Very little is known about how people in malaria endemic areas interpret policy makers' decision to replace existing anti-malarials, such as sulphadoxine-pyrimethamine (SP) with "new" treatment regimens, such as ALu or other formulations of ACT. This study was conducted to examine community level understandings and interpretations of ALu's efficacy and side-effects. The paper specifically examines the perceived efficacy of ALu as articulated by the mothers of young children diagnosed with malaria and prescribed ALu.New malaria treatment guidelines in Tanzania have led to the large-scale deployment of artemether-lumefantrine (CoartemParticipant observation, six focus group discussions in two large villages, followed by interviews with a random sample of 110 mothers of children less than five years of age, who were diagnosed with malaria and prescribed ALu. Additionally, observations were conducted in two village dispensaries involving interactions between mothers/caretakers and health care providers.While more than two-thirds of the mothers had an overall negative disposition toward SP, 97.5% of them spoke favourably about ALu, emphasizing it's ability to help their children to rapidly recover from malaria, without undesirable side-effects. 62.5% of the mothers reported that they were spending less money dealing with malaria than previously when their child was treated with SP. 88% of the mothers had waited for 48 hours or more after the onset of fever before taking their child to the dispensary. Mothers' knowledge and reporting of ALu's dosage was, in many cases, inconsistent with the recommended dosage schedule for children.Deployment of ALu has significantly changed community level perceptions of anti-malarial treatment. However, mothers continue to delay seeking care before accessing ALu, limiting the impact of highly subsidized rollout of the drug. Implementation of ACT-based treatment guidelines must be complemented with educational campaigns to insure that mothers seek prompt help for their children within 24 hours of the onset of fever. Improved communication between health care providers and mothers of sick children can facilitate better adherence to ALu's recommended dosage. Community level interpretations of anti-malarials are multifaceted; integrating knowledge of local beliefs and practices surrounding consumption of anti-malarials into programmatic goals can help to significantly improve malaria control interventions. These n malaria .Documenting community level understandings and interpretations of ALu's efficacy and side effects is important for several reasons. First, the deployment of ACT on a large scale marks a major shift in global discourses on malaria control, mainly because ACT is a very expensive therapy that is unsustainable in poor countries like Tanzania without substantial donor funding ,6. SeconDetermining the efficacy and side effects of anti-malarials is a complex task, both in biomedical and behavioural terms. This is especially true when the patients are young children who have been treated with anti-malarials, and mothers do the reporting on their children's behalf . Many rewaganga), two licensed private practitioner's clinics, and 14 drug stores (duka la dawa baridi), which are managed by people without adequate formal training.This study was conducted in the Chamazi administrative ward of Temeke District, Dar es Salaam (population: 3.5 million), Tanzania's commercial capital, which comprises of three independently governed municipalities - Temeke, Ilala and Kinondoni. Temeke district, with a population of 886,529 in 2007, and an area of 656 sq.km. is the largest of the three districts that comprise Dar es Salaam. Chamazi ward, which is located some 25 km south of Dar es Salaam's central business district, has two large villages -- Chamazi proper  and Mbande . A number of small villages and hamlets surround these two large villages. 85% of the local residents are Muslims. While the majority of the local residents identify themselves as Zaramo, there are substantial numbers of people in these villages who identify themselves as Makonde, Matumbi, Mpogoro, Ndengereko, Ngindo, Nyamwezi, Msukuma and Myao, among others. Cash income is scarce for many of the local residents whose economic base is subsistence-oriented farming. The completion of the all-weather road in 1996, which connects the trading town of Mbagala with Mbande village, marked the beginning of a new wave of migrants into this region, mostly from north-western and south-eastern Tanzania. The road facilitated the rapid transportation of people and goods between the villages and the city. The local health arena is pluralistic as villagers have access to municipal dispensaries - one of which is located in Chamazi village and the other in Mbande village. Both dispensaries are generally well staffed and well stocked. Additionally, in Chamazi ward, there are more than ten registered "traditional healers" (Data were gathered during four months of fieldwork (May to August of 2007) in the Chamazi ward. Additional follow-up research led by the second author was undertaken during the months of July and August 2009. Data were gathered using a combination of participant observation in the villages and at the health facilities, exploratory focus group discussions (FGDs) and semi-structured interviews with mothers of children below five years of age, who were diagnosed with malaria and treated with ALu less than two weeks prior to the interview. All interviews were conducted in Kiswahili with the help of an experienced female research assistant. The first author interacted with all the interviewees and was present during all the interviews. The second author conducted follow-up interviews, organized FGDs, made observations at the dispensaries, and translated the interviews from Kiswahili to English.FGDs were conducted in Mbande and Chamazi with six groups of six to eight mothers . Two field assistants who were local residents initially approached mothers of young children who were diagnosed with malaria and treated with ALu at the local municipal dispensary, and invited them to participate in the FGDs. Those who were recruited at the dispensaries and were willing to participate in the FGDs were given further details about the study, the venue for the FGDs, and their role in generating important information for the study through discussions in small groups with the help of a moderator. Some of the key questions and topics addressed in the focus groups were: symptoms that prompt mothers to take their sick children to the dispensary; reasons why mothers delay seeking prompt treatment for their children when they have high fever; participants' opinion regarding the changes they have noticed in the quality of treatment for malaria since SP was replaced with ALu as the first-line drug at public health facilities; their perceptions regarding the side-effects associated with SP and ALu; their perceptions regarding the cost of dealing with childhood malaria; measures taken by members of the community to protect their children from contracting malaria; whether in their view, the malaria situation in their village has improved since the introduction of ALu, and what according to them needs to be done to minimize malaria's impact on their community.After reviewing the data from the FGDs and refining the semi-structured interview schedule, detailed interviews were conducted with 110 mothers whose children were treated with ALu for malaria during the past two weeks, in Mbande, Chamazi, and three adjoining villages -- Wembebamia, Kiponza and Kisewe. Mothers were randomly selected from a list that was prepared following initial contacts with them at the dispensaries. Only those who were willing to participate in the study were interviewed. Mothers were interviewed regarding the child who was under five years of age, diagnosed with malaria and prescribed ALu at the local municipal dispensary less than two weeks before the interview. Two children in the study sample had been treated with ALu followed by another anti-malarial, such as quinine (QN) or antibiotics.madhara) after being treated with ALu, and if so, to describe the side effects. Mothers were also asked to describe their experience of treating their children with ALu, and how these compared with their experiences of SP. Finally, as a closing question, they were asked to express their thoughts on why they believed malaria persisted in their respective villages.Mothers were asked to describe the symptoms that had prompted them to take their child to the municipal dispensary; the time between the onset of symptoms and their decision to take the child to the dispensary; the advice they had received from the doctor or the nurse at the dispensary; the period they had waited before concluding that their child had recovered from his or her illness; whether their child had experienced any undesirable bodily side effects . The Behavioural Research Ethical Board, University of British Columbia, and the Medical Research Coordinating Committee of the National Malaria Research Institute, Dar es Salaam gave ethics clearance for this study. Additional research and ethics clearance was obtained from the University of Dodoma.Demographic information on the 110 mothers interviewed for this study is presented in Table ® (paracetamol) a small number (2.5%) had used Panadol® in combination with SP. Another 2.5% had given their child SP albeit to no avail. 14% of the mothers had not given their child any medication before taking him/her to the dispensary.While early detection and access to prompt, affordable and effective treatment is regarded as the cornerstone of a successful malaria control strategy, in the homa kali (high fever) or mwili ulichemka (the body temperature was very high), (22.5%), homa haishuki (the fever wouldn't come down), (34%), homa ilikuwa inatisha (the fever was frightening) (6%), hali iliharibika zaidi (the condition worsened) (20%), hali ya mtoto haikuboreka (the child's condition did not improve), (7.5%), and alichoka na kutapika (he/she was exhausted and vomited) (2.5%). The remaining 7.5% had taken their child to the dispensary "without thinking too much about it," mainly to get the doctor's advice. Thus, the key symptom prompting mothers to consider taking their sick child to the dispensary is high fever that does not subside following treatment with an antipyretic. However, observational data gathered at the two municipal dispensaries revealed that in addition to persistent high fever, mothers also mentioned alitapika (vomiting), aliharisha (diarrhoea) and ananyongea (bodily weakness) as other symptoms that had prompted them to take their sick child to the dispensary.Nearly 90% of the mothers mentioned high fever or persistent fever as the most important symptom influencing their decision to take their child to the dispensary. They denoted key symptoms by using terms and phrases such as ® or Panadol syrup (Panadol ya maji), 15% of them reported that their child was prescribed only ALu. The remaining 5% of the mothers said that their child was prescribed ALu along with Panadol® and either a cough syrup or a packet of oral rehydration solution (ORS). Significantly, 92.5% of the mothers had taken their child to the dispensary only once. The remaining 7.5% of the mothers had gone to the dispensary at least twice because their child's condition had not improved even after completing the dosage. Four children in the study sample had received further treatment at the district hospital, and two others were taken to the Muhimbili National Hospital (MNH) located 30 km away.While 80% of the mothers reported that their child was prescribed ALu along with an antipyretic, often recognized as Panadoldawa inafaa, amepona kabisa, anaendelea vizuri tu  (55%); anaendelea vizuri, anacheza na anachangamka vizuri  (42.5%) to indicate that their child had completely recovered following treatment with ALu. Two mothers were uncertain if their child had completely recovered. Significantly, the data in Table Focusing on perceptions of ALu's curative efficacy, mothers were asked whether they believed ALu was an effective drug in the treatment of malaria. Responses to the above question, coded retrospectively as positive or negative revealed that 97.5% of the mothers had an overall positive disposition toward ALu. They confidently stated active) 4.5% to inmadhara or "undesirable" side effect in their child who was most recently treated with ALu. They emphatically stated that they had not seen any side effects and the drug had helped the child to recover completely. The remaining five mothers mentioned various side effects including the worsening of the child's fever. By contrast, when asked to compare their experiences with SP, a majority of the mothers recalled a range of undesirable madhara they believed were caused by SP such as inachokesha, mtoto ana legea sana , homa haishuki, haipungui, iko pale pale wiki nzima (the fever does not go away for a week), inaleta vipele mdomoni, mapele mwilini (it results in mouth ulcers and rashes on the body), kuwashwa washwa  and anakuwa na marengerenge (he/she developes impetigo), mtoto hatulii (the child becomes restless). A 40-year-old mother of three children explained:94% of the mothers reported that they had not noticed any My son became very weak after he was treated with SP. I thought to myself "Have we treated the illness or have we worsened it?!" It took about two weeks for him to return to his normal self. But last week when he had malaria, he was treated with ALu; he woke up in the morning and started playing as usual and his condition returned to normal.Perceptions of ALu's efficacy were closely tied to the perceived cost of dealing with a child's malaria episode. More than 90% of the mothers emphatically stated that ALu was far superior to SP because of its long-lasting effect, and also because the process of treatment-seeking was less expensive. A 27-year-old mother of a three-year old child contextualized her experience with ALu and SP as follows:It's a lot better now because ALu really helps. Earlier you had to pay to get SP, which in any case did not help; the fever wouldn't go away so you had to take your child to the dispensary three or four times. By then you'll have exhausted all your money. But now it's different; the medicine is good. At the dispensary they also do a blood test. If your child is treated with ALu, he'll get better right away. The medicine of today is genuine (dawa za uhakika). If you use it once, you get better right away, so there's no need to go to the dispensary again and again.Perceptions of the drug's efficacy were also reflected in statements about the expenses incurred in the treatment of childhood malaria. 62.5% of the mothers reported spending less money treating malaria than when SP was the first-line drug. Owing to the fact that ALu was prescribed to them free of cost at the local dispensary and treatment seeking did not involve multiple trips to the health facilities, they could "afford" to deal with a malaria episode. However, 30% said that dealing with malaria had become more expensive than before, although many of them added that while SP was a relatively inexpensive drug, they could not trust it because it took a lot longer for the drug to work and they had to make multiple trips to health facilities. The remaining 7.5% of the mothers said that they did not find any significant difference in the expenses incurred during the SP era and now.® to their child and to return to the dispensary if the fever did not go away after three days. Another 17.5% were specifically told to keep their house and surroundings clean by clearing the grass, and to use an ITN to minimize mosquito bites. The remaining 5% of the mothers stated that they had not received any advice from the dispensary staff. Observational data revealed that the dispensary staff, usually one of the nurses, spent an average of two minutes advising the mothers on how to use ALu and reminding them that they need to give their child plenty of water to drink during the treatment period.95% of the mothers had received at least some advice from the dispensary staff along with a prescription for ALu. 77.5% reported that they were told to continue giving Panadolmalaria iko nