comparisons for baseline characteristics of patients with or without severe malaria are reported in 9/µL, presence of schizonts in the peripheral blood smears, and albumin concentration of <3.5 g/dL at the time of admission  patients, a clinical diagnosis of severe malaria was made because of the presence of clinical jaundice with evidence of other vital organ dysfunction including acute kidney injury , shock , alteration of consciousness , and hypoxemia ; in 27 (26.0%) because of evidence of shock; in 21 (20.2%) because of pulmonary edema; in 18 (17.3%) because of dark colored urine excretion; in 14 (13.5%) because of impaired consciousness; in 6 (5.8%) because of abnormal spontaneous bleeding; and in 6 (5.8%) because of an acidotic breath. Laboratory findings that indicated a diagnosis of severe malaria included abnormally high hemoglobinuria in 51 (49.0%) patients; hyperparasitemia in 44 (42.3%); renal impairment in 20 (19.2%); severe metabolic acidosis in 17 (16.3%); severe anemia in 3 (2.9%); and hypoglycemia in 2 (1.9%). Majority of clinical and laboratory diagnosis of severe malaria was found at admission, but some of the patients with renal failure , shock , pulmonary edema  were diagnosed after hospitalization.The sensitivity of 2010 WHO criteria for diagnosing severe malaria, for requiring ICU admission was low, but the specificity of the criteria for requiring ICU admission was high with the exception that the criteria of jaundice with evidence of other vital organ dysfunction, hemoglobinuria, and hyperparasitemia had lower specificity. However, severe falciparum malaria patients who met ≥3 of the 2010 WHO criteria had sensitivity (95% CI) of 79.2% (64.6–89.0) and specificity (95% CI) of 81.8% (68.7–90.5) for requiring ICU admission .In this study, no in-hospital mortality among patients with falciparum malaria was observed, thus, the surrogate endpoint of ICU admission was used for determining severity of malaria in patients. The median (IQR) for the duration of ICU admission in patients with severe falciparum malaria was 4.0 (2.0–4.0) days. Patients with severe falciparum malaria who had ethnicity of Thai or Myanmar, referred from other hospitals, no a prior history of malaria, and serum albumin <3.5 g/dL were more likely to require ICU admission. Patients with severe falciparum malaria who were admitted at the ICU received higher total dosages of artesunate and higher adjusted total dosages of artesunate per kilogram of body weight compared to those without ICU admission. Further, patients with severe falciparum malaria who were admitted at the ICU had significantly longer fever and parasite clearance times, periods of hospitalization, and a significantly higher proportion of hospital acquired infections  than thoWe used a univariate logistic regression analysis to ascertain which of the baseline characteristics, clinical symptoms, and laboratory findings associated with the requirement of ICU admission in patients with severe falciparum malaria. All factors that were significantly associated with severe malaria requiring ICU admission were included in the univariate logistic regression analysis. The results identified the following factors associated with severe malaria requiring ICU admission: (1) referrals, (2) ethnicity of Thai or Myanmar, (3) no a prior history of malaria, and (4) serum albumin <3.5 g/dL .P≤0.2 in the univariate logistic regression analysis were then further analyzed by the stepwise multiple logistic regression analysis using the backward selection method in order to determine the independent clinical factors that associate with the requirement for ICU admission in patients with severe falciparum malaria. We found the following clinical factors to be independently associated with the requirement for ICU admission in patients with severe falciparum malaria (1) referred patients, (2) ethnicity of Thai or Myanmar, (3) no a prior history of malaria, and (4) serum albumin <3.5 g/dL  the presence of schizonts in peripheral blood smears, and (6) albumin levels of <3.5 g/dL. In addition, patients with severe falciparum malaria had longer duration of hospitalization and more likely to developed hospital acquired infections than those with non-severe malaria. However, all patients with falciparum malaria included in our study were treated with an artesunate-based regimen. Patients were given a dose of 2.4 mg/kg of artesunate at the time of diagnosis and at 12 and 24 h post diagnosis. Treatment was continued at a dose of 2.4 mg/kg per day until parasitemia disappeared in peripheral blood smears P. falciparum to treatment with artemisinin and artemisinin derivatives are declining in the areas around the Thai-Cambodia and the Thai-Myanmar border In this study, we identified the following clinical characteristics and laboratory findings that were more likely to develop severe falciparum malaria: (1) referrals, (2) no a prior history of malaria, (3) a body temperature of >38.5°C, (4) white blood cells counts >10×10Using the 2010 WHO criteria for diagnosing severe malaria, 104 patients had severe falciparum malaria in this study. The presence of clinical jaundice with evidence of other vital organ dysfunction (64.4%) was the most common complications followed by hemoglobinuria (49.0%), shock (26.0%), pulmonary edema (20.2%), renal impairment (19.2%), severe metabolic acidosis (16.3%), cerebral malaria (13.5%), abnormal spontaneous bleeding (5.8%), severe anemia (2.9%), and hypoglycemia (1.9%). In addition, severe falciparum malaria patients who met ≥3 of the WHO criteria had sensitivity and specificity for requiring ICU admission of 79.2% and 81.8%, respectively. The other clinical factors that were associated with the requirement for ICU admission in hospitalized patients with severe falciparum malaria included (1) referrals, (2) ethnicity of Thai or Myanmar, (3) no a prior history of malaria, and (4) serum albumin <3.5 g/dL.Previous study showed that referred patients were at risk to develop severe malaria due to delay in diagnosis and late effective treatment Previous studies showed that shock (27–49%) was one of the common complications of severe malaria patients who were admitted at the ICU 9/µL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL were more likely to develop severe malaria. Using ≥3 of the 2010 WHO criteria, the sensitivity and specificity of the criteria for requiring ICU admission were 79.2% and 81.8%, respectively. Multivariate analysis identified the following clinical factors as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai and Myanmar, (2) referrals, (2) no a prior history of malaria, and (3) albumin concentrations of <3.5 g/dL. However, the clinical parameters, management and outcomes of severe falciparum malaria patients requiring ICU admission who developed shock and those without shock were similar.Our findings show that patients with falciparum malaria who present with (1) referred, (2) no a prior history of malaria, (3) body temperatures of >38.5°C, (4) white blood cell counts >10×10Our findings are important for the clinical management of patients with malaria as it contributes to the early identification of these patients. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of patients with severe falciparum malaria.