Plasmodium falciparum and almost never for Plasmodium vivax. We conducted a longitudinal cohort study in a Karen population of 3,484 individuals in a region of mesoendemic malaria, Thailand from 1998 to 2005. The number of P. falciparum and P. vivax clinical cases and the parasite density per person were determined. Statistical analyses were performed to account for the influence of environmental factors and the genetic heritability of the phenotypes was calculated using the pedigree-based variance components model. The genetic contribution to the number of clinical episodes resulting from P. falciparum and P. vivax were 10% and 19% respectively. There was also moderate genetic contribution to the maximum and overall parasite trophozoite density phenotypes for both P. falciparum (16%&16%) and P. vivax (15%&13%). These values, for P. falciparum, were similar to those previously observed in a region of much higher transmission intensity in Senegal, West Africa. Although environmental factors play an important role in acquiring an infection, genetics plays a determinant role in the outcome of an infection with either malaria parasite species prior to the development of immunity.The majority of studies concerning malaria host genetics have focused on individual genes that confer protection against rather than susceptibility to malaria. Establishing the relative impact of genetic versus non-genetic factors on malaria infection and disease is essential to focus effort on key determinant factors. This relative contribution has rarely been evaluated for  Plasmodium falciparum and Plasmodium vivax are the most common. These two species co-exist in many parts of the world including Thailand. Despite major attempts over the past century to control malaria, this infection remains the most important human parasitic disease worldwide. P. falciparum is estimated to cause about half a billion episodes per year P. vivax, P. malariae and P. ovale. Malaria parasites are becoming increasingly drug resistant and despite considerable effort over the last decade, there is as yet no sight of an effective vaccine.Human malaria is caused by four species of erythrocyte-invading plasmodial parasites, of which P. falciparum parasite density and chromosome 10p for the number of malaria episodes of approximately 35% Human genetic resistant factors for malaria were first discovered half a century ago, largely as a result of Haldane's insight that malaria was the likely evolutionary driving force behind common erythrocyte variants in tropical populations Plasmodium infection and malaria disease are well known to be strongly influenced by environmental factors. In addition, there are human non-genetic factors such as age and gender that also have a major effect on both incidence and outcome of infection. Establishing the relative impact of genetic versus non-genetic factors on infection and disease is essential to focus effort on key determinant factors. This relative contribution has been rarely evaluated 2h) in infection and disease. Estimation of heritability requires reliable information on family structure and sufficient data on well-defined malaria related phenotypes all the while controlling for heterogeneity in both the human genetic background and the force of infection. Longitudinal community-cohort studies are thus required.The ultimate goal of genetic epidemiology is to identify critical molecular pathways of pathogenesis and immunity, thereby generating novel strategies for treatment and prevention. Yet, both P. falciparum and P. vivax malaria in Thailand.In this study, we have estimated the contribution of host genetic factors to malaria relative to environmental factors in a Karen population located near the Thai-Myanmar border, which is an endemic area for A community-based cohort study was carried out from June 1998 to May 2005. The study was conducted in a mountainous area of Suan Phung district, Ratchaburi province, Thailand. Suan Phung is a small district situated near the Thai-Myanmar border surrounded by the long Tanaosri ranges on the western side. The Tanaosri subdistrict is located at the southern part of Suan Phung, approximately 163 km west of Bangkok. Suan Phung has a total population of 5,368 living in 7 hamlets, of which 3,484 villagers of all ages participated in the study. This community is made up of a group of 4 closely related ethnic groups, the majority of which are Karen (85%), some Thai (14%) and the rest are Mon and Burmese (1%). The project protocol and objectives were explained to the population and informed consent was individually obtained from all subjects either by signature or by thumbprint on a voluntary consent form written in Thai, the local language. Such consent was obtained in the presence of the school director, an independent witness. For very young children, parents or designated tutors signed on their behalf. The recruitment procedure has been previously detailed Family structures were constructed by using a questionnaire, interviewing each individual or key representatives of the household to obtain both demographic information such as birth date, age, sex, day of fever, and genetic relationships between children, their parents, and sometimes their grandparents or non-relatives in the same household, and other households. Pedigree structures were checked in 928 individuals, from whom we obtained DNA samples, by identity by state allele sharing of each relative pair from genotyping results of 400 microsatellite markers used during genome screening using IBS_check program . There was <5% discordance including labelling errors. Pedigrees were modified accordingly.The total pedigrees are comprised of 2,427 individuals, including absent or deceased relatives. There were 238 independent families containing 603 nuclear families; the majority are 2 generation-families with family size range from 3 to 958 . For estP. falciparum and from 79 to 28 for P. vivax. P. falciparum prevalence rates varied from 1–7% seasonally and from 1–4% for P. vivax. There was good concordance in the proportion of fevers that were found to be positive for malaria parasites and the fraction of fevers attributable to malaria. Thus, in this site, virtually all infections lead to febrile episodes. Peak incidence occurred in an earlier age group (5–9 years old) for P. vivax than for P. falciparum (10–15 years old). Parasite densities of either species peaked in the <10 years old age group. Bed nets had no effect on incidence rates.The epidemiology of malaria in this site has been described elsewhere P. falciparum, P. vivax, P. malariae and P. ovale parasites at any density. To determine the correct number of clinical episodes, consecutive presentation at the clinic with blood-stage malaria parasite of the same species within 30 days after treatment of the initial infection was considered as the same episode. Consecutive presentation with non-malaria fever (or aforementioned symptoms) within 7 days following first presentation was likewise considered as the same episode. In addition, initial parasite negative visits were considered as part of a malaria episode if followed by a parasite-positive visit within the subsequent two days. All positive malaria cases were treated with appropriate antimalarial treatment according to the recommendation of the Malaria Division, Ministry of Public Health, as previously described The installation of a health clinic enabled passive case detection of malaria episodes and a record of non-malaria presentation. We defined clinical malaria episodes as measured fever (axillary temperature>37.5 °C) or fever-related symptoms including headache, back pain, chills, myalgia, nausea and vomiting associated with a slide positive for blood-stage trophozoite Two categories of malaria phenotypes were considered in the study.P. falciparum (Acronym PFA) or P. vivax (PVA) or which were negative i.e. number of non-malaria fever visits (Acronym NMF), (2) The proportion of visits that was positive for P. falciparum (Acronym PPFA) or P. vivax (Acronym PPVA). Using proportion in addition to number is an attempt to take into consideration differing tendency to visit the clinic; although virtually all malaria parasite infections are symptomatic, individuals who tend to visit the clinic very frequently may present with the few asymptomatic infections. In addition, this measure provides an independent measure that implicitly takes into account the time in the study.We studied the phenotypes related to clinical malaria episode in 2 different ways. (1) As the number of clinical visits that were positive for P. falciparum and mx-PVD for P. vivax), (2) All trophozoite parasite densities excluding zero parasite density values i.e. excluding uninfected individuals . Maximum density was chosen to assess whether there were individual human differences in the control of parasite density independently of parasite genotype. “All densities” was chosen to consider overall variation in the human-parasite interaction, taking into account variation due to parasite genotype.For each species, we studied 2 different phenotypes related to parasite density during malaria clinical attacks. (1) Maximum trophozoite density  with a Poisson error structure (loglinear regression). For phenotypes with repeated measures for the same individual , a Generalized Linear Mixed Model (GLMM) was fitted with individual person as a factor in the random model. For number of episodes  and parasite density, a Poisson error structure was implemented, thus yielding a loglinear regression. For proportion of visits that were positive for either species, a binomial error structure was implemented (thus a logistic regression). Explanatory factors include year (1998-2005), month, hamlet (7), and gender, with age factored into eight groups . Age was additionally refitted as a factor with two groups (<15 and ≥15 years of age), and as a continuous variable, but both proved less explanatory (lower adjusted r2) than age defined as eight groups. Initially a full model including all interactions possible was fitted and then the model was refined to include only statistically significant parameters and interactions. Inclusion of such interaction terms was considered important as it has been reported on numerous occasions that gender and age-specific behaviour can influence exposure and this can vary locally depending on the nature of the environment. Date was only considered as a main effect and not as an interaction with the other variables . The seasonality of malaria transmission in this region varies annually and thus month was considered only via its interaction with year (i.e. as a factor month-year). Analysing using date as a factor with just four levels (trimesters) proved less explanatory (lower adjusted r2). Because the data were over-dispersed a dispersion parameter was estimated. F-statistics in the GLM and Wald statistics, which approximate to a χ2 distribution, in the GLMM were established. In the analyses of the number of non-malaria or parasite species episodes, the time in the study was added as a weight; 89% of individuals were recruited from the outset and 97% by the end of the first year. In the analyses of maximum parasite density, the number of parasite density data points per individual was used as a weight.The residual variance not explained by the “environmental” factors was generated. Because a non-normal error distribution was used, Pearson rather than standardized normal residuals were generated. For the phenotypes concerning number and proportion of episodes, the sum of the residuals per person were then calculated and used in the genetic analysis. For “all parasite densities”, all residual values for any individual (who had repeated parasite density measures) were then used in the genetic analysis. Only phenotypes from individuals for whom family structure was available were analysed for heritability.2h of “single value” phenotypes  by using the SOLAR software package (version 2.1.4) 2h), individual or environmental (2e) and house (2c) effects. We tested for the heritability of the phenotype by comparing likelihood between the reduced model, where total variation is due to environmental variation only, and the full model where total variation is composed of environmental and genetic effects estimated from the genetic relationship coefficient of each pair of individuals. When the null hypothesis was rejected, heritability (2h) was then estimated as the percentage of genetic variance of the total. Although SOLAR can additionally incorporate measured covariates (e.g. explanatory variables), a normal distribution is assumed. For this reason we took into account the contribution of such variables in an initial statistical analysis (section above) and generated residual value for the phenotypes. The relative contribution of genetic factors to phenotype variation is then estimated by the heritability (2h), defined by the ratio of genetic variance component to the residual phenotypic variance tdist option, which creates an extra parameter in the model to describe the distribution of the phenotype, was applied in all analyses. An additive model, which is a general model, making no assumptions of the dominant or recessive nature of the gene, was used to avoid multiplying tests.To determine the contribution of genetic factors to clinical episodes and parasite density phenotypes, we evaluated the Household can confound the estimation of the genetic contribution to a trait, because related individuals often live in the same house. A household or shared environment effect can be added by an additional variance component with a coefficient matrix (H) whose elements (house) are 1 if the relative pair in shares the environmental exposure or 0 otherwise. Genetic effect (i.e. heritability) is estimated using matrix of correlation coefficients for identity by descent (IBD) allele sharing in various types of family relative pairs, whose elements (phi2) provide the predicted proportion of genes of the whole genome that a pair of individuals share at least 1 allele P. falciparum and P. vivax), we evaluated heritability by using the classic repeated measures model  b]+Za+Zpe+Zh+e where y is the residual parasite density value, b is the fixed effects vector , a is the additive genetic effects vector, pe is the permanent environment effects vector , h is the common house effect vector and e is the residual effects vector; × is the design matrix relating observations to fixed effects and Z are design matrices relating observations to random effects. The model was fitted using ASREML vers. 2 P, which is partitioned into VA, additive genetic variance, VPE, variance due to permanent environmental effects, VH, common-house variance and VR, residual variance. Heritability (2h) is again VA/VP.For the phenotypes for which there were multiple residual values  P. falciparum, presented by 1,120 individuals . There were 1,280 cases of P. vivax, presented by 636 individuals , 30 cases of P. malariae and 11 cases of P. ovale. There were reliable P. falciparum trophozoite density data for 1,795 slides from 949 individuals and for 975 slides from 517 individuals for P. vivax; mixed infections were excluded. Only 38 P. falciparum-P. vivax and 3 P. falciparum-P. malariae mixed infections were observed. A total of 3,710 infections by any malaria parasite spp. were presented by 1,370 individuals presented with infection at any time over the period of study.From 1998 to 2005 (7 years) there were 19,162 independent clinical presentations by 2,545 individuals . Of these 2,430 were positive for P. falciparum presented by 996 individuals and 1,118 cases of P. vivax by 555 individuals. There were 36 mixed P. falciparum-P. vivax infections and 3 mixed P. falciparum-P. malariae infections. Excluding mixed species infections, trophozoite density data was available for 857 individuals for P. falciparum and 470 for P. vivax. Of the 2,304 individuals for whom family structure was established, 2,018 came at least once to the clinic presenting a total of 16,926 visits. There were 2,179 cases of Plasmodium spp. and has a higher negative correlation coefficient with the number of clinical P. falciparum attacks (R =  −0.78) than of P. vivax (R =  −0.43), (ii) Number of clinical P. vivax attacks shows a highly significant positive correlation with maximum or overall P. falciparum parasitemia  but negligible or no significance with P. vivax parasite density phenotypes , (iii) Maximum P. falciparum density shows a positive correlation with maximum P. vivax density . Not surprisingly, the proportion of presentations with P. falciparum were very strongly correlated with the number of presentations with P. falciparum (R>0.90) and yielded very similar results in the statistical analyses. The same was true for P. vivax. These two proportional phenotypes yielded no additional information and were thus later removed from the final correlation analyses to reduce the overall number of pair-wise comparisons made.2h) of the number of clinical cases of P. falciparum and P. vivax were 12.4% (s.e. 5.7) and 20.8% (s.e. 3.6) respectively. There was a significant house effect for the number of clinical cases of P. falciparum (4.6% P = 0.05) but not for P. vivax. Similar values were obtained when considering the proportion rather than the number of all visits that were due to P. falciparum and P. vivax. The total number of non-malaria visits was greatly influenced by house  but not significantly so by genetics . Exclusion of house from the heritability analyses increased the genetic contribution to the number of P. falciparum cases to 19.7% and to the number of non-malaria cases to 18%.Heritabilities  and P. vivax (19.1% and 14.9%). There were no effects of house on these phenotypes.There was moderate heritability of parasite trophozoite density phenotypes for both P. falciparum phenotypes but the insignificant effect on clinical P. vivax phenotypes, (ii) the higher genetic effect for P. vivax clinical phenotypes than for P. falciparum and (iii) the very strong effect of age on P. falciparum parasite density.We have partitioned the total variation in the number of clinical malaria and parasite density phenotypes into its genetic, house and environmental components . Of partWe screened for Duffy negative blood group using polymorphism (DuffyT-46C) P. falciparum and P. vivax after controlling for environmental effects. For all malaria phenotypes considered, with the exception of P. falciparum maximum parasite density, genetic effects explained more of the phenotypic variability than any environmental effect. The parasite species-specific genetic contributions varied according to the phenotype, but were similar for both species. Although direct comparison of genetic contributions between sites is confounded by the differing importance of environmental factors, it is notable that the total variation in number of malaria species episodes explained by host genetic factors was similar in both this study and the previous study in Sri Lanka P. falciparum and P. vivax episodes respectively, compared to 15% for either species in Sri Lanka. Moreover, the host genetic contribution to P. falciparum parasite density was similar in both sites; only the genetic contribution to P. vivax parasite density was found to be different here (13%) as compared to 0% in Sri Lanka.Our clinical malaria longitudinal cohort study, conducted in the western province of Thailand, focused on analyses of the number of clinical attacks and parasite density of both P. vivax than on P. falciparum. In our study, there was only a marginal effect of house on P. falciparum and no effect on P. vivax. Such clustering of P. falciparum cases has been previously observed in Thailand Anopheles gambiae and An. funestus are predominantly endophilic, whereas the major vectors here, An. maculatus and An. minimus, are exophilic. House structure and proximity to mosquito breeding and resting sites would therefore be expected to be less important in Thailand than in Africa.One pertinent difference between the two studies is the relative importance of house as a covariate. In the Sri Lanka study, house had a more significant influence on P. vivax cases suggests that some additional factor impacts upon P. vivax compared with P. falciparum. Differential Plasmodium species-specific vectorial competence and behaviour could result in differences in house clustering. An alternative explanation may reflect malaria species interactions. The capacity of one Plasmodium species to suppress another within the human is to some extent dependent on the infection status of each species, where both P. falciparum and P. vivax can dominate the infection. However, P. falciparum generally dominates when present P. vivax cases occurred following treatment of P. falciparumP. vivax infections may be likewise clustered but do not become patent because of suppression by P. falciparum. The overlapping age-incidence curves of both parasite species observed here may generate more potential for species interactions than in Sri Lanka where peak incidence of disease does not overlap.The absence of clustering of P. falciparum cases (PFA) suggests that illness due to non-malaria pathogens may protect from P. falciparum infection or disease and vice versa. The incidence of both diseases is concentrated annually in a very short time span, thus enabling such negative interactions. This is unlike the annual distribution of P. vivax cases (PVA) that is less concentrated into one peak, most probably because of its ability to relapse; this could then explain the lower negative correlation of PVA with NMF.The strong negative correlation between the number of non-malaria fevers (NMF) and P. vivax episodes (PVA) and P. falciparum parasite density, especially maximum density (mx-PFD) (and to a lesser extent between PVA and mx-PVD) suggests that common mechanisms are involved in determining these phenotypes. To investigate this, we examined the effect of having a P. vivax episode during the first three years of study on the mx-PFD for each corresponding individual during the last 4 years of the study, taking into account environmental parameters. Those previously infected with P. vivax (1998–2000) had higher mx-PFD in 2001–2004 (P<0.001). They also had a greater number of P. vivax episodes (P<0.001), but did not have significantly different mx-PVD from those not previously infected with P. vivax (P = 0.16). This suggests that common mechanisms do govern susceptibility to infection by P. vivax and control of P. falciparum density but not control of P. vivax density. There was, however, a strong positive correlation between maximum P. vivax parasite density (mx-PVD) and mx-PFD. Given the differences in red blood cell (rbc) tropism , although blood disorders may be implicated, the involvement of shared immunological mechanisms is likely. It has been suggested that the two species may negatively impact upon one another through the development of cross-species immunity The positive correlation between the number of P. vivax infection is the effect of Duffy blood group antigen on the inability of P. vivax merozoites to invade Duffy negative red blood cells P. vivax erythrocyte infection P. vivax in this study, although we cannot exclude other mutations in DARC gene coding for Duffy blood group. Other inherited blood disorders occurring regionally, such as the thalassemias, HbE and Glucose-6-Phosphate Dehydrogenase may be involved in determining susceptibility to P. vivax infection . Numerous population studies have confirmed the important protective role of such genetic disorders P. falciparum even when studied in South-East Asia To our knowledge, the only known human genetic influence on resistance to P. falciparum phenotypes in Senegal found that parasite density was under a similar genetic effect to PFA (2h 21% vs. 28%) 2h 20% vs. 20% [excluding house effect]). The similarity of these values is all the more remarkable given the very differing transmission intensities observed in the two areas and differing human genetic background (i.e. ethnicities). The marginally lower heritability of number of episodes in this study site likely reflects lower transmission intensity that has lead to increased heterogeneity in exposure. The difference in the contribution of genetics is, however, small given the very large difference in transmission intensity. The statistical analyses that take into account environmental factors prior to genetic analyses seemingly effectively account for differences in transmission heterogeneity (risk of being infected). The high similarity of the genetic contributions to the parasite density is independent of acquired immunity, accounted for by use of age. It should be noted, however, that there may be a genetic contribution to acquired immunity, as previously shown A recent study on heritability of Finally, it is important to note that we estimated only the additive genetic effect. The non-additive genetic effect may have an important contribution to overall estimates of heritability. However, the study population is inbred, which could lead to bias in estimation of non-additive genetic effect: the chance to be homozygous at particular regions of the genome is higher than under panmixia. In addition, positive selection could lead to an increase in frequency of some polymorphisms or mutations, which would contribute to higher heritability of the traits than here estimated.P. falciparum and P. vivax. Although environmental factors also play some role, their importance varies according to the infection phenotype considered. Environment does seemingly play an important role in acquiring an infection, but genetics plays a determinant role in the outcome of an infection with either malaria parasite species prior to the development of immunity.In conclusion, our study clearly shows that there are major human genetic effects influencing both clinical response to infection and parasite parameters describing the infection status of both


It is estimated that over 70% of patients with suspected malaria in sub-Saharan Africa, diagnose and manage their illness at home without referral to a formal health clinic. Of those patients who do attend a formal health clinic, malaria overdiagnosis rates are estimated to range between 30–70%.This paper details an observational cohort study documenting the number and cost of repeat consultations as a result of malaria overdiagnosis at two health care providers in a rural district of Mozambique. 535 adults and children with a clinical diagnosis of malaria were enrolled and followed over a 21 day period to assess treatment regimen, symptoms, number and cost of repeat visits to health providers in patients misdiagnosed with malaria compared to those with confirmed malaria (determined by positive bloodfilm reading).Diagnosis based solely on clinical symptoms overdiagnosed 23% of children (<16y) and 31% of adults with malaria. Symptoms persisted (p = 0.023) and new ones developed (p < 0.001) in more adults than children in the three weeks following initial presentation. Adults overdiagnosed with malaria had more repeat visits  compared to those with true malaria. There was no difference in costs between patients correctly or incorrectly diagnosed with malaria. Median costs over three weeks were $0.28 for those who had one visit and $0.76 for ≥ 3 visits and were proportionally highest among the poorest (p < 0.001)Overdiagnosis of malaria results in a greater number of healthcare visits and associated cost for adult patients. Additionally, it is clear that the poorest individuals pay significantly more proportionally for their healthcare making it imperative that the treatment they receive is correct in order to prevent wastage of limited economic resources. Thus, investment in accurate malaria diagnosis and appropriate management at primary level is critical for improving health outcomes and reducing poverty. In sub-Saharan Africa over 70% of patients with suspected malaria diagnose and manage their illness at home with traditional remedies or drugs bought from local shops . AttendaMalaria misdiagnosis can either result in over estimating or under estimating the burden of disease and in both instances the burden of malaria misdiagnosis is likely to fall most heavily on the poor and vulnerable because they have less resilience to cope with the effects of prolonged ill health . There iThis study documented the number and cost of repeat consultations with health providers in rural Mozambique specifically as a result of malaria overdiagnosis. Such information is important because many countries in sub-Saharan Africa are introducing, or have recently introduced, new malaria drug policies based on combination therapies. These combinations are more expensive than previous first-line drugs such as chloroquine and sulphadoxine-pyrimethamine (SP) and using them to treat illnesses that are not truly malaria will be a significant waste of resources as well as having a detrimental impact on health outcomes and drug resistance. Recommended malaria treatment in Mozambique at the time of this study was amodiaquine and SP (AQ/SP).High rates of overdiagnosis of malaria are well recognised but have been tolerated because first-line antimalarial drugs, such as chloroquine, were cheap. The introduction of more expensive malaria treatments prompts the need for better knowledge about the consequences of malaria misdiagnosis to improve targeting of malaria drugs to those who truly have malaria illness and to make sure that the poorest benefit from these highly effective medicines .Plasmodium falciparum. Mocuba town has a district hospital and two health centres – one government-owned and one private – which only deliver out-patient care. The study was conducted at both health centres in order to maximise patient enrolment.The study was conducted in Mocuba in central Mozambique, a rural town 80 km from the Zambézia Provincial capital of Quelimane with a population of 66,000. Malaria is Mozambique's leading cause of morbidity and mortality, with an estimated 44–67,000 deaths per annum across all age groups [This was an observational study to document the impact on patients of malaria overdiagnosis that occurs during routine malaria care. Patients who attended the clinics were seen by a triage nurse and those with suspected malaria were diagnosed clinically and treated according to local protocols; all attempts were made not to interfere with local practice, allowing clinic staff to diagnose and treat patients as was customary. A full patient history and temperature reading was taken from all patients. Only first-line treatment with anti-malarial drugs is administered at this level; severe cases are transferred to the district hospital. Patients that had recently been diagnosed with malaria and were returning to the clinic for follow-up tests were excluded from the study. All patients who had a clinical diagnosis of malaria, and who consented to take part in the study, provided a finger prick blood sample for malaria slides for microscopy. This was the only change we made to the routine care of patients at the clinic and was necessary to enable us to determine retrospectively which patients truly had malaria; customarily, slides were only taken in less than 10% of cases. Results from any slides requested as part of routine clinical care were processed as normal and results given to triage nurse on the same day. However the results of the blood slides did not appear to alter the treatment regime as patients with negative results were still prescribed anti-malarials based on the clinical diagnosis. The proportion of clinically diagnosed malaria patients who had malaria parasites was determined by microscopy of Giemsa-stained thick blood films by two independent researchers. Discrepant results were resolved by an independent third microscopist.This study compared the household costs associated with repeated health-related consultations between patients correctly and incorrectly diagnosed with malaria in the two health centres. Individuals attending either health clinic in May-June 2004 with malaria clinically diagnosed by the triage nurse, and resident within 10 km of the health clinic (to facilitate patient follow up) were invited to enrol in the study. Clinical diagnosis was based on the presence of symptoms and fever at the time of enrolment, according to the local protocol. Both adults and children were enrolled, with children defined as those under 16 years of age and dependent on parental support.Permission for inclusion in the study was sought from all adult participants, and from carers of child patients. The Liverpool School of Tropical Medicine Research Ethics Committee, the Director of Mozambique's National Malaria Control Programme and the Zambézia Provincial Medical Director granted ethical approval for the study.After discussions with local health workers, a mean difference of 0.15 health consultations per patient per month was considered to have important implications for local public health. This figure was based on two malaria-related health consultations per patient per annum. For the study to have a 90% power (1-probability of a type II error) to detect a difference of this size at a significant level of 5% (probability of a type I error) the total number of study patients required was 466.Three questionnaires were used for data collection. The first at day 0 when the patient first presented at the clinic and then on days 7 and 21 when subjects were visited at home. For children, questionnaires were completed by the accompanying carer. Three weeks was chosen as the period for follow up because this was sufficient time for any self-limiting infection to have resolved and for any underlying non-malarial cause of fever to have become manifest and to have promoted repeat clinic visits. The baseline questionnaire collected data on: age, sex, education level (adults only), occupation (adults only), symptoms, transport and costs, missed wages , treatments and cost, and monthly household outgoings. Monthly expenditure rather than monthly income was collected as a pilot study indicated that many individuals were unwilling to provide reliable income data and that expenditure information would be more accurate. Although chosen to be more accurate, household expenditure remains a very difficult entity to measure  and dataThe number of HIs by correctly and incorrectly diagnosed individuals over the 21-day study period were compared by fitting them to a Poisson model using the maximum likelihood method and adjusting for overdispersion. P values that were close to the significance threshold are presented as a range with the lower value representing no overdispersion and the higher value representing adjustment for maximum overdispersion. Costs were compared by Mann-Whitney Tests. Analyses were conducted in SigmaStat version 3.1 and STATA version 8.535 patients were enrolled in the study; 170 from the private clinic and 365 from the government clinic. After the 21 day follow up and exclusion of those who did not meet the inclusion criteria  were correlated with the readable slide information, data was available for 312 patients. Children constituted the majority of study participants  with most individuals enrolled at the government clinic  (Table 2 test) (Table 2 test).All 535 individuals enrolled in the study at day 0 had clinically diagnosed malaria but only 396 (74%) had microscopically confirmed parasitaemia. Clinical diagnosis of malaria at both clinics was more reliable for children than adults. 77% of malaria diagnoses in children and 69% in adults were confirmed by microscopy  Table . Althoug2 test). 51 (25%) children and 41 (38%) adults had developed new symptoms such as anaemia, wheezing, cough, convulsions, diarrhoea, vomiting, constipation and earache by day 7 and these persisted to day 21 in 26 (24%) of adults and 15 (7%) of children. The chance of new symptoms occurring was significantly greater in adults compared to children at day 7  and day 21 . There was no significant difference in persistence of original symptoms or in recurrence of new symptoms between patients (either adults or children) with a correct or incorrect diagnosis of malaria at either 7 or 21 days.75 (37%) children and 43 (40%) adults still had their original symptoms at day 7 but this had fallen to 33 (16%) and 30 (28%) respectively by day 21. At day 21 but not at day 7, prevalence of original symptoms was significantly higher in adults than in children  to nine . For the 53% of patients who had more than one HI, the median number of attendances was two . The median and 25–75Monthly household expenditure varied widely from $9–174 and did not differ significantly between those attending the private and government clinics . Median monthly expenditure was lowest in the households of children presenting to the private clinic (US$36.96) and highest in the households of adults presenting at the private clinics (US$41.3). Health costs over the 21 day study period ranged from $0.1–19.3 (median 1.1) and were proportionally significantly higher  in patients with a household expenditure of ≤ $40/month (i.e. the lowest quintile of household expenditure) compared to those with household expenditure $40–200  sought further health advice after their initial consultation and 25% of these interactions took place with traditional medicine outlets or shops rather than the clinic they had originally attended. Three weeks after the initial consultation, 28% of adults still had their original symptoms and one week after initial consultation 38% of adults had developed new symptoms. These clinical outcomes were significantly worse in adults than in children who overall had a higher proportion of correct malaria diagnoses. Adults who had been overdiagnosed with malaria had significantly more health consultations during the following three weeks than those who had been correctly diagnosed. There was no difference in expenditure on health care during this period between patients correctly or incorrectly diagnosed with malaria and this may have been related to inconsistencies in antimalarial treatment for both groups as well as wide variation in health expenditure between individuals. Total health costs and lost earnings as a proportion of monthly expenditure during the three weeks were significantly higher in the poorest households.The rates of malaria overdiagnosis using clinical features alone in this study and the inconsistencies in anti-malarial treatment are very similar to findings from other studies ,2,10. ThPublished studies about the household costs of malaria illness highlight the need to take account of both direct and indirect costs and the disproportionately high economic burden that malaria places on the poorest households . SeveralBecause this was an observational study and confirmation of true malaria infections was done retrospectively, there were no alterations in clinical practice at the health clinics. The inconsistent prescribing of anti-malaria drugs for clinically diagnosed malaria patients, and the occasional use during follow up visits of malaria microscopy results of unknown quality, meant that it was difficult to draw conclusions from data that compared outcomes and costs between those with true and overdiagnosed malaria. Although patients reporting to the formal health clinics in Mocuba were accessed, during the study period, it is recognized that individuals who never access these formal health services may have been excluded and that these individuals are, potentially, most at risk from the consequences of malaria infection.Health-seeking behaviour was not specifically examined in this study but could confound our results about frequency and cost of health interactions as it is influenced by many factors such as the patient or carers'  ability to make decisions about types of health care and to access money . IneffecThe true costs of overdiagnosis for malaria negative patients cannot be strictly inferred as there is no way of knowing what would have happened to these individuals if a confirmed negative diagnosis had been available. That is to say, if a slide negative individual had been investigated and treated for a non-malaria cause of their symptoms, what the costs of their diagnosis and subsequent treatment would have been and whether their symptoms would have resolved and how this would have affected the number of repeat visits. It was not possible to answer these questions in our study because it was an observational study so changes to regular practice were not feasible and the health facilities had very limited resources to investigate non-malaria causes of symptoms.The malaria overdiagnosis rates and costs presented in this study may not be transferable to other contexts where malaria transmission patterns, health-seeking behaviour and health systems differ from those in rural Mozambique. However, the consequences of failing to confirm clinically diagnosed malaria infections, particularly on the poorest households, will be common to many communities in sub-Saharan African countries.Malaria overdiagnosis rates using clinical features alone continue to be unacceptably high in all patients except young children living in areas of intense transmission . MalariaIt has been postulated that possible consequences of malaria misdiagnosis include prolonged ill health, increased costs and loss of faith in formal health services but there is very little evidence to support these suggestions . In thisIt is those who are unable to access sophisticated diagnostic services for malaria who are hardest hit by the invisible burden of malaria misdiagnosis. Much more needs to be done to improve and promote access to accurate malaria diagnosis for the poorest communities. For them, the consequences of malaria misdiagnosis contribute to a vicious cycle of increasing ill-health and deepening poverty.IB and GB conceived and designed the study. TM and GB carried out field work with support from LA and ES. JCCH collated and analysed the data and drafted the manuscript with IB. All authors read and approved the final manuscript.


P. falciparum malaria is presented, who was successfully treated with voriconazol and caspofungin. This is the first survival of malaria-associated invasive aspergillosis.Invasive fungal infection is rarely reported in association with malaria, even though malaria-associated inhibition of phagocyte function is a well-known condition. Invasive aspergillosis is frequently found in severely immuno-compromised patients but not in healthy individuals. Here, a case of pulmonary invasive aspergillosis in a previously healthy patient with severe  Plasmodium falciparum malaria with subsequent transient immunosuppression can lead to opportunistic infections in previously immunocompetent patients [There is strong evidence that malaria can lead to altered immune response via modulation of both humoral and cell-mediated immunity. Therefore, patients .Aspergillus spp.) is a major cause of morbidity and mortality in immuno-suppressed hosts, such as patients with haematological malignancies and transplant recipients. Aspergillus spp. is found ubiquitously in the environment worldwide and reaches the alveoli by airborne transmission. In healthy persons, the spores are eliminated by mucociliary clearance and pulmonary macrophages. Although infection in healthy individuals can occur, invasive aspergillosis is extremely uncommon in immuno-competent hosts. There are only four reported cases of invasive aspergillosis complicating falciparum malaria in immuno-competent hosts, all with fatal outcome [Aspergillosis , creatinine 6.09 mg/dl, urea 208 mg/dl, LDH 805 U/l, GOT 69 U/l, GPT 197 U/l. Blood culture, urine culture and serology for hepatitis A, B, C and HIV were negative. Chest x-ray and ultrasound were unremarkable except for mild hepatosplenomegaly. Pharyngitis sicca was diagnosed by the ENT consultant.On admission, he was sleepy, but fully oriented, afebrile and pale. No signs of meningitis were found. The lungs were clear and the heart sounds normal. BP was 150/85 mmHg and the pulse rate 95/min. Blood films showed Treatment with quinine i.v. was initiated with a loading dose of 7.0 mg/kgBW and continued at a rate of 10 mg/kgBW every eight hours for four hours over 10 days. Parasitaemia decreased to 2.7% within three days and was cleared by day five. He was put on intermittent hemodialysis after developing acute renal failure. Antibiotic coverage with imipenem was started.Aspergillus fumigatus was cultured abundantly. The circulating antigens of 0.8 remained below the positive cut-off of 1.0 given