Health record-based observations from several parts of Africa indicate a major decline in malaria, but up-to-date information on parasite prevalence in West-Africa is sparse. This study aims to provide parasite prevalence data from three sites in the Gambia and Guinea Bissau, respectively, and compares the usefulness of PCR, rapid diagnostic tests (RDT), serology and slide-microscopy for surveillance.Cross-sectional surveys in 12 villages at three rural sites were carried out in the Gambia and Guinea Bissau in January/February 2008, shortly following the annual transmission season.A surprisingly low microscopically detectable parasite prevalence was detected in the Gambia , and Guinea Bissau , with low parasite densities . In comparison, PCR detected a more than three times higher proportion of parasite carriers, indicating its usefulness to sensitively identify foci where malaria declines, whereas the RDT had very low sensitivity. Estimates of force of infection using age sero-conversion rates were equivalent to an EIR of approximately 1 infectious bite/person/year, significantly less than previous estimates. The sero-prevalence profiles suggest a gradual decline of malaria transmission, confirming their usefulness in providing information on longer term trends of transmission. A greater variability in parasite prevalence among villages within a site than between sites was observed with all methods. The fact that serology equally captured the inter-village variability, indicates that the observed heterogeneity represents a stable pattern.PCR and serology may be used as complementary tools to survey malaria in areas of declining malaria prevalence such as the Gambia and Guinea Bissau. Plasmodium falciparum may be on the decline in parts of sub-Saharan Africa. Longitudinal health record-based datasets have recently indicated a significant reduction of the burden of disease in the Gambia [Although still considered a major international health problem, accumulating evidence indicates that malaria caused by e Gambia , in Kenye Gambia ,3 and Ere Gambia  occurrinIn February 2008, the Gambian Government launched a policy that malaria should be eliminated as a public health problem. The effectiveness of such efforts needs to be monitored carefully requiring an adequate surveillance system. It has already been recognized that dependent on the aim (control or elimination) and transmission intensity different surveillance methods are likely to be needed . DerivinAlthough surveillance is defined as an ongoing continuous collection of data , for praTaken together, these considerations indicate the need to employ further tools in addition to or instead of slide microscopy to fully capture parasite prevalence and transmission dynamics at the community level in areas where malaria appears to be on the decline.P. falciparum malaria parasite prevalence in the Gambia and Guinea Bissau, and to validate the usefulness of PCR, and serology as tools to monitor malaria endemicity in comparison to slide microscopy, and rapid diagnostic tests (RDT). To this end, cross-sectional surveys were carried out in 12 villages at three different sites enrolling a total of 2659 individuals at the end of the malaria transmission in January/February 2008.The aim of this study was to obtain up to date information on th - 15th January), and Farafenni (31st Jan - 7th Feb), and in Guinea Bissau, in the area around Caio (19th - 28th Feb), where the MRC supports a research laboratory. In both countries, malaria transmission is highly seasonal, occurring from August till January, with a peak in November. The study sites have been described in detail elsewhere [Consecutive cross-sectional surveys were carried out in the Gambia shortly after the end of the malaria transmission season 2008 in the areas around Basse  under oil immersion. 10% of slides were read twice as a quality check. An RDT for malaria (OptiMAL) was performed on site, following the manufacturer's instructions. The remaining sample was stored into an EDTA K2  tube. Blood was separated by centrifugation  into plasma and red blood cells which were subsequently stored separately at -20°C. Upon completion of the survey, DNA was extracted from the pellets using a Corbett Robotics X-Trator Gene™ , and detection of parasite DNA was performed based on nested PCR amplification of the multi-copy 18s rRNA gene as previously described [Thick blood films were performed in the field and Giemsa-stained in MRC laboratory facilities at Farafenni, Basse and Caio. Slides were examined for the presence of escribed . Positiv19, , fused to glutathione S transferase were measured by indirect ELISA as described [IgG antibody levels binding to MSP-1escribed . Sera weescribed .19 sero-prevalence data from each site stratified into age groups by using standard maximum likelihood methods, based on a binomial error distribution, as described [Taking the age stratified nature of the data into account estimates for the parasite prevalence in the population at each site were calculated as weighted averages of the slide microscopy results in the study population based on the age distribution in each of the villages. To model malaria endemicity based on malarial antibody responses, a reversible catalytic conversion model was fitted to the measured MSP-1escribed ,18. Thisescribed  a fixed 19 antibodies, allowing for the clustering between sites. Initially, variables that were likely to be associated or had already been shown to be associated with malaria prevalence in univariable analysis, such as age, gender, village, ethnicity and bed net use were included in the model. Subsequently, only variables with p-values < 0.05 were kept in the model, which applied to age and gender. In a second step possible additional risk factors such as ethnicity and bed net use were adjusted for.To investigate the potential differences in parasite or sero-prevalence within and between sites, separate mixed effect models were fitted for i) parasitaemia assessed by slide microscopy, ii) by PCR, and iii) for anti-MSP-1The cross-sectional surveys of malaria prevalence described here, carried out in January/February 2008, shortly after the end of the malaria transmission season showed that the parasite prevalence assessed by slide microscopy for those enrolled into the survey was 11.2% (CI 95%. 9.2-13.7%) in Farafenni, 10.4% (CI 95%: 8.6-12.5%) in Basse, and 4% (CI 95%: 2.8-5.5%) in Caio. Taking the age distribution at each village into account, the estimated parasite prevalence in the population in Farafenni, Basse and Caio is 10.9% (CI 95% 8.7-13.1%), 9.0% (CI95%: 7.2-10.8%), and 4% (CI95%: 2.6-5.4%), respectively. Compared to data from similar surveys carried out in the past ,25-34, tWhen parasite prevalence was assessed by qualitative PCR, a three-fold higher percentage  of the study population tested positive. The microscopy: PCR prevalence ratio is thus 0.33, indicating that parasite prevalence detected by microscopy is 66% lower than that detectable by PCR.P. falciparum in our laboratory, the detection limit of the RDT was between 140 to 1,400 parasites/μl , which is consistent with previous reports indicating considerable loss of sensitivity at levels below 500/μl [The RDT detected 8 fold 22 fold) less parasite carriers (1.1% CI 95%: 0.8 - 1.6%) compared to slide microscopy  spleen rates are displayed in a similar way. For all shown parameters, there is considerable variation between villages within a study site, but less variability between study sites. Using mixed effect models adjusting for age and sex and applied to either slide results, PCR or sero-prevalence data there was no clustering by study site , indicating that the variation of malaria prevalence between study sites is not significant.In Figure However, significant inter-village variability was observed for parasite prevalence assessed by slide microscopy in all sites , and by PCR in Basse (p < 0.0001) and Caio (p = 0.03). Likewise, significant inter-village variability was noted for sero-prevalence in Basse (p < 0.0001) and Farafenni (p < 0.0001). Even after additional adjusting for bed net usage and ethnicity, variability for parasite prevalence within a study site remained significant in Basse (p = 0.001) when assessed by slide reading, and in Basse (p = 0.0002) and Caio (p = 0.02) when assessed by PCR, while the variability for sero-prevalence remained significant in Basse (p < 0.0001) and Farafenni (p < 0.0001). However, since the association of village and malaria prevalence or sero-positivity was highly confounded with ethnicity  and those who didn't, overall bed net-usage had a protective effect with regard to microscopic parasite prevalence . Especially, children not using a net had a 3.3 fold , and 1.7 fold  higher parasite prevalence Figure . For chiThe presented surveys revealed surprisingly low levels of microscopically detectable malaria parasite prevalence at all study sites. Although direct comparison to previous studies is considerably limited by differences in study design (e.g. age groups surveyed) and the fact that surveys were carried out at different months of the year (shortly after the end of the transmission season versus peak of transmission season) and in different villages, the parasite prevalence values reported here are amongst the lowest published, remaining below the level reported from surveys carried out at the end of the non-transmission season. The data presented are thus in line with the decline in disease burden documented previously for the Gambia . The renNot surprisingly, the RDT, having a sensitivity threshold of around 500 parasites/μl, which is designed to detect clinical malaria, proved unsuitable to identify parasite carriers in this low transmission setting, where the geometric mean parasite density, amongst those with microscopically detectable parasitaemia, was as low as 104 parasites/μl (CI 95%: 76 - 143).Thick film slide microscopy, still considered as the diagnostic gold standard for malaria, missed out 66% of parasite carriers identified by nested PCR that has a detection limit of ~1 to 10 parasites/μl ,14. ThisClearly, the considerable number of individuals with low density parasitaemia missed by microscopy constitutes a major challenge for a malaria surveillance system: given that gametocyte prevalence amongst parasite carriers may be as high as 90% when assessed by Pfs25 QT-NASBA , and thaThe decline in burden of disease documented previously , and the19 antibodies was evident, both models with a fixed ⌊ [To assess whether interventions had a sustained impact, surveillance should therefore be able to capture long and mid-term trends of transmission too, which could be achieved by age profiles of anti -malarial antibody prevalence ,19. A su fixed ⌊   data available for 2000  and 2005Regardless of the method used , significant variability of parasite (or sero)-prevalence amongst villages of the same site were observed, whereas the variability between sites did not differ significantly. This remained true even after adjusting for the potentially confounding effects of bed net use and ethnicity, suggesting that other factors may contribute to the variability.The difference in the distance to malaria breeding sites that has been identified previously as a risk factor for malaria in the Gambia , as wellIn view of the planned malaria elimination efforts that will require adequate surveillance, the spatial heterogeneity of malaria transmission observed locally also constitutes a considerable challenge as it renders the identification of a sentinel village that ought to be representative for surveillance purposes of a larger area difficult. Alternative approaches, such as surveys carried out in schools and/or at health facilities which combine PCR/serology with geographic information (GIS) will become invaluable to identify remaining foci of transmission.Compared to previous reports, a shift of peak parasite prevalence (both by PCR and slide reading) towards the group of 11-15 years old was observed. This is in sharp contrast to a survey carried out in rural Gambia in the dry season of 1950 where over 95% of children < 5 years and 89% of children aged 6-10 years were found to be parasitaemic . Still iWhile age prevalence profiles of malaria parasitaemia may shift to older age groups with decreasing transmission regardless of its cause, the most obvious reason in our study area probably is the comparably high level of bed net usage in children < 5 years in the Gambia, currently being amongst the countries with the highest coverage of ITNs in children < 5 . As expeIn summary, the data presented here suggest that particularly in areas of declining malaria prevalence efficient surveillance requires methods able to reliably identify sub-microscopic levels of parasitaemia in order to identify remaining foci of transmission for targeted interventions. Where malaria eradication is the aim the considerable variability in malaria prevalence observed in a confined space calls out for a rather dense network of sentinel sites or alternative approaches. This will require highly sensitive, high throughput techniques such as PCR that show promise even on non-invasive samples . PCR wouThe authors declare that they have no competing interests.JS contributed to study design, conducted the fieldwork and the laboratory assays, and contributed to data analysis and drafting the manuscript; BW carried out data management and data analysis; CD analysed serological data, contributed to data interpretation and drafting the ms; DN and EO provided technical expertise and help with the PCR assays; SC helped organize the fieldwork and performed ELISAs; PC provided expertise with the ELISA, and the mathematical modelling; DC helped design the study, and critically revised the ms for important intellectual content; MW conceived, designed and planned the study, helped with the analysis and drafted the ms; All authors have given final approval to the ms.Demographic characteristics of the study population at all sites broken down to areas and villages within the areasClick here for file


Individuals homozygous for α+-thalassaemia have microcytosis and an increased erythrocyte count. α+-Thalassaemia homozygosity confers considerable protection against severe malaria, including severe malarial anaemia (SMA) (Hb concentration < 50 g/l), but does not influence parasite count. We tested the hypothesis that the erythrocyte indices associated with α+-thalassaemia homozygosity provide a haematological benefit during acute malaria.The heritable haemoglobinopathy α+-thalassaemia against severe malaria were reanalysed to assess the genotype-specific reduction in erythrocyte count and Hb levels associated with acute malarial disease. We observed a reduction in median erythrocyte count of ∼1.5 × 1012/l in all children with acute falciparum malaria relative to values in community children (p < 0.001). We developed a simple mathematical model of the linear relationship between Hb concentration and erythrocyte count. This model predicted that children homozygous for α+-thalassaemia lose less Hb than children of normal genotype for a reduction in erythrocyte count of >1.1 × 1012/l as a result of the reduced mean cell Hb in homozygous α+-thalassaemia. In addition, children homozygous for α+-thalassaemia require a 10% greater reduction in erythrocyte count than children of normal genotype (p = 0.02) for Hb concentration to fall to 50 g/l, the cutoff for SMA. We estimated that the haematological profile in children homozygous for α+-thalassaemia reduces the risk of SMA during acute malaria compared to children of normal genotype .Data from children living on the north coast of Papua New Guinea who had participated in a case-control study of the protection afforded by α+-thalassaemia may contribute substantially to their protection against SMA. A lower concentration of Hb per erythrocyte and a larger population of erythrocytes may be a biologically advantageous strategy against the significant reduction in erythrocyte count that occurs during acute infection with the malaria parasite Plasmodium falciparum. This haematological profile may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage.The increased erythrocyte count and microcytosis in children homozygous for α +-thalassaemia-homozygous children against severe malaria anaemia.Karen Day and colleagues show that increased microcytic erythrocyte count may contribute substantially to the protection of α +-thalassemia, which is common in Africa and Southeast Asia, provides protection against malaria, a parasitic disease that occurs in tropical and subtropical parts of the world. α+-Thalassemia is caused by the loss of one or more of the genes that encode the α chains of hemoglobin, the red blood cell (erythrocyte) protein that carries oxygen around the body. These α chains are normally encoded by four genes, two on each Chromosome 16 . People with heterozygous α+-thalassemia lack one copy of the α chain gene and have a –α/αα genotype (genetic makeup). People with homozygous α+-thalassemia lack one copy of the gene on each chromosome (they have a –α/–α genotype) and have mild “microcytic anemia,” a condition characterized by increased numbers of abnormally small erythrocytes (microcytosis) that contain reduced amounts of hemoglobin.Mutations (changes in the DNA that encodes proteins) continually arise within human populations. Harmful mutations that affect an individual's ability to reproduce usually disappear, but most other mutations persist at a low frequency. Some mutations, however, protect their human carriers against specific disease-causing organisms, and consequently occur at high frequencies in human populations that live in places where these organisms are common. For example, the inherited blood disorder α+-thalassemia causes mild anemia, it provides protection against severe malarial anemia, a potentially fatal complication of malaria. Malaria parasites cause anemia because they multiply inside erythrocytes and rupture them. Scientists originally thought that α+-thalassemia protects against malaria by interfering with the parasite's ability to infect erythrocytes, but the evidence collected so far does not support this hypothesis. In this study, therefore, the researchers have investigated whether the microcytosis and increased erythrocyte count associated with α+-thalassemia might be responsible for the protection that this blood disorder provides against severe malarial anemia. Specifically, they asked whether this hematological (blood) profile protects against severe malarial anemia because people with the –α/–α genotype lose less hemoglobin for a given degree of malaria-induced erythrocyte loss than do those with the normal genotype.Paradoxically, although homozygous α+-thalassemia) showed that homozygous α+-thalassemia protects against severe malaria. To investigate why, the researchers re-analyzed the genotype-specific reduction in erythrocyte counts and hemoglobin levels associated with acute malarial disease in these children and developed a simple mathematical model to predict hemoglobin levels after malaria infection. They found that when malarial infection reduced the number of erythrocytes per liter of blood by more than 1.1 × 1012 , children with homozygous α+-thalassemia lost less hemoglobin than did those with the normal genotype. Furthermore, children with homozygous α+-thalassemia needed a 10% greater reduction in their red blood cell count than children with the normal genotype for their hemoglobin levels to fall below the value that defines severe malarial anemia.A study done in the mid 1990s in children living on the north coast of Papua New Guinea , but speculate that the hematological profile associated with α+-thalassemia might also prevent other Plasmodium species causing anemia. Futhermore, they suggest, other mutations that increase the erythrocyte count and cause microcytosis might protect against severe malaria anemia in a similar fashion.These findings suggest that the increased number of abnormally small erythrocytes associated with homozygous αhttp://dx.doi.org/10.1371/journal.pmed.0050056.Please access these Web sites via the online version of this summary at thalassemia and on malaria (in English and Spanish)The MedlinePlus encyclopedia contains pages on US National Heart Lung and Blood Institute, from the US National Human Genome Research Institute, from the Cooley's Anemia Foundation, and from MedlinePlusDetailed information is available on thalassemia (including useful links to other resources) from the malaria (in English and Spanish)The US Centers for Disease Control and Prevention provide information on malaria Information is also available from the World Health Organization on  In other words, children homozygous for α+-thalassaemia would require 10% greater reduction in erythrocyte count than children of normal genotype, for Hb concentration to fall to 50 g/l. This result should be considered in relation to our observation that similar degrees of reduction in erythrocyte count were found among α+-thalassaemia genotypes in acute disease, relative to community controls  erythrocyte loss was higher in homozygous α+-thalassaemia homozygotes, we applied the reduction of 2.35 × 1012/l erythrocytes  to all children living in the community. The proportion of community children predicted to develop SMA, defined by the genotype-specific erythrocyte count definitions for SMA, was lower in children who were homozygous for α+-thalassaemia  than in heterozygous children , and those of normal genotype . Compared with normal children, the risk of a community child being defined as having SMA was 0.52  in α+-thalassaemia homozygotes and 0.74  in heterozygotes for this degree of reduction in erythrocyte count. This simple model based on observed haematological data predicted that children who were homozygous for α+-thalassaemia would be 48% less likely to develop SMA than children of normal genotype.To determine whether microcytosis and increased erythrocyte count could explain the protection observed against SMA in α+-thalassaemia may be a haematological advantage in the face of the estimated >30% reduction in erythrocyte count, which we observed in PNG children with acute malaria.The reasons for the microcytosis and relatively high erythrocyte count in carriers for alpha or beta thalassaemia are not understood. It has been proposed that it reflects an increased number of terminal cell divisions during erythropoiesis, due to the combination of defective haemoglobinisation of the erythrocytes and a highly proliferative bone marrow . We inve12/l) at which microcytosis and an increased erythrocyte count becomes an advantage to the host. This erythrocyte cutoff is considerably lower than the median erythrocyte reduction we estimated to be associated with acute malaria (∼1.5 × 1012/l). This result would account for the reversal of Hb concentrations in children with acute malaria , a phenomenon that has been noted previously , 3.27 × 1012/l [2.11–4.44]; mild malaria, 90 g/l [67–103], 4.03 × 1012/l [3.12–4.84]; community children, 100 g/l [88–109], 4.78 × 1012/l [4.24–5.24].Hb is positively associated with erythrocyte count, (158 KB JPG)Click here for additional data file.


The objective was to study the seasonal effect on the spatial distribution of the incidence of malaria in children under 10 years old living in the Manhiça district, Mozambique.The data of the clinical malaria incidence were obtained from a study of two cohorts of children followed from December 1996 to July 1999. The cases were obtained by the active detection method. Hierarchical Bayesian models were used to model the incidence of malaria, including spatial correlation nested to climatic season. The models were compared with the deviance information criterion. The age and gender of the children were also taken into account.The incidence of malaria is associated with age, period and climate season. The incidence presents a clear spatial pattern, with a higher incidence in the neighbourhoods situated in the north and northeast of the Manhiça area. The transmission of malaria is highest during the wet season but the spatial pattern of malaria does not differ from that during the dry season.The incidence of malaria in Manhiça presents a spatial pattern which is independent of the seasonal climatic conditions. The climate modifies the incidence of malaria in the entire region but does not change the spatial pattern of the incidence of this disease. These findings may be useful for the planning of malaria control activities. These activities can be performed taking account that the neighbourhoods with more incidence of malaria do not change over the annual climate seasons. Although the disease has been eradicated in most temperate zones, it continues to be endemic in most of the countries of sub-Saharan Africa and particularly, in the area of Mozambique where this study was performed. The study of malaria incidence conducted by Saute et al  in the aet al  located Anopheles). In this area of study the main species is Anopheles funestus  and then [et al .The main aim of this study was to evaluate whether the spatial patterns of the incidence of malaria in children under the age of 10 years living in an endemic area of the Manhiça district (in southern Mozambique) vary between the wet and dry seasons. The variables of age and sex were also considered in the analysis.The study was performed in the Manhiça district (Province of Maputo) in Southern Mozambique where the Centro de Investigaçao Saúde de Manhiça (CISM) is located.2. It is located in a well-drained sandy elevate plane, surrounded by the Incomati River meanders . The maps were scanned and georeferenced the limit of the neighbourhoods by CISM.Data on the incidence of clinical malaria episodes were obtained from a study performed by the CISM in this area . Two cohPlasmodium falciparum parasites). The children diagnosed with malaria were treated and excluded from the following period of 28 days. This study falls within the National Ethical Clearance granted to the malaria epidemiological studies of the CISM/National Institute of Health 1996.Each child visit was counted as one-person week at risk (PWAR). Clinical malaria was defined as fever  with a positive blood slide . The confidence intervals of the incidence density were calculated assuming that the number of new malaria episodes are Poisson distributed. A preliminary Poisson regression analysis was carried out to assess the relationship between the incidence of malaria and the covariates: sex, age groups, period, and climate season. The association between the covariables and the incidence was evaluated using the log-likelihood ratio test. This bivariate Poisson regression was fitted in the SAS programme with the GENMOD procedure . The depThe inclusion of regional random effects with a conditional autoregressive structure in the model for fitting the data would be called the spatial model . The regNext, this model was extended to include other regional random effects without spatial structure. This assumes independence on incidence density of the neighbourhoods and induces 'global' smoothing. With this spatial + non structured model, it is possible to check if part of the variability is not spatial . FinallyThe details of the models applied are explained in Additional file et al [The parameters of the regression were estimated by the Bayesian methods using Gibbs Sampling  and thret al . The creA total of 2,006 children were followed from December 1996 to July 1999. The average incidence density was six cases per 1,000 PWAR. The average seasonal rainfall and monthly mean temperature were of 1328.67 mm (SD: 374.59 mm per season) and 25.33°C (SD: 0.68°C) during the wet season and 372.10 mm (SD: 85.27 mm per season) and 20.80°C (SD: 0.58°C), respectively, in the dry season.The crude incidence density for each level of sex, age, period and climate season are presented in Table Figure The incidence density for each neighbourhood is plotted in Figure According to the exploratory data analysis with the Moran's I test , the hypothesis of the existence of spatial association is compatible with our data.The estimate and the credibility intervals for the parameters of the non spatial model, spatial model, spatial+non structured model and spatial seasonal model are presented in table Model comparison showed that the spatial model had a small DIC value and is, therefore, the model that best fit the data. The spatial seasonal model, that incorporates regional random effects nested within climate seasons, had only a slightly higher DIC value. Similarly, when the spatial model is extended with non-structured random effects, the DIC value is also slightly higher. The spatial variance of this extended model  is very similar to the variance of the spatial model  and the non-structured variation is very close to 0 .During the first period the incidence of malaria in the wet season is not different to that of the dry season . However, the incidences in the following two period intervals are greater in the wet than in the dry season. In the second period interval the density ratio is 1.775  being slightly higher in the third period: incidence density ratio = 2.481 .To illustrate the spatial distribution of the incidence of malaria, Figure The objective of this study was to determine the variation that seasonal effects can produce on the spatial distribution of malaria in children under the age of 10 years living in an area of the Manhiça district. The results of this study confirm that the incidence of malaria in Manhiça has a clear spatial pattern, with a high incidence in the neighbourhoods situated in the north and northeast. The variation in climate season influences the incidence in malaria but does not modify the spatial patterns.In this study, the incidence of malaria was obtained by a weekly follow up of a cohort of children in each neighbourhood. This method reinforces the reliability of the estimation of the incidence rates for each neighbourhood and eliminates the uncertainty involved in trials studying cases detected by passive detection methods.There was no difference in the incidence of malaria between the dry and wet seasons in the first period (Dec 96 – Oct 97). In contrast, in the second (Nov 97 – Oct 98) and third (Nov 98 – July 99) periods there was a greater incidence in the wet season. The results of this first period may have been the consequence of this serial data since the standard effect is for the incidence to be higher in the wet season. The interaction between period and climate season shows that the climate season varies along years. In addition to the climate season and period, our model incorporated the demographic characteristics of the children. Hence, this model was able to obtain the associations between the incidence in malaria and the seasons, adjusting for confounding demographic effects. In relation to demographic factors, young children up to five years of age are at the highest risk of malaria infection, probably they have not yet developed sufficient immunity, making these early years particularly susceptible.The covariates age group, period, and climate seasons, do not explain all the variability in the data, because there is a part of variability (overdispersion) that is captured by the regional random effects. It should also be taken into account that this overdispersion can be caused by the occurrence of more zero observations than expected by a Poisson distribution. To discard an excess of zeros in the data a zero inflated Poisson regression is also used . HoweverIn contrast to the spatial-temporal studies performed in Zimbabwe , the spaet al [et al [P. falciparum produced 90% of the cases of malaria in the region and the prevalence of infection by P. falciparum was higher in the north and northeast. The capacity of flight dispersion of the An. funestus (about 1 km – 2 km) [P. falciparum.According to these results, the neighbourhoods in the north and north-east, which had a higher risk of malaria, were located close to the Incomati River, concretely, on the edge of the swampy area of the river which is normally flooded during the wet season and seldom dries up after previous rainy season. In the study by Aranda et al , this arl [et al  found th – 2 km)  agreed wThe incidence of malaria in Manhiça area presents a spatial pattern which is independent of the seasonal climatic conditions. The climate modifies the incidence of malaria in the entire region but does not change the spatial pattern of the incidence of this disease. Children under five years of age are at the highest risk of malaria infection.These findings may be useful for the planning of malaria control activities. These activities can be performed taking account that the neighbourhoods with more incidence of malaria do not change over the annual climate seasons. Thus, these results suggest the need to plan interventions which continue over time in the areas with the highest incidence to control the transmission and thereby maximize the reduction in incidence.The authors declare that they have no competing interests.RA and CA were responsible for statistical analysis and the preparation of the manuscript. JA, FS and PA conceived and designed the study. FS, DN and AN were responsible for fieldwork and the geographical information system of Manhiça. JA was responsible for data cleaning and manuscript preparation.Description of the model formulation in terms of the malaria data set.Click here for file


The World Health Organization has recommended that anaemia be used as an additional indicator to monitor malaria burden at the community level as malaria interventions are nationally scaled up. To date, there are no published evaluations of this recommendation.To evaluate this recommendation, a comparison of anaemia and parasitaemia among 6-30 month old children was made during two repeated cross-sectional household (HH) and health facility (HF) surveys in six districts across Malawi at baseline (2005) and in a follow-up survey (2008) after a scale up of malaria control interventions.HH net ownership did not increase between the years (50.5% vs. 49.8%), but insecticide treated net (ITN) ownership increased modestly from 41.5% (95% CI: 37.2%-45.8%) in 2005 to 45.3% (95% CI: 42.6%-48.0%) in 2008. ITN use by children 6-30 months old, who were living in HH with at least one net, increased from 73.6% (95% CI:68.2%-79.1%) to 80.0% (95% CI:75.9%-84.1%) over the three-year period. This modest increase in ITN use was associated with a decrease in moderate to severe anaemia (Hb <8 g/dl) from 18.4% (95% CI:14.9%-21.8%) in 2005 to 15.4% (13.2%-17.7%) in 2008, while parasitaemia, measured as positive-slide microscopy, decreased from 18.9% (95% CI:14.7%-23.2%) to 16.9% (95% CI:13.8%-20.0%), a relative reduction of 16% and 11%, respectively. In HF surveys, anaemia prevalence decreased from 18.3% (95% CI: 14.9%-21.7%) to 15.4% (95% CI: 12.7%-18.2%), while parasitaemia decreased from 30.6% (95% CI: 25.7%-35.5%) to 13.2% (95% CI: 10.6%-15.8%), a relative reduction of 15% and 57%, respectively.Increasing access to effective malaria prevention was associated with a reduced burden of malaria in young Malawian children. Anaemia measured at the HF level at time of routine vaccination may be a good surrogate indicator for its measurement at the HH level in evaluating national malaria control programmes. The unprecedented increase of investment in the control of malaria in Africa means that over the coming years, there should be a substantial reduction in malaria-related morbidity and mortality in these settings. Unfortunately, there are still few affordable and reliable indicators that can be used to measure short-term, malaria-specific impact of the scale-up of various interventions at the community level. Malaria related mortality, an important overall malaria control target, is difficult to diagnose, define and measure at the population level . Its meaet al (2004) showing that, in areas of stable malaria transmission, moderate-to-severe anaemia (haemoglobin <8 g/dl) is more sensitive than is parasite prevalence, and may respond more quickly than mortality to changes in malaria exposure due to increasing coverage of malaria interventions such as insecticide-treated bed nets (ITNs), malarial prophylaxis, and indoor residual spraying [The World Health Organization (WHO) and Roll Back Malaria (RBM) Partnership have recommended that anaemia be used as an additional indicator to monitor malaria burden at the community level as interventions are nationally scaled-up . This reet al 200 showing Other reasons to include anaemia as an indicator for malaria control include: (a) its association with malaria in areas of high malaria transmission, (b) its ability to be measured and quantified in the field, (c) it has a morbidity burden, and (d) severe malarial anaemia, for young children and pregnant women, is on the pathway to mortality, which is the overall RBM target.To date, there is no published literature evaluating this recommendation, or whether measurement of these indicators at time of routine immunizations could serve as a comparable substitute to measurement at the household level. This paper presents the utility of two approaches to monitoring and evaluation recommended in the WHO/RBM guidelines. The success of interventions implemented under the National Malaria Control Programme (NMCP) and the Roll Back Malaria Malawi country plan were assessed using both parasitaemia and anaemia. The evaluation was also designed to determine if measurement of these indicators at the health facility level at time of Expanded Programme on Immunization (EPI) visits could be a comparable substitute for measurement at the household level, information that is potentially very useful to Malawi and other malaria-endemic countries as it is logistically easier and cheaper to conduct health facility surveys than household surveys.The study consisted of two population based cross-sectional surveys conducted in six sentinel districts Figure  across MRecognizing that malaria is a major public health problem, the government of Malawi is focusing on the rapid scale up of malaria interventions so as to significantly reduce malaria morbidity and mortality in the country Figure . Three sThe study protocol was approved by Institutional Review Boards at the University of Malawi and the US Centers for Disease Control and Prevention.For the 2005 HH survey, 30 enumeration areas (EAs) were selected with probability proportional to estimated size (PPS) in each of the six districts. The sampling frame was developed through the use of global positioning system (GPS) linked with hand-held computers (PDAs) to allow rapid preliminary mapping of the EA from which a simple random sample of households was selected. This method involved: a) mapping every household in the selected EA using PDAs equipped with GPS, b) selecting a random sample of 11 households, and c) using the GPS to navigate back to the households and perform the interview . This trIn the 2008 HH survey, a modified EPI cluster survey method, as described by Turner, was used to select households . A diffeThe EPI-HF survey component was conducted in three of the six districts selected for the HH survey  immediately following the respective HH surveys in those districts. In each of the three districts, government health facilities, which provide EPI services, were chosen to attain a sample size of 540 children per district. Due to differences in enrolment rates, five health facilities were chosen in Blantyre, three in Rumphi and four in Lilongwe in order to meet the required sample size. The same health facilities were used in both the 2005 and 2008 surveys. Children 6-30 months of age attending the EPI clinic for a routine visit were systematically sampled from the clinic and invited to enrol.Sample size was calculated based on assumption that 21% of all children 6-30 months old in the baseline survey would have Hb <8 g/dl (based on results from a 2003 household survey in Blantyre district). Sample sizes were calculated using 95% confidence limits, 80% power, and 10% adjustment for non-response to detect a 33% reduction in the prevalence of Hb <8 g/dl between two cross-sectional surveys. Using this estimate, a sample size of 1,900 households (712 children) was needed for the HH survey assuming 37% of households had children 6-30 months of age (DHS 2000) and a design effect of 1.32. A sample size of 1,617 children was required for the EPI-HF survey assuming a design effect of 3.0. The same sample size was applied for the 2005 and 2008 surveys.®) (2008). All children with anaemia were treated with iron and albendazole as per national guidelines.Structured interviews were conducted with household heads and/or parent/guardian of all children in selected households. Information was gathered on ownership of household assets, household net ownership, net use and health of children under five years of age; blood smears and haemoglobin results were obtained for children 6-30 months of age. Per national policy, children with an Hb<8 g/dl, or fever, or a history of fever in the previous two weeks, were treated with sulphadoxine-pyrimethamine (SP) (2005) or with artemether-lumefantrine . During the survey, data were backed up onto a secure digital card every evening. Upon survey completion, PDAs were returned to Blantyre, where data were aggregated into a Microsoft Access database.. Thick blood smears were prepared for microscopic diagnosis of malaria parasites. Blood slides were stained with field stain and read by an expert microscopist at the College of Medicine. Slides were declared negative if no asexual parasites were found after examining 100 high-power fields. Slide reading was blinded and the external quality control consisted of having all positive slides and 10% of randomly selected negative slides read again by an independent microscopist.Blood samples were collected from each child for on-the-spot measurement of haemoglobin concentration Household was defined as a family, comprising the head of household (man or woman), husband or wife, children and immediate family members who shared income. An ITN was defined as any bed net that had been treated with insecticide in the previous 12 months or a long-lasting insecticidal net (a net treated during the manufacturing process). Adherence to net/ITN was defined as a child sleeping under a net/ITN the previous night in a HH that owns a net/ITN. Parasitaemia was defined as the presence of any asexual malaria parasites detected on a thick peripheral blood smear. A child with haemoglobin concentration 5 to <8 g/dL was classified as moderately anaemic, while those with haemoglobin <5 g/dL were classified as severely anaemic. For the purpose of analysis, anaemia was defined as any haemoglobin level <8 g/dL. Clinical malaria was defined as a documented axillary temperature ≥ 37.5°C in the presence of any asexual parasitaemia. Household wealth scores were developed using loading factors from the 2004 Malawi Demographic and Health Survey [Estimates of frequencies were calculated using SAS survey procedures (SAS v9.1.3) which use a Taylor series expansion to account for stratification, cluster sampling and unequal probabilities of selection. All analyses of HH data used weights equal to the inverse probability of selection. The EPI-HF survey was not a probability sample and could not be weighted. The outcome variable anaemia was considered as dichotomous (Hb < 8 g/dL or Hb >= 8 g/dL) and parasitaemia was measured as presence or absence of asexual parasites.Multivariate logistic regression models were performed in SAS using Generalized Estimating Equations (GEE) with an exchangeable working correlation matrix to produce empirically corrected standard error estimates that account for clustering and unequal selection probabilities. Rao-Scott chi-square values were used to detect associations between the outcome variable and the predictors of anaemia such as net use, ITN use, and prompt and appropriate treatment of fever. To observe whether there were any statistically significant differences between HH and EPI-HF measurement in change of anaemia from 2005 and 2008, an interaction term between study year and an indicator variable identifying the survey type (HH or EPI-HF) was created. The final models for anaemia and parasitaemia were adjusted for age, ethnicity, SES, clinical malaria (in the case of anaemia), current fever (for parasitaemia), district, and whether or not the child had slept under an ITN the previous night.The 2005 HH survey approached 1,739 HHs for participation over a four-week period, and recruited 926 children between the ages of 6-30 months; immediately following the HH survey, 12 EPI-HFs were surveyed over a two-week period to yield 1,637 children in this same age group. Children included in the HH survey were older, had a different distribution of ethnic groups, were less likely to have sought treatment within 24 hrs, and were less likely to have concurrent fever than children included in the EPI-HF survey  quintile than the 2008 survey. Overall, HH net ownership (regardless of treatment status) did not increase between the years (50.5% vs. 49.8%), but ITN ownership increased modestly from 41.5% (95% CI: 37.2%-45.8%) in 2005 to 45.3% (95% CI: 42.6%-48.0%) in 2008; ITN ownership was significantly increased in the poorest quintile  in 2005 to 15.4% (95% CI: 13.2%-17.7%) in 2008, while parasitaemia prevalence, measured by microscopy, decreased from 18.9% (95% CI: 14.7%-23.2%) to 16.9% (95% CI: 13.8%-20.0%), a relative reduction in crude prevalence from baseline of 16% and 11%, respectively. In EPI-HF surveys, anaemia decreased from 18.3% (95% CI: 14.9%-21.7%) to 15.5% (95% CI: 12.7%-18.2%), while malaria decreased from 30.6% (95% CI: 25.7%-35.5%) to 13.2% (95% CI: 10.6%-15.8%), a relative reduction of 15% and 57%, respectively  were distributed and a new malaria treatment policy was implemented countrywide, with artemether-lumefantrine replacing SP as the first-line drug. A modest, non-statistically significant increase in ITN household ownership (from 41.5% to 45.3%) and a statistically significant increase in adherence to any net (regardless of treatment status) were observed between 2005 and 2008. The results also show a moderate reduction in the odds of anaemia; the prevalence of anaemia decreased by a modest 15-16% from baseline, depending on the type of survey. In light of these data, and the very recent introduction of ACT, it can be argued that anaemia is a sensitive indicator to the scale up of malaria interventions and may thus be used as a proxy indicator to track the burden of malaria and demonstrate timely impact of malaria interventions in areas of high malaria This finding is important because existing impact indicators are either hard to measure at the national scale  or not sensitive enough to changes in malaria transmission . In contrast, anaemia is measurable and quantifiable in the field with the portable HemoCue haemoglobinometre using small volumes of capillary blood, it requires a smaller sample size, and gives more precise results than does measurement of all-cause mortality for children under-five years of age. However, anaemia may have limited utility in areas of lower malaria transmission where the attributable fraction of anaemia due to malaria is too low to reflect changes in malaria transmission/control.Parasitaemia, another common indicator used to assess malaria control, was also evaluated in this study. The prevalence of parasitaemia was reduced by 11% in the HH survey and 57% in the EPI-HF survey. This observation supports the finding above that reduction in both anaemia and parasitaemia could have been due to the modest scale up of malaria interventions. The dramatic reduction in parasitaemia observed in the EPI-HF survey was unexpected but could be due to accidental inclusion of some sick children during the EPI-HF survey in 2005. This is supported by the data showing that the proportion of children who had fever at the time of recruitment into the EPI-HF survey was significantly decreased from 11.7% to 4.6% between 2005 and 2008 (p < 0.0001). A corresponding decrease was also observed in children presenting to the clinic with a history of fever in the past 48 hours (p < 0.0001) measured at time of routine immunizations may be a good surrogate indicator for its measurement at the household level, and should be evaluated/verified in other settings.The authors declare that they have no competing interests.DPM was responsible for the design of the study, implementation of the study protocol, data analysis/interpretation, drafting of the manuscript and critical revision of the manuscript. CHC was responsible for the study concept, design and interpretation of the data. JVE was responsible for the statistical analysis/interpretation of the data and drafting of the manuscript. AW, RB, GJM and DA were responsible for implementing the protocol, supervision of the data collection and critical revision of the manuscript. MD was responsible for the study concept/design, analysis/interpretation of the data and revision of the manuscript. All authors read and approved the final manuscript.Table A. Characteristics of children participating in the 2005 and 2008 household and EPI-health facility surveys. The table demographic information on children recruited during the 2005 and 2008 surveys.Click here for file


Plasmodium falciparum malaria.Severe and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in The utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral  and severe  malaria  from uncomplicated malaria . Comparative statistics are reported using a non-parametric univariate analysis . Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups , adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma.ANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates . Further, there was a significant decrease in ANG-1 levels in patients with SM  versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM  or UM and CM .These results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes. Plasmodium falciparum malaria progress to severe and potentially fatal forms of infection [Only a small proportion of individuals with nfection ,2. The dnfection . These fnfection . In studnfection . A rapidEndothelial activation and dysfunction have been implicated in the pathogenesis of severe and cerebral malaria -11. The Based on the hypothesis that dysregulation of angiopoietins is associated with severe malaria syndromes, ANG-1 and ANG-2 levels were examined in malaria-infected patients to determine if they would distinguish between uncomplicated, severe and cerebral malaria. This study focused on the predictive value of ANG-1 and ANG-2 levels in whole blood, since unprocessed whole blood obtained by finger prick is a preferred clinical specimen for point-of-care testing .Plasmodium falciparum diagnosis was confirmed by PCR as previously described [Individuals (≥ 13 years of age) presenting with falciparum malaria to Hospital for Tropical Disease clinics  were eligible for enrolment into this study. Patients were classified into uncomplicated (UM), cerebral (CM) or severe  malaria (SM) according to WHO criteria . Whole bescribed . The insWhole blood concentrations of ANG-1 and ANG-2 were measured by ELISA  according to the manufacturers' instructions. Concentrations were interpolated from 4-parameter-fit standard curves generated using a standard curve of recombinant human angiopoietin proteins. The lower and upper limits of detection for each assay were as follows: ANG-1  and ANG-2 .Statistical analysis was performed using SPSS v17.0. Comparative statistics are reported from a non-parametric univariate analysis . Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups , adjusting for differences due to ethnicity, age, parasitaemia and sex. Spearman's correlation was used to examine correlations between angiopoietin levels and parasitaemia or cumulative organ injury scores. Receiver operating characteristic (ROC) curves and area under the ROC curves were generated using SPSS v.17.0. Optimal test thresholds were derived mathematically from the ROC curves. To adjust for multiple comparisons at a family-wise simultaneous error rate of α = 0.05, Bonferonni corrections were applied as appropriate.P. falciparum malaria were enrolled during the study. Patients were classified as having UM, (n = 70), SM (n = 36) or CM (n = 87) [193 patients with (n = 87) . In patiWhole blood ANG-2 levels were significantly higher in individuals with SM or CM compared to individuals with UM  that differs significantly from that of a chance result (AUC: 0.5) ) were determined for ANG-1, ANG-2, and the ratio of ANG-2:ANG-1. Based on sensitivity and specificity, ANG-1 was best able to discriminate between UM and SM (cut-off (12.38 ng/mL): sensitivity: 0.861, specificity: 0.857, LR(+): 6.028, LR(-): 0.162)); whereas ANG-2 was better at discriminating between UM and CM (cut-off (1.33 ng/mL): sensitivity: 0.759, specificity: 0.771, LR(+): 3.319, LR(-): 0.313)). The ratio of ANG-2:ANG-1 was best at discriminating between complicated disease of mixed phenotype (SM+CM) (cut-off 0.08): sensitivity: 0.789, specificity: 0.829, LR(+): 4.60, LR(-): 0.264))  and a negative correlation between ANG-1 and parasitaemia . Furthermore, ANG-2 and ANG-1 were negatively correlated with one another . Multivariate logistic regression analysis revealed that the observed differences in ANG levels between groups remained even after adjusting for differences due to ethnicity, age, gender, and parasitaemia. There remained a significant increase in ANG-2 (and the ratio of ANG-2:ANG-1) in SM vs. UM, and CM vs. UM, with no significant difference between SM and CM. For ANG-1, there was a significant decrease in SM vs. UM, CM vs. UM, and SM vs. CM (p < 0.0001).To address whether ANG levels are a measure of disease severity, an organ injury score ranging from 0-5 was assigned to each participant based on the number of organ systems involved. A value of 1 was added to the organ injury score for each participant based on the presence of each of the following: acute renal failure requiring haemodialysis, clinically apparent jaundice, severe anaemia requiring a blood transfusion, circulatory collapse with a systolic pressure <80 mmHg, and coma. ANG-2 and the ratio of ANG-2: ANG-1 were positively correlated with cumulative organ injury . Interestingly, ANG-1 also trended towards a positive correlation  despite its ability to discriminate between individuals with cerebral vs. severe non-cerebral malaria. To investigate this further, a post-hoc analysis was performed to examine how ANG-1 correlated with the organ systems included in the analysis. There were no associations observed between ANG-1 levels in participants with hepatic dysfunction, severe anaemia or circulatory collapse, although the latter was underpowered. However, ANG-1 was positively associated with renal dysfunction  and coma .These results demonstrate that whole blood ANG-1 and ANG-2 levels are robust biomarkers of severe and cerebral malaria. ANG-2, ANG-1 and the ratio of ANG-2: ANG-1 were significantly different between UM and both SM and CM Figure . Of partThe ability of angiopoietins to discriminate between uncomplicated and complicated malaria has now been confirmed in three distinct geographic regions and populations . These proteins have previously been measured in serum ANG-1, ANG-2) , plasma , ANG-2 ,ANG-1 and ANG-2 are stable proteins able to withstand repeated freeze thaws without significant changes in protein levels, and are readily detectable, as this study demonstrates, in whole blood. Furthermore, the angiopoietins, which are linked with inflammation, angiogenesis, and endothelial function and integrity, play a central role in many of the disease processes implicated in the pathogenesis of severe malarial syndromes. ANG-2 exists pre-stored within WP bodies and is rapidly released upon, and may further contribute to, endothelial activation . As a reIn the current study, ANG-2 levels were elevated in severe malarial syndromes compared to uncomplicated malaria but were also correlated with scores of cumulative organ injury in the cohort of severe malaria patients. This confirms other studies suggesting that ANG-2 is a marker of disease severity in conditions associated with endothelial activation ,11,13,14The use of biomarkers is inherently limited by their specificity for the disease in question. Dysregulation of angiopoietins may occur in a number of severe infectious syndromes associated with endothelial activation and dysfunction. As a result, it will be important to consider the implications of co-infections, including bacterial and viral (e.g. HIV) infections if these proteins are to be used as prognostic indicators for malaria.Finally, based on our study design it was not possible to examine the predictive value of angiopoietins and future prospective studies are needed to examine the kinetics of angiopoietin levels and their relation to symptom onset, clinical history of malaria, and response to treatment. However, should angiopoietins fail to distinguish between malaria and other infectious diseases, these proteins may still be informative biomarkers to facilitate clinical decision-making  and optimal allocation of health resources for the treatment of severe infections associated with endothelial dysfunction.In summary, these data suggest that ANG-1 and ANG-2 are promising biomarkers of severe malaria. Furthermore, the ability to robustly detect angiopoietin levels in whole blood makes them attractive candidates for potential integration into point-of-care diagnostic/theranostic devices of disease severity.The University Health Network holds intellectual property related to the role of angiogenic factors in the pathogenesis of infectious disease. The authors declare that they have no competing interests.ALC performed data analysis and drafted the manuscript. FEL contributed to the study design and performed the experiments. EIL performed the experiments. NT and SK contributed to the study design and collection of patient samples. WCL and KCK conceived of the idea, contributed to study design and helped write the manuscript. All authors read and approved the final manuscript.


Given that helminth infections are thought to have strong immunomodulatory activity, the question whether helminth infections might affect responses to malaria antigens needs to be addressed. Different cross-sectional studies using diverse methodologies have reported that helminth infections might either exacerbate or reduce the severity of malaria attacks. The same discrepancies have been reported for parasitemia.Plasmodium spp infections, and the incidence of malaria episodes are recorded. In vitro cultures of whole blood, stimulated with a number of antigens, mitogens and toll like receptor ligands provide relevant immunological parameters at baseline and following 1 and 2 years of treatment rounds. The primary outcome of the study is the prevalence of Plasmodium falciparum and P. vivax infection. The secondary outcome will be incidence and severity of malaria episodes detected via both passive and active follow-up. The tertiary outcome is the inflammatory cytokine profile in response to parasite antigens. The project also facilitates the transfer of state of the art methodologies and technologies, molecular diagnosis of parasitic diseases, immunology and epidemiology from Europe to Indonesia.To determine the effect of geohelminth infections and their treatment on malaria infection and disease outcome, as well as on immunological parameters, the area of Nangapanda on Flores Island, Indonesia, where malaria and helminth parasites are co-endemic was selected for a longitudinal study. Here a Double-blind randomized trial will be performed, incorporating repeated treatment with albendazole (400 mg) or placebo at three monthly intervals. Household characteristic data, anthropometry, the presence of intestinal helminth and The study will provide data on the effect of helminth infections on malaria. It will also give information on anthelminthic treatment efficacy and effectiveness and could help develop evidence-based policymaking.This study was approved by The Ethical Committee of Faculty of Medicine, University of Indonesia, ref:194/PT02.FK/Etik/2006 and has been filed by ethics committee of the Leiden University Medical Center. Clinical trial number:ISRCTN83830814. The study is reported in accordance with the CONSORT guidelines for cluster-randomized studies. Ascaris lumbricoides), whipworms (Trichuris trichiura), and/or hookworms  [Worldwide, more than a billion people are infected by geohelminths, with a majority harboring roundworms  -3. Such odenale) ,5. Anothodenale) . Their aodenale) . The latAlthough the immunological consequences of helminth infections primarily reflect responses directed towards helminth antigens, there may be spill-over effects on responses to unrelated antigens ,8. WhereMalaria itself is one of the most serious infectious diseases, infecting 5-10% of the world's population, with 300-600 million cases and more than 2 million deaths annually . In areaA. lumbricoides might suppress malaria symptoms [Co-infection is the norm in nature ,17, sincsymptoms . Since tsymptoms  or reducsymptoms -24 the ssymptoms ,25.http://www.immunospin.org has been initiated. As part of the project ImmunoSPIN, the helminth-malaria sub project  was designed to determine whether and how helminth infections may affect the course of a malaria infection. This study is a double-blind randomized trial of placebo and anthelminthic treatment to elucidate the impact of helminth infections on malaria longitudinally in an area where helminth infections and malaria coexist. It is expected to provide new information and contribute to our understanding of how regulatory responses that may be induced by chronic helminth infections affect inflammatory conditions, especially on malaria infection.Although helminth infections are considered as pathogens, it is clear that their course of infection is relatively free of overt clinical manifestations and in recent years several reports have shown their suppressive effect on diseases such as allergy ,26, autoThis paper presents the rationale of the ImmunoSPIN-Malaria study that, at the clinical and biological level, aims to discern plausible and meaningful interactions between these infections.A. lumbricoides, hookworms and T. trichiura) and malaria parasites . The sub-district is divided into villages of which those located near the primary health centre (Puskesmas), Ndeturea, Ndorurea1, and Ndorurea are the focus of ImmunoSPIN-Malaria study.Nangapanda is a sub-district of the Ende district, Flores Island, Indonesia and is situated in a coastal area with a population of about 22000 Figure . SituateThe majority in the study area is Ende tribe and migrants from neighbouring district of Bajawa that have populated the island for more than 300 years. Most individuals work as farmers and grow their own food. Recently also government employees and individuals owning small businesses moved to the area. Individuals with Chinese and Arabic ethnicity have come to the area about 200 years ago and most of them, run business corporations with other tribes. There are very few other tribes from outside of Flores and most of them were brought from outside by marriage with Flores natives. The Nangapanda population is dynamic and individuals shift address within the area as well as move in and out of the area for purposes of studying, marriage, or searching for jobs.Baseline mapping of houses by Global Positioning System (GPS) system has allowed maps to be generated using ArcGIS 9,1 software . In order to perform spatial analyses based on individual data, geographical coordinates were assigned to each individual as well as centroid coordinates to households.Community workers were recruited and trained to do questionnaires, follow-ups, malaria surveys (finger prick), and distribute treatment or placebo, as well as doing health promotion among the population. Each community worker will be in charge of a certain number of households that they visit monthly for recording movement and active follow-up of malaria symptoms, and three monthly for finger pricking and distributing the treatment.The study is designed as a double-blind randomized trial with two arms. One arm is treatment with albendazole (single dose of 400 mg) , while tOn approval of individuals in the area, peripheral blood will be collected from a subset of individuals over 4 years of age that were randomly selected for whole blood culture based on households. The assay will be done at baseline, 1 and 2 years after treatment. For those who are not included in whole blood assays, malaria evaluation by finger pricking will be obtained aiming to include the whole population in the study area.Every household receives a card identifying all family members. This card is used when community workers visit homes and when family members visit the Puskesmas. Data on socioeconomic status and health status is collected at baseline and 2 years after treatment using questionnaires in Bahasa Indonesia. Monthly data such as birth, death and migration will be recorded. Newborns and individuals who enter the study area will be registered with a newly assigned ID number as well as their geographical coordinates. Active follow-up includes a questionnaire with questions on clinical complaints in general and clinical malaria in particular and will be conducted monthly by community workers. In addition to the questionnaire, blood slides will be collected at three monthly intervals to test for plasmodia. For examination of intestinal helminths, stool samples will be collected yearly. Samples will be used for microscopy and PCR analyses. Passive follow-up will be held in collaboration with the Puskesmas that keeps clinical records of individuals that visit either for consultation or for overnight admission, including information on diagnosis and treatment. If malaria is suspected, two blood slides will be collected: one to be examined by the Puskesmas staff and another for re-examination by the research team in Jakarta at the Department of Parasitology, University of Indonesia (UI).All blood samples , blood culture supernatants, as well as stool samples for PCR, will be kept at -20°C in a temperature recorded freezer which is checked twice a day and will be sent to Jakarta on dry ice for storage -20°C or -80°C.P. falciparum and P. vivax up to 2 years after treatment, the secondary outcome is the incidence and severity of malaria recorded as a result of passive and active follow-up up to 2 years after treatment. The tertiary outcome is the inflammatory cytokine responses to P. falciparum antigens 1 and 2 years after treatment.The study aims to determine whether and how helminth infections may affect the course of infection with malaria parasites and disease outcome. Therefore we will monitor disease in individuals treated with albendazole compared to placebo. The primary outcome is the prevalence of infection with P. vivax ≥ 250/ul, P. falciparum ≥ 1000/ul). Asymptomatic carriers have a blood slide positive for P. falciparum and/or P. vivax asexual forms, as well as positive result by PCR and no concomitant clinical symptoms. Suspected malaria cases are defined as individuals who report symptoms typical of malaria but for whom no slide is available at the time of presentation with symptoms.Helminth and plasmodia infections will be defined by the presence of parasites detected by microscopic examination of stool and blood samples respectively and will be confirmed by molecular (PCR-based) methods -38. A maThe Harada Mori method will be carried out on fresh stool samples to detect hookworm larvae. A certain amount of each stool sample is preserved in formalin (4%) and kept at room temperature for microscopic examination. The formol-ether acetate concentration method  is perfo3 PFU/ml Phocine herpes virus 1 (PhHV-1) is added to serve as an internal control [For DNA isolation from stool, approximately 100 mg unpreserved faeces (that was kept at -20°C) are suspended in 200 μl PBS containing 2% polyvinylpolypyrolidone . DNA isolation and setup of the PCR reactions are performed using a custom-made Hamilton robot platform (made in Germany). After heating for 10 min at 100°C suspensions are treated with sodium dodecylsulphate-proteinase K for 2 h at 55°C. DNA is isolated using QIAamp DNA-easy 96-well plates  . Within  control .A. duodenale-, N. americanus-, and S. stercoralis-specific primers and probes are used as described previously [A. duodenale specific XS-probe is labelled with Texas Red and the S. stercoralis-specific probe is labelled with Quasar-705. The A. lumbricoides-specific primers and probe are chosen using Primer Express software , from the internal transcribed-spacer-1 (ITS1) sequence of A. lumbricoides (GenBank accession ALJ000895). The A. lumbricoides-specific primers, Alum96F 5'-GTAATAGCAGTCGGCGGTTTCTT-3' and Alum183R 5'-GCCCAACATGCCACCTATTC-3' amplify an 87-bp fragment of the ITS1 sequence and the XS-probe Alum124T Yakima Yellow-5'-TTGGCGGACAATTGCATGCGAT-3'-XS is used to detect the A. lumbricoides-specific product. The real-time PCRs were optimized first as monoplex assays with 10-fold dilution series of A. duodenale, N. americanus, A. lumbricoides, and S. stercoralis DNA, respectively. The monoplex real-time PCRs were thereafter compared with the multiplex PCR with the PhHV internal control. The cycle threshold (Ct) values obtained from testing the dilution series of each pathogen in both the individual assay and the multiplex assay were similar, and the same analytical sensitivity was achieved. The multiplex ANAS PCR showed 100% specificity when tested against 145 DNA controls derived from a wide range of intestinal microorganisms [Sequences of the eviously ,38 with rganisms .2, 2.5 μgram Bovine Serum Albumin , 5 pmol of each A. duodenale-specific primer, and of each N. americanus-specific primer, 2 pmol of each A. lumbricoides-specific primer, 2.5 pmol of each S. stercoralis-specific primer and 3.75 pmol of each PhHV-1-specific primer, 1.25 pmol of each N. americanus-specific XS-probe, A. lumbricoides-specific XS-probe, S. stercoralis-specific double-labelled probe, and PhHV-1-specific double-labelled probe, and 2.5 pmol of the A. duodenale-specific XS-probe, and 5 μl of the DNA sample.Amplification reactions are performed in white PCR plates in a volume of 25 μl with PCR buffer , 5 mM MgClAmplification consists of 15 min at 95°C followed by 50 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Amplification, detection, and analysis are performed with the CFX real-time detection system (Bio-Rad laboratories). The PCR output from this system consists of a cycle-threshold (Ct) value, representing the amplification cycle in which the level of fluorescent signal exceeds the background fluorescence, and reflecting the parasite-specific DNA load in the sample tested. Negative and positive control samples are included in each amplification run.The amplification is considered to be hampered by faecal inhibitory factors if the expected cycle threshold (Ct) value of 33 in the PhHV-specific PCR is increased by more than 3.3 cycles.P. falciparum, P. vivax, P. malariae, and P. ovale, blood slides (thick and thin) are stained with Giemsa [To detect h Giemsa  followedh Giemsa .DNA was isolated from 200 μl blood with QIAamp DNA-easy 96-well plates according to the manufacturer's recommendations. DNA isolation and setup of the PCR reactions are performed using a custom-made Hamilton robot platform.Plasmodium-specific primers and the P. falciparum, P. vivax, P. ovale, and P. malariae-specific probes are used as described previously[P. falciparum-specific XS-probe is labelled with Yakima Yellow, the P. ovale-specific XS-probe is labelled with Texas Red and the P. malariae-specific probe is labelled with Quasar-705. An additional P. ovale-type 2 XS-probe (Texas Red 5'-TCCAAAAGGAATTTTCTTATT-3'-XSQ) is used for sensitive detection of the P. ovale genetic variant type 2 (GenBank accession X99790/J001527).Sequences of the reviously,44 with P. falciparum, P. vivax-, P. malariae-specific XS-probes, and PhHV-1-specific Cy5 double-labelled detection probe, and 2.5 pmol of each P. ovale-specific XS-probes (Biolegio), and 5 μl of the DNA sample were used.Amplification reactions of each DNA sample are performed in white PCR plates, in a volume of 25 μl with PCR buffer (HotstarTaq master mix), 5 mmol/l MgCl2, 12.5 pmol of each Plasmodium-specific primer and 15 pmol of each PhHV-1-specific primer, 1.5 pmol of each Amplification, detection, and analysis are performed as described for the faecal PCR.2. Stimulations were performed with medium/control, PHA , LPS , Pam3Cys , PolyIC , Ascaris antigen  using Liquichip analyzer software .Blood samples (6 ml) are drawn into Vacutainers  containing sodium heparin as anticoagulant. Within 6 hours, blood cultures are set up according to methods that were optimized and tested under field conditions during pilot studies. The heparinized blood is diluted 1:4 with RPMI 1640 medium   and cultured in 96 well round bottomed plates in 37°C with 5% CO E et al ), iRBC (therlands), uRBC (herlands ). SupernTotal IgE will be measured as described previously . BrieflyP. falciparum or P. vivax infections in terms of presence of infection and intensity. The effect of anthelminthic treatment will be assessed 1 and 2 years after treatment by analyzing the prevalence ratios as well as the incidence ratios of malaria infection . In addition, treatment will be compared with placebo for reduction of helminth infection and is based on an intention to treat principle, in anticipation of individual movement between treatment groups. Cytokine profiles in response to parasite antigens will be analyzed for levels of pro- and anti-inflammatory cytokines.A database using MS Access is developed for this study. At baseline, we will analyze whether helminth infected subjects are at a higher risk of having The characterization of immune responses to helminth infections, malaria infections and co-infections will be assessed prior to treatment using linear regression . As cytoMultiple regression analysis will be used to determine 1) the association between helminth infections  with malaria parasites ; 2) the effect of decreasing helminth infection on plasmodia parasitaemia and incidence of malaria cases and 3) the effect of decreasing helminth infection on immunological responses to malaria antigen. All analyses will be adjusted for confounders such as socioeconomic status, body mass index, age and sex. During the study any other confounders that are identified will be used in our analyses.P. falciparum and P. vivax was 12% in the target population and the helminth infection  in the population was 60%. As study activities such as active follow-up and prompt treatment will have an effect on the prevalence, we expect the malaria prevalence to decrease to an estimated 6%. Based on these findings a power calculation  gave a sample size of 749 study subjects needed, in order to detect a 50% reduction or increase in the prevalence of plasmodia after 2 years of anthelminthic treatment. The effect of treatment will presumably be present in helminth carriers only and as an estimated 60% will be carriers of helminths during study period, giving an estimated 1248 subjects in each study arm will be needed. Allowing for an estimated 20% lost to follow-up due to movement and refusal, we would need to include 1495 participants in each arm.Unpublished microscopy data from the area showed the combined prevalence of This study was approved by The Ethical Committee of Faculty of Medicine, University of Indonesia, ref: 194/PT02.FK/Etik/2006 and registered as clinical trial ref: ISRCTN83830814 and has been filed by ethics committee of the Leiden University Medical Center. The study is reported in accordance with the CONSORT guidelines for cluster-randomized studies .The regional health authorities in Ende, the regional capital, were informed of the study and gave their agreement and support. Socialization took place over a two year period prior to the study involving staff at the public health centre (Puskesmas) and 50 community workers, including training for follow-up of study subjects keeping the community well-informed and well-engaged. Through many organized sessions, the village heads were involved in passing on information about the study, including the benefits and risks involved. The longitudinal nature was explained and information sheets and consent forms (in Bahasa Indonesia) were distributed. Inhabitants of the area were invited to participate in the study, consented either by written signature or thumb print and were informed that they may withdraw from the study at any time, for any reason and without consequences. For children below 15 years old of age also parent consent was obtained. Probable illness by burden of helminth infection will be taken into account by three study doctors that will be present in the area as well as by collaboration with the Puskesmas. Severe cases will be treated directly. The medical doctors will also give support and treatment to the Puskesmas. After completion of the study the whole population will be adequately treated for helminth infections ,50.The study has so far provided data that are shown in a flow chart given in Figure Table The traditional source of income in the area is farming and fishing while some individuals engage in jobs at government offices with a few in the private sector Figure . The eduSince this large community-based trial provides an important possibility to undertake a series of evaluations on the effect of helminth infections on malaria as well as the control of helminth and malaria at the community level, our study could help develop evidence-based policymaking. This study is unique in that it will provide data on anthelminthic treatment efficacy and effectiveness in a defined large population in a developing country. In conclusion the ImmunoSPIN helminth-malaria study is the first and currently the only longitudinal study of helminth and malaria co-infections in Indonesia. The study has received enthusiastic support from the authorities in Ende. At the same time, the study facilitates the transfer of state of the art technologies in immunology, molecular biology, epidemiology and statistics to Indonesia.IgE: immunoglobulin E; IL: interleukin;The authors declare that they have no competing interests.AEW Medical doctor in charge of the field study, involve in setting up, supervising gathering of data, clinical care, and follow up of the study populationMAP Medical doctor in charge of the field study, involve in setting up, supervising gathering of data, clinical care, and follow up of the study populationFH Medical doctor in charge of the field study, involve in setting up, supervising gathering of data, clinical care, and follow up of the study populationLW Medical doctor in charge of the field study, involve in setting up, supervising gathering of data, clinical care, and follow up of the study populationBL Medical doctor who is the advisor on databases, epidemiological and statistical aspects of the studyIA Medical doctor who is the advisor on databases, epidemiological and statistical aspects of the studyHWU Statistician who is developing methods to analyze the complex data generated during the lifetime of the projectHW Parasitologist and field study expert who is in charge of the process of data selection, storage, safeguarding randomization, and privacy of the study subjectsYD Medical doctor who advises on the immunological aspects of the studySW Medical doctor who is supervising of study set upIS Medical doctor who is a specialist on malaria and advises on clinical malaria and in the studyLM Immunoepidemiologist who is advising on databases maintenance, epidemiological, statistical, and immunological aspects of the studyAJFL Immunologist who specializes in malaria immunology and advises on malaria responses in the studyJJV Molecular parasitologist who is involved in the molecular diagnosis of parasitic infectionsES Immunoparasitologist who is involved in coordinating the study and advising on parasitological and immunological aspects of the studyMY Immunologist who has developed the study and is the Dutch coordinator of the ImmunoSPIN programTS Parasitologist who has developed the study and is the Indonesian coordinator of the ImmunoSPIN programAll authors read and approved the final paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/77/prepub


In areas in which malaria and HIV-1 are coendemic, adults with severe malaria who show no clinical signs of immunosuppression are likely to be infected with HIV-1. To determine whether HIV-1 infection and HIV-1–related immunosuppression were risk factors for severe malaria in adults with some immunity to malaria, we conducted a case–control study in Luanshya, Zambia, during December 2005–March 2007. For each case-patient with severe malaria, we selected 2 matched controls . HIV-1 infection was present in 93% of case-patients, in 52% of controls with uncomplicated malaria, and in 45% of asymptomatic controls. HIV-1 infection was a highly significant risk factor for adults with severe malaria compared with controls with uncomplicated malaria  and asymptomatic controls . Persons with severe malaria were more likely to have a CD4 count <350/µL than were asymptomatic controls . The degree of interaction between HIV-1 infection and malaria emerged during 1999–2009 and has been extensively reviewed for both nonpregnant and pregnant adult women  or history of fever in the previous 48 hours, Plasmodium falciparum monoinfection with a density of at least 100 parasites per 200 leukocytes  in the absence of any other evident causes of illness, and at least 1 of the following signs: impaired consciousness (Glasgow Coma Score <10), multiple grand mal convulsions, jaundice, hypoglycemia (glycemia <2.5 mmol/L), hyperparasitemia , renal impairment, and cardiorespiratory distress. We collected information about anemia but did not include anemia in the case definition because it can be HIV-1 related, resulting in possible bias, and because uncomplicated and severe malaria episodes can be misclassified in HIV-1–infected persons.The study was conducted at Thompson Hospital, a government district hospital serving Luanshya district in the Copperbelt province of Zambia. In this district, 99% of the population is at risk for malaria , and 30%Patients with severe malaria were treated according to the national guidelines for severe malaria in Zambia , with adequate supportive treatment  . For each participant, a blood sample (0.5 mL) was collected for HIV-1 testing and CD4 cell count, and for hemoglobin (1 mL), by venipuncture. Neither the study staff nor the patient had access to the HIV-1 test results. HIV-1 testing followed an unlinked anonymous procedure: blood samples were sent to the hospital laboratory, where they were processed anonymously. Determine HIV1/2/O  was the first test. If test results were negative, the patient was considered not infected. If results were positive or indeterminate, the blood sample was tested with Unigold Recombinant HIV-1/2 ; if results were positive, the patient was classified as HIV-1 infected. If results were negative, the outcome was considered indeterminate, and the sample was further tested with ELISA and Western blot. Patients, after recovering from malaria, and controls were counseled about HIV-1 and offered the opportunity for voluntary counseling and testing.Laboratory technicians were blinded to patient identity and to patient-related parameters. We used microscopy to screen case-patients and community controls for malaria using Giemsa staining 10% for 5 min. A thin blood film was examined to identify type of parasite; a thick film was taken for quantification. Computation of parasite density took into consideration the actual leukocyte count. In asymptomatic controls, we screened for CD4 counts were determined by flow cytometry on a CyFlow  within 5 hours after collection, and multiset software was used to obtain the absolute counts and the CD4+ lymphocyte ratio , were described by using summary statistics, means, and count and were tested by using Wilcoxon signed rank test for matched variables.Crude associations between the primary outcome measures and potential risk factors, including HIV-1 status and HIV-1–related immunosuppression  . Case-paEighty-three percent of case-patients had a CD4 count <350/µL, compared with 79% of controls with uncomplicated malaria (p = 0.76) and 8% of asymptomatic controls (p<0.0001). Controls with uncomplicated malaria were more likely than asymptomatic controls to have a CD4 count <350/µL . Case-patients were more likely to have a CD4 count <350/µL than were asymptomatic controls  but not controls with uncomplicated malaria .The extremely high proportion of low CD4 count in both case-patients and controls with uncomplicated malaria might be confounded by a temporary malaria-induced reallocation of specific T-cells (–Streptococcus pneumoniae or Salmonellaenterica serovar Typhimurium infection (In Luanshya, Zambia, an area where malaria is mesoendemic, HIV-1 infection is an important risk factor for severe malaria in adults, primarily in those with a low CD4 count. The increased risk for severe malaria in HIV-1–infected persons already has been reported from areas of low and unstable transmission (The increased risk for uncomplicated malaria in HIV-1–infected patients with a low CD4 count is consistent with information in several cohort studies (Almost half of malaria cases throughout the world occur in areas where the disease is holoendemic (