by the laboratory. The anti-Aspergillus antibody titre remained below the positive cut-off of 1:160 – 1:320. Therapy with voriconazol and caspofungin was started immediately.On day 5 of hospitalization, respiration deteriorated and the patient was intubated. The chest x-ray revealed patchy infiltrates of the upper left lobe and on bronchoscopy bronchial obstruction with viscous mucus was seen. This was cleared and in the material obtained Aspergillus spp. (Figures In the following days respiration improved. On day 7 of artificial ventilation, however, severe haemoptysis suddenly precipitated with cardio-pulmonary arrest. He was successfully resuscitated and pulmonary bleeding spontaneously stopped. A thoracic CT scan showed multiple confluent ground glass-infiltrates of both lungs and enlarged mediastinal and hilar lymph nodes Figure . Broncho Figures  to 1G. WP. falciparum malaria has only been described in four cases, none of which survived [P. falciparum malaria had disseminated candidiasis and cryptococcal meningitis [Aspergillosis, in particular the invasive course of the disease, is only seen in patients with severe immuno-suppression and has a very poor prognosis. In individuals without severe immuno-suppression, invasive fungal infections are extraordinarily rare. Till date invasive aspergillosis during survived -4. Two oningitis ,6.Falciparum malaria can lead to suppression of both humoral and cell-mediated immunity. Humoral immunity is impaired through reduced production of specific antibodies, while cell-mediated immunity is modulated by inhibition of dendritic cells, which is crucial as they initiate all adaptive and several innate immune responses, and also impairment of T cell and macrophage function plays a role [s a role ,7,8.in vitro and in vivo studies showed that large proportions of resident macrophages as well as circulating monocytes and leucocytes are loaded with haemozoin and consequently lose their ability to phagocytose and kill ingested germs such as fungi [Aspergillus spp., as they are eliminating spores from the lungs [Aspergillus spp. is the most frequent fungal pathogen and pneumonia the major cause of death, because here phagocytes fail to generate reactive antimicrobial oxidants which impairs oxidative burst [Inhibition of key enzymes involved in oxidative burst by the malaria-pigment haemozoin seems to be one of the most important mechanisms contributing to the disruption of macrophage function. Both as fungi . Especiahe lungs . The cenve burst . The malve burst . While hIn the case presented, as in the four other published cases, inhibition of macrophages leading to aspergillosis was most likely malaria-associated as no other cause was found. Other mechanisms such as inhibition of humoral and antigen-specific T-cells response are unlikely to be a strong factor for malaria-induced immunosuppression in the acute phase of the disease in a patient without prior malaria infection or semi-immunity. Prolonged and profound neutropenia, the single most important and common risk factor for invasive aspergillosis, was neither present in this case nor in the patients published.Aspergillus infection is not clear. The Aspergillus antigen detection with a magnitude below 1.0 was interpreted as negative despite extensive Aspergillus growth in the culture. However, in many settings now a positive cut-off of 0.5 is used, therefore the maximal antigen magnitude of 0.8, which was measured after the positive culture result, would meet the criteria for a positive result. There are also several studies which showed that galactomannan ELISA is not as sensitive in invasive aspergillosis as anticipated [Aspergillus antibody titre could be an additional marker for severely impaired immune response to Aspergillus due to malaria-induced immunosuppression.In contrast to the previously described fatal cases, no other malaria-related complication such as cerebral malaria or ARDS was seen in this case, but in two of the other published cases. The patient responded quickly to malaria treatment with rapid clearance of parasites. Invasive aspergillosis became clinically apparent only after malaria parasites had been eradicated and after the patient had started to improve already which is not surprising, as it is known that malaria-induced immunosuppression occurs late in the acute phase of the disease. Whether the initial presentation of pain upon swallowing was already related to the beginning of the icipated . The negVoriconazole is a triazol antifungal that is considered first-line-treatment for invasive aspergillosis . BecauseAs malaria mostly occurs in low-resource countries with limited access to diagnostic tools, underreporting of fungal disease complicating malaria may be a problem. With only four cases reported so far, the incidence seems to be very low, however. For practical purposes invasive fungal disease should be considered in malaria patients deteriorating after successful anti-malarial treatment. This case shows that early recognition and immediate treatment in principle can avert a fatal outcome.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.IE collected all information and wrote the first version of the manuscript. IE and DG reviewed the literature. RE and PS supplied macroscopic and microscopic photographic material. All authors gave their input into the final version of the manuscript. DE, CE and TJ were the physicians responsible for the patient. All authors read and approved the final manuscript.


Plasmodium falciparum malaria are based on the clinician classifying the patient as uncomplicated or severe according to the clinical and parasitological findings. This process is not always straightforward.The treatment options for acute P. falciparum trophozoite count  from malaria blood smear, consistent with a diagnosis of uncomplicated P. falciparum infection. However, the physician on duty treated the patient for severe malaria based on the reported P. falciparum schizont count, which was very high (0.3% IRBC), noticeably in relation to the trophozoite count and schizont:trophozoite ratio 0.25:1. On intravenous artesunate, the patient deteriorated clinically in the first 24 hours. The trophozoite count increased from 1.2% IRBC at baseline to 20.5% IRBC 18 hours following the start of treatment. By day three, the patient recovered and was discharged on day seven having completed a seven-day treatment with artesunate and mefloquine.An adult male presented to a clinic on the western border of Thailand with a physical examination and P. falciparum to sequester in the microvasculature. In severe malaria, high schizont counts are associated with worse prognosis. In low transmission areas or in non-immune travelers the presence of schizonts in the peripheral circulation is an indication for close patient supervision. In this case, an unusually high schizont count in a clinically uncomplicated patient was indicative of potential deterioration. Prompt treatment with intravenous artesunate is likely to have been responsible for the good clinical outcome in this case.The malaria blood smear provides only a guide to the overall parasite biomass in the body, due to the ability of  The 2006 WHO malaria treatment guidelines  laid outA 28 year old male from the eastern border of Burma walked to a malaria clinic on the Thai side of the river that marks much of the border between the two countries. He complained of a three-day history of fever, nausea, headache and dizziness. He denied ever having had malaria in the past. He was fully conscious, able to answer all questions appropriately and had an aural temperature of 37.5°C, pulse 88 beats/min, respiratory rate 20/min and blood pressure 110/60 mmHg. His liver could just be palpated, 1 cm below the costal margin in the mid-clavicular line and his spleen was not palpable. Examination was otherwise unremarkable. In particular, he had no signs suggestive of severe malaria [®Pf) result was strongly positive and, consequently, a malaria blood smear was taken. The malaria smear result (12:20 hrs) was Plasmodium falciparum trophozoites 12/1,000 RBC . Plasmodium falciparum schizonts were present on the slide and the count was high: 3/1,000 RBC . Plasmodium vivax trophozoites were also noted at low density: 8/500 White Blood Cells (WBC) (or a parasitaemia of 128/μL). Scarce malaria pigment was observed in < 1% of polymorphonuclear leucocytes (neutrophils) , and these were all just within normal limits. The patient felt very well on the 3rd day of treatment but did have a spontaneous nose bleed on this day. The treatment was changed to oral artesunate and mefloquine (15 mg/kg on the 6th day and 10 mg/kg on the 7th day of treatment). Fever clearance time was 66 hours and time to parasite clearance (based on 24 hourly blood smears) was 144 hours. This patient had a negative thalassaemia screen, mild anaemia and thrombocytopaenia, by complete blood count (taken on the 4th day of treatment): white blood cell 8.9 × 103/μL, haemoglobin 11.2 g/dL and platelet count 125 × 103/μL,The patient exhibited no signs of severe malaria but was admitted to hospital and treated according to the WHO severe malaria protocol  on the bThis man had a presenting falciparum trophozoite parasitaemia of 51,245/μL and a very high schizont parasitaemia of 15,072/μl (Table Table P. falciparum schizont releases an average of 16 merozoites, each of which can theoretically infect another RBC causing the infective biomass to expand exponentially. Modelling data of real patients with acute uncomplicated falciparum malaria, White and co-workers [High parasite densities are frequently equated with severe disease but the reverse is not always true. There may be wide differences between the number of parasitized cells in the peripheral blood smear and the number sequestered in the microvasculature,8. This -workers  demonstrAlthough antimalarials are frequently referred to as blood schizontocides, in fact mature trophozoites are more susceptible to the drugs and formed schizonts are relatively drug- resistant . Young rThis report comes from an area of low transmission. In areas of low transmission where immunity remains poor across all age groups, or in returned travelers, even a low schizont count is an indicator for close patient observation. The advantages of access to high quality microscopy with parasite staging are shown here but would be considered a luxury in many malaria endemic settings , where aP. falciparum malaria one needs to account for all stages reported from the malaria blood smear bearing in mind it provides only a guide to the total parasite biomass. Any clinical deterioration in a patient on oral treatment necessitates a change to the severe malaria protocol. High parasite counts, as anticipated in this patient, are best treated with artemisinin derivatives.When treating KML was responsible for the treatment of the patient and consulted RM and FN, SP confirmed the blood smear findings and provided photographic evidence of the diagnosis, EAA and KS reviewed and verified data on schizonts in large treatment trials; RM, EAA, and FN and coordinated the manuscript. All authors read and approved the final manuscript.


Malaria incidence has been reported to decrease substantially in parts of sub-Saharan Africa, including the Zanzibar Archipelago in East Africa. A cohort study with an intensive follow-up on Pemba Island just before the onset of the highly successful malaria control intervention was conducted. The reported estimates of parasite prevalence and incidence can serve as a robust baseline to evaluate the effect size of the successful interventions and the potential contribution of quality controls and other factors associated with research studies in the decreased estimate of transmission.In a rural clinic, two successive cohorts of 537 children total aged 2-23 months were followed for six months each with an intensive visitation schedule of bi-weekly follow-up. Robust estimates of incidence and prevalence according to four different malaria definitions were obtained.Malaria incidence and prevalence placed Pemba Island in a hyperendemic rather than holoendemic setting for the years 2003-2005. Overall parasite prevalence was estimated to be 39% - with monthly estimates varying between 30% and 50%. Incidence of malaria varied between 2.3 and 3.8 malaria episodes per year based on a diagnosis of fever and various microscopy-based parasite thresholds and between 4.8 and 5.7 based on a diagnosis of fever and 100 parasites/microliter analogous to detection by rapid diagnostic tests. Both parasite densities and malaria incidence increased with age and rainy season. Malaria incidence also varied substantially between the individual villages within the study area.Pemba Island was previously considered holo-endemic for Malaria. The data suggest that the transmission situation on Pemba Island was significantly lower in 2003-2005 suggesting a hyper-endemic or meso-endemic transmission environment. The figures were obtained just before the onset of the highly successful malaria control intervention by impregnated bed nets and IRS on the Zanzibar Archipelago and provide robust estimates of the malaria transmission situation prior to the control programme. Together with other published data, the results suggest that malaria transmission had started to decrease before the onset of the control programme. The local heterogeneity in malaria incidence highlights the importance of a micro-epidemiological approach in the context of malaria control and elimination. Malaria incidence has reportedly decreased substantially in parts of sub-Saharan Africa, including the Zanzibar Archipelago in East Africa. A number of reasons have been proposed to explain this positive trend - among them successful control interventions, more specific diagnostic capabilities, and demographic patterns including better access to care. However, the extent of the decrease is still somewhat surprising and leaves room for speculation if the full range of explanations is being properly evaluated.Plasmodium falciparum malaria is the leading cause of morbidity and mortality in children on the island of Pemba [The results are reported of a cohort study that was carried out on Pemba Island, which is located north of Zanzibar Island and is part of the Zanzibar Archipelago. The island is densely populated with approximately 300,000 people in an area roughly 70 km long and 30 km wide. Malaria had almost been eradicated on the island in the 1950's. By 1979, 11 years after the suspension of the control programme, malaria had re-established itself on the island ,3, with of Pemba ,5. The sof Pemba . ArtesunIncidence and prevalence estimates of the pre-intervention period are essential to evaluate the impact of the current programme. Robust estimates of parasitaemia prevalence including quality control of microscopy and robust estimates of incidence according to three laboratory-based malaria definitions and the presumptive IMCI definition were calculated. In addition to seasonal variation of parasite prevalence and malaria incidence using four different definitions, the local heterogeneity of incidence in the micro-epidemiological context of central Pemba Island was described.The study took place between December 2003 and January 2005 in three adjacent administrative units ("shehias") on Pemba Island, Zanzibar, Tanzania. The shehias of Kiungoni, Pandani and Finya are located in Wete and Michweni District, about halfway between Chake Chake and Wete Towns. Kiungoni, Pandani and Finya are comprised of 17 sub-villages distributed over an area of around 65 square kilometers. The area has a rural character with fishing, farming and clove production as main income generating sources.The design was a prospective cohort with bi-weekly active follow-up over a period of 12 months. Two cohorts of 298 and 253 children each, aged 1-23 months at enrollment, were recruited and followed for six months each over the period of one year total. The children enrolled represented 80-90% of the total children per village in the age range of 3-15 months.Informed consent was sought with the village committee first and after that with individual caregivers. Written informed consent was acquired from every guardian before a child was enrolled into the study. None of the guardians of a child declined consent at enrollment. Ethical clearance was obtained from the Johns Hopkins Bloomberg School of Public Health Institutional Review Board and the Zanzibar Health Research Council.The study was nested within a larger zinc/iron supplementation trial with a two-by-two-factorial design that was recently completed on Pemba Island, Tanzania . All nec537 children aged 1-23 months at enrollment were included in the analysis. Table Children were censored from the study when their caregivers either actively refused further participation or when the children missed four consecutive visits that were scheduled every two weeks. Altogether 67 children (12.2%) were lost to follow-up - most of them due to refusals (N = 37) or outmigration (N = 15). Data on these children were used for analysis up to the point of censorship. 13 children had to be censored completely from the analysis because they only presented for the enrollment visit. One child was removed from the analysis because of ineligible age at enrollment (49 months). Two children were referred for further medical treatment by the study team and eight children died during the observation period.At baseline, a clinical examination and a structured questionnaire were administered and a capillary blood specimen was taken. After the baseline examination, every two weeks a follow-up clinical examination and a capillary blood specimen for malaria smear, full blood count, including haemoglobin, and a PCR-filter paper for later genotyping of the parasites, were conducted.A rural clinic was held every day except weekends. Mothers were informed about the results of the blood smear, haemoglobin and white blood cell counts from the previous day and given malaria treatment if appropriate for their children according to WHO recommendations and guidelines of the Ministry of Health (MOH) in Zanzibar. Mothers/caretakers of the children were given a scheduled date of return approximately two weeks after their last visit each time they attended the clinic even in the absence of any health problem. The mothers were encouraged, however, to attend the clinic any day of their choice in case of a health problem with their child. The clinic was open five days a week for children up to five years of age, so that elder siblings of the children enrolled in the study were eligible for free treatment. Free treatment was also given for non-malarial illnesses like diarrhoea, pneumonia and ARI, skin infections  or otitis media, which could be treated in a peripheral health care setting. If hospitalization was necessary, the child was given free transport to the main hospital of the island on the same day. If a child was investigated for a health problem at a given day, the next visit was scheduled two weeks from that date of investigation.Blood slides for malaria parasites were prepared according to standard protocols and read against 200 white blood cells ,8. StandThree laboratory-supported definitions of a clinical malaria episode and the presumptive IMCI definition of clinical malaria were used:1. A child presenting with 5,000 parasites per microliter plus history of fever (last 48 h) or fever (≥ 37.5°C) at presentation .2. A child presenting with either a) documented fever ≥ 37.5°C) and a parasite count of more than 1,000 per microliter; b) history of fever (last 48 hours) and a parasite count of more than 3,000 per microliter; or c) a parasite count of more than 8,000 per microliter. This definition was developed and used in the zinc/iron trial [7.5°C and3. A child presenting with ≥ 100 parasites per microliter plus history of fever (last 48 h) or fever (≥ 37.5°C) at presentation. This definition represents the performance of most malaria rapid diagnostic tests (RDT) or "malaria dipstick test". It is also very close to a definition occasionally used in the literature of 'any parasitaemia plus history of fever (last 48 h) or fever (≥ 37.5°C) at presentation'.4. A child presenting with history of fever (last 48 h) or fever (≥ 37.5°C) at presentation and no sign of invasive bacterial disease  by clinician's judgement (IMCI definition).The monthly malaria incidence rate was calculated using a person-time approach. For a conservative estimate missed days (defined as any days exceeding the retrospective assessment of 14 days prior to a visit to the clinic) were subtracted from the denominator as well as days not under risk if a child was diagnosed with a malaria episode (where the following seven days were subtracted from the denominator). The incidence rate was calculated just based on the total follow up time per child (defined as the period between first and last visit). Both estimates give a plausible range for the true malaria incidence rate per malaria definition.P. falciparum. Documented fever (temperature ≥ 37.5°C) was present in 26% of the parasite-positive visits and in 11% of the parasite-negative visits. Fever either by history or documented was present in 48% of the parasite-positive visits and in 28% of the parasite-negative visits. Asymptomatic parasitaemia was defined as any parasite load (by microscopy) greater than zero without fever (≥ 37.5°C) or negative history of fever in the prior 48 hours. Asymptomatic parasitaemia was confirmed in 19.1% of all visits and in 5.8% and 2.5% with parasite densities above 1,000 and 5,000 parasites per microliter respectively. Only 39% of the children in this study were reported to sleep under a bed net at the time of enrollment  and compliance was very good. Eight children died with unknown cause . All deaths were followed up with verbal autopsies within four weeks of the death. In one case, the child died immediately after enrolment indicating that the illness was already in progress. In the majority of the other cases a probable diagnosis leading to death could be made. An acute fever episode with or without convulsions was the most frequent clinical picture leading to death, which results in malaria and/or pneumonia being the most likely diagnoses. Measles was included in the cause of two deaths. A total of 6,125 visits were counted over total follow-up time. At every visit, except 14 with missing data , a blood-slide for malaria parasites was taken and analysed according to standard procedures [Parasite prevalence per two-week period ranged between 30-50%. Absolute parasite prevalence as well as relative parasite densities were highest during and shortly after the rainy season in April/May and November/December  were compared to the presumptive IMCI definition. By the rather stringent definition of malaria with a parasite threshold of 5,000 per microliter, malaria incidence is estimated to lie between 2.3  and 2.6 malaria episodes per child per year in Figure If incidence is calculated separately in three age groups over the study period  a seasonal effect is present as with parasite prevalence villages within the study area mapped in Figure Estimates of malaria incidence estimates are highly dependant on the definition used. By using four different definitions to provide a plausible range of incidence in order to make comparisons across sites was attempted. In cases where the absolute number of episodes per year is difficult to compare across sites, the age pattern of incidence can still be a good point for comparison. Incidence rates in holo-endemic regions as in the mainland of Tanzania are reported to peak towards the end of infancy and then decrease . A pattePemba Island was previously defined as a holo-endemic area for Malaria -5 similaRegarding the successful malaria control intervention on the Zanzibar Archipelago, the estimate is questioned of the effect size that is implied when the baseline for the highly successful control strategy for Pemba Island is assumed to be that of a holo-endemic setting as the evidence suggests that transmission intensity was decreasing prior to the onset of the intensive malaria control programme. The lower parasite prevalence and incidence figures in our study could be caused by a number of reasons - some of which are directly related to the research study itself. The intensive follow-up including the provision of transport actually provided a better access to care. In addition, a quality control component for malaria diagnosis was implemented, which in itself could lead to lower estimates. Another contributing factor could be, by closely observing and treating malaria episodes as they occur in a cohort study, a situation similar to that of "intermittent presumptive treatment" was created, which as a control strategy was proven to decrease the number of malaria episodes in children . As onlyThe micro-epidemiology on Pemba Island shows considerable heterogeneity of incidence and, therefore, presumably also heterogeneity in transmission. Figures were based on an area of approximately 20 square kilometres in the centre of Pemba Island (see Figure Surveillance and micro-epidemiology of malaria has become increasingly important as pockets of malaria remain despite the intensive efforts and success of the malaria control programme . The detPemba Island was previously considered holo-endemic for malaria. The data collected in this study suggest that the transmission situation on Pemba Island was significantly lower in 2003-2005 suggesting a hyper-endemic or meso-endemic transmission environment. These figures were obtained just before the onset of the highly successful malaria control intervention by impregnated bed nets and IRS on the Zanzibar Archipelago and provide robust estimates of the malaria transmission situation prior to the control programme. Together with other published data, these results suggest that malaria transmission had started to decrease before the onset of the control programme. The local heterogeneity in malaria incidence highlights the importance of a micro-epidemiological approach in the context of malaria control and elimination.The authors declare that they have no competing interests.TJ, SS and DS conceived and designed the study. TJ, AD, SD, MR, MO and SS performed the study. TJ, RG, JT, SS and DS analyzed analysed the data. TJ and RG produced figures and tables. TJ, JT, SS and DS drafted the manuscript. All authors contributed to the writing of the manuscript and approved the final version.


Intermittent preventive treatment for malaria in children (IPTc) is a promising new intervention for the prevention of malaria but its delivery is a challenge. We have evaluated the coverage of IPTc that can be achieved by two different delivery systems in Ghana.IPTc was delivered by volunteers in six villages (community-based arm) and by health workers at health centres or at Expanded Programme on Immunisation outreach clinics (facility based) in another six communities. The villages were selected randomly and drugs were administered in May, June, September and October 2006. The first dose of a three-dose regimen of amodiaquine plus sulphadoxine-pyrimethamine was administered under supervision to 3–59 month-old children (n = 964) in the 12 study villages; doses for days 2 and 3 were given to parents/guardians to administer at home.The proportion of children who received at least the first dose of 3 or more courses of IPTc was slightly higher in the community based arm . Completion of the three dose regimen was high and similar with both delivery systems (91.6% and 91.7% respectively).Seasonal IPTc delivered through community-based or facility-based systems can achieve a high coverage rate with the support and supervision of the district health management team. However, in order to maximise the impact of IPTc, both delivery systems may be needed in some settings.NCT00119132ClinicalTrials.gov  Seasonal intermittent preventive treatment for malaria in children (IPTc) is a promising new intervention for the prevention of malaria in children. Seasonal IPTc is safe and can reduce the burden of malaria substantially in countries of Sahelian West Africa. Dicko and colleagues in Mali showed that two treatments with suphadoxine-pyrimethamine (SP) given two months apart at the height of the malaria transmission season reduced the incidence of malaria by 40% in children 6 months to 10 years old A recent study in Senegal showed that IPTc using SP + AQ given for 3 days had the best protective efficacy compared to three other drug regimens. The adjusted hazard ratio for the incidence of malaria was 0.50  for SP (1 dose) +AQ given for 3 days, 0.90  for SP (1 dose) + AS given for 3 days 1.13  and AQ+AS given for 3 days when compared to SP +AS each given for one day Although IPTc with AQ and SP is safe and efficacious, there are concerns over how it can be delivered in an effective and sustainable way. In order to be effective, the delivery system must be accessible to the maximum number of target children, the SP/AQ must be offered to the correct children by the provider, and the correct regimen must be adhered to by the carer of the child. In terms of sustainability, the first consideration is whether it is possible to achieve effective delivery through routine public sector delivery systems. Delivery of IPTc during routine EPI/growth monitoring clinic visits must, therefore, be considered as one possible delivery system. However, there are two reasons to suspect that delivery of IPTc through this system may not reach the maximum target numbers in Ghana and other countries of West Africa. Firstly, the coverage of EPI vaccination is low in several countries of West Africa Another option is a community based, volunteer delivery systems of the kind that has been used successfully for mass treatment of onchocerciasis Ensuring that the correct amount of drugs are delivered to targeted children at the most appropriate time of the year and sustaining adherence to the treatment regimen through any delivery system is a challenge. However, these challenges are not insurmountable and different ways of delivering IPTc need to be explored. In this paper we describe an evaluation of two different approaches to the delivery of IPTc in Ghana.The protocol of this trial and supporting CONSORT checklist are available as supporting information see The study took place in the Jasikan district of Ghana from May to October 2006. The Jasikan District is within the semi-equatorial climatic zone and has an extended rainy season (April – November) with peaks in May-July and September-October. The district has two hospitals, 9 health centres, 17 reproductive and child health static clinics, 3 private maternity homes and 2 private clinics. The district also has 80 Expanded Programme on Immunisation (EPI) outreach clinics. In children under the age of five years resident in the study area in 2006 who were treated for clinical malaria the day28 PCR corrected adequate clinical and parasitological cure rate was 75%  for SP (n = 241) and 85%  for AS+AQ (n = 241) .The study had two arms; IPTc was delivered either at the outpatient department of health centres and at EPI outreach clinics [health facility-based IPTc] or by community volunteers [community-based IPTc] . SamplinMembers of the district health management team and the principal investigator (MK) explained the objectives of the study to leaders of the study villages and obtained their consent for the trial. All children aged 3–59 months (1039) resident in the 12 study villages were then enumerated in May 2006. Children whose parents gave written consent (964) were enrolled into the study after a physical examination. Only three children who had a history of drug allergy were excluded. An identification card (ID card) with a unique ID number was issued to all enrolled children. The dates when IPTc was to be administered and the dose required were indicated on the ID card. Parents were advised to refer to the ID cards for information on the dates of IPTc drug administration and on whether they should go to the community-based volunteers or to the health facility to receive the study drugs. A finger prick blood sample for examination of malaria parasite was obtained at enrolment and at the end of the six month observation period.District Health Management Team (DHMT) staff distributed drugs to the health centre staff and community-based volunteers and supervised the administration of drugs to children in May, June, September and October. In the facility-based arm, health workers who were providing outpatient treatment for sick children gave IPTc to the study children on the scheduled IPTc days and EPI nurses administered IPTc during the monthly out-reach vaccination and growth monitoring clinic. In the community based arm, children were invited to a central point in the village on the scheduled IPTc days once a month for four months and the community based volunteers administered the IPTc. Children in each arm received a three day regimen of AQ plus a single dose of SP in May, June, September and October. The first dose of the drug was administered under supervision by a health worker or by a community-based volunteer. Subsequent doses were dispensed by parents/guardians at home after training. Tablets of AQ (150 mg) and (SP) (500 mg sulfadoxine/25 mg pyrimethamine) were given as follows: ¼ tablet of each drug to 3 to 5 month-old infants, 1/2 tablet of each drug to 6 to 11 month-old infants, 3/4 of each tablet to children aged 12 to 23 months and 1 tablet of each drug to those aged 24 months or more. Tablets were crushed and mixed with water and sometimes sweetened with sugar. Drugs were administered based on age not on exact weight. Five days after administration of the first dose of IPTc, field workers visited a randomly selected 20% sample of the children in every village to ask about side effects of drugs, detect any left over medication and to enquire about the use of bednets. The number of children surveyed after each course of IPTc ranged from 73 to 91 in the community based arm and from 68 to 85 in the facility-based arm.Community volunteers received a payment of $10 and facility-based staff a salary supplement of $10 every month for undertaking the additional work required to deliver and monitor coverage of IPTc. Further details of the economics of the two approaches to drug delivery will be presented elsewhere .Carers were asked to take their child to the nearest health facility for examination and treatment if he or she developed any adverse event or any illness. Malaria episodes in study children were monitored through a passive surveillance system at the health centres and hospitals within the study area for a period of six months after the last dose of the first course of drug had been given (May - October 2006). A finger prick blood sample was collected from all children who presented with a history of fever or any signs suggestive of malaria before treatment with AS + AQ. Axillary temperature was measured using an electronic thermometer when a child reported at the health facility.Blood slides were read twice at a central laboratory to give a definitive diagnosis. Laboratory assistants examined thick blood films for parasitaemia. A sample was considered negative only after 200 high power fields had been read. Parasite counts were converted to parasites per micro liter (µl), assuming a white blood cell count of 8000 leukocytes per µl of blood. If there was a discrepancy in the findings in a slide between the two initial technicians (positive or negative or a 50% or more difference in parasite density) a third, more senior microscopist read the slide and his reading was deemed to be the correct reading. A senior microscopist from the Noguchi Memorial Institute of Medical Research  examined all the positive blood films and a 20% random sample of negative blood slides for quality control. Haemoglobin (Hb) concentration was measured using a Hemocue ® Photometer  in children who attended study health facilities for suspected malaria or anaemia. Children with an Hb<8.0 g/dl received treatment for anaemia with iron 12.5 mg and folic acid 50 µg tablets daily for 14 days and those with an Hb = <6.0 g/dl were referred for hospital management.The number of villages required for the trial was based on the following assumptions. It was assumed that coverage of IPTc in the community-based arm would be 80%, that the coefficient of variation in the IPTc coverage between villages would be 0.4 and that the number of children per village would be between 60 and 100 (average 80). On the basis of these assumptions a study with 12 clusters would have 80% power to detect a relative difference in coverage between groups of 35% at a 5% level of significance. It was also assumed that the incidence of malaria in the study area before the intervention would be 19 per 1000 child weeks and it was expected that there would be a 42% reduction in the incidence of malaria post intervention, that the coefficient of variation in the malaria incidence between villages would be 0.4 and that the number of children per village would be between 60 and 100 (average 80). On the basis of these assumptions the study had 80% power to detect a relative difference in incidence before and after the intervention of 42% at a 5% level of significance. The sample size was calculated using the formula suggested by Smith & Morrow P. falciparum clinical malaria was defined as a history of fever during the past 72 hours or an observed temperature ≥37.5° C plus the presence of peripheral parasitaemia of any density. High parasite density malaria was defined as parasitaemia >7000 per µl in the presence of a history fever during the past 72 hours or a temperature ≥37.5°. Anaemia was defined as an Hb <8.0 g/dl.The primary outcome of the trial was coverage with IPTc. Acceptable coverage was defined as the proportion of children who received at least the first dose of 3 or more courses of IPTc. Poor coverage was defined as the proportion of children who received the first dose of two or less courses of IPTc. Secondary outcomes were (1) adherence to treatment (2) the incidence of clinical malaria, (3) the incidence of anaemia, (4) the prevalence of malaria parasitaemia at the beginning and at the end of the rainy season and (5) the incidence of adverse events. Adherence to the IPTc regimen was classified as follows; highly adherent - took first supervised dose of all 4 courses; moderately adherent - took first supervised dose of three courses; poorly adherent - took the first supervised dose of two or less courses. An episode 2test.For the analysis of coverage of IPTc, an intention to treat analysis was used in which all children who were enrolled and received the first dose of IPTc course one were included in the analysis. Statistical significance of the difference in the coverage of IPTc between the two arms was tested using logistic regression with robust standard errors to take in to account the cluster effects. The secondary outcome of adherence was defined as the proportion of children who received all three doses of each course of IPTc in the 20% random sample of children who were visited at home. Statistical significance of the differences in the adherence between the groups was tested by chiFor the analysis of the effect of IPTc on the incidence of malaria and anaemia, we calculated person time at risk from the date of enrolment to date of the final cross sectional survey (6 months after the start of intervention). In addition, if a child received an antimalarial drug (AS+AQ) during the follow up period, we subtracted 28 day post treatment period from the person time at risk. To compare the effect of ITPc on the incidence of malaria between the two arms of the study, we used the random effects Poisson regression model and robust standard errors to allow for intra-cluster correlation and to adjust for the effect of age, and bed net use.To compare the prevalence of malaria parasitaemia between the two arms of the study, we used a logistic regression model to adjust for the effect of clustering in the communities using robust standard errors and for the effect of age, bednet use and the number of IPTc courses administered. All statistical analysis was done using STATA version 10. (©Statacorp LP).The trial was approved by the ethics committees of the London School of Hygiene & Tropical Medicine and of the Ghana Health Service/Ministry of Health (GHS/MOH).The trial was registered on the NIH clinical trials database as an amendment to the number ClinicalTrials.gov NCT00119132.At enrolment, children in the two study groups were similar in regard to age, gender, anthropometric indices and malaria parasite prevalence . The numThere was no statistically significant difference in the proportion of households who owned an insecticide treated net (ITN) between villages in the community based arm and the facility based arm . Similarly, there was no statistically significant difference in the proportion of children who slept under an ITN on the night before the survey between villages in the two arms (11.4% vs 16.3% p = 0.196).The proportion of children who received 3 or more courses of IPTc ranged from 72.9% to 94.8% in the community-based arm and from 58.5% to 94.9% in the facility-based arm villages. The mean coverage of IPTc was slightly higher in the community based arm than in the facility-based arm  . The proAmong the 650 children who were assessed for adherence to IPTc doses, adherence to 3 doses of any course of IPTc ranged from 85% to 100% and there was no difference in the adherence to IPTc between the community based arm and the facility based arm .Forty-four percent (110/248) parents/guardians interviewed reported that they had used the ID card to determine the IPTc drug administration day and where they should take their child to receive treatment. Those who did not use the ID card gave the following reasons: forgot 80 (32.3%), could not read 41(16.5%) and did not understand what was written on the card 17 (6.9%).After the administration of each course of IPTc, drug distributors were asked to record on the ID card that the child had received the first dose of drug under supervision. Eighty-one percent (783/962) of ID cards were available to assess the record of IPTc administration. Recording of IPTc drug administration on ID cards was significantly higher for children who received IPTc from community-based volunteers than for children who received their IPTc from facility-based health workers (65% vs 39% respectively) OR = 2.90 .No serious adverse event attributable to use of the study drugs was recorded. One death occurred during the observation period. This child became unwell before the fourth course of drug administration. The probable cause of death ascertained by verbal autopsy was a brain abscess.Some children spat or vomited drugs immediately after their administration due to the bitter taste of amodiaquine. In the villages where IPTc distribution was community based, 3.5% (23/650) of the children required a repeat dose of IPTc because of vomiting compared to 2.5% (16/650) of children resident in villages where IPTc distribution was facility-based.One hundred and fifty-three of the 650 children (23.5%) visited at home five days after administration of the first dose of IPTc experienced at least one adverse event . Common The incidences of clinical malaria, microscopically confirmed malaria of any parasite density and high parasite density were lower in children in the facility based arm than in the community based arm . There wThis study has shown that the coverage achieved for four courses of IPTc was slightly higher employing a community-based rather than a health facility-based delivery system. Both systems achieved more than 60% coverage for all four courses and over 80% coverage for 3 or more courses. However, this required good supervision and support from a District Health Management Team and the study team.In Ghana there are many communities without a static health facility and, during the rainy season which is also the farming season, approximately 25% of the population is not regularly reached by EPI outreach clinics. Thus, although a facility-based delivery system had a relatively high coverage (86.6%) in this study, a substantial proportion of children would not have access to if IPTc is delivered exclusively through the facility-based approach.It is likely that children who would not have access to IPTc through the health facility-based delivery system would be those living in the most inaccessible and deprived areas of the district and would be those most at risk from malaria. Delivering IPTc through the routine health system will require extra effort to reach the deprived population and adequate supervision to ensure that records are kept properly. In this area of Ghana where the study was done, a combination of health facility-based and community-based approach might be needed to maximise the impact of IPTc. In other areas, different approaches may be appropriate to achieve maximum coverage.Although no serious adverse event attributable to the administration of IPTc with AQ + SP was observed, a significant proportion of caretakers (24%) reported mild adverse events within five days of administration of IPTc. However, in the absence of a placebo group it cannot be determined how many of these reported symptoms were due to the drug and how many to associated minor illnesses. Nevertheless, this is a concern if IPTc is to be rolled out on a national scale. The main adverse events observed in this study were bodily weakness and drowsiness and vomiting of drugs (18.9%). The reported incidence of adverse events fell over time after educating caretakers to feed children with food containing sugar at the time of drug administration. Appropriate health education on adverse events and actions to mitigate them should be a priority for any national IPTc programmes.Most children (66/75) who reported at the health facility with a history of fever received treatment for malaria but only 27% (20/75) of the children had malaria parasitaemia. This high use of antimalarial drugs is partly due to the current treatment guidelines in Ghana that all children <5 years of age who report at the health facility with fever should be treated for malaria unless there is a clear alternative cause for the fever.The reasons for the lower incidence of malaria in the facility based arm than in the community based arm are not clear. We speculate that background incidence of malaria was higher in the rural villages of the community based arm than the villages with a static health facility. However, both arms of the study had rural and semi-rural villages and the prevalence of fever and malaria parasitaemia was not different between the groups at the baseline.We conclude that seasonal IPTc using community-based or facility-based delivery system can achieve high coverage in children aged 3 to 59 months. However, a facility based approach alone would not reach children living in areas without access to formal health care system. Adverse events such as drowsiness, general bodily weakness and vomiting of drugs may be concern for using SP +AQ for IPTc. However, IPTc using four courses of SP+AQ in areas with a prolonged seasonal transmission probably can reduce the burden of malaria.Checklist S1CONSORT Checklist(0.05 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(0.61 MB DOC)Click here for additional data file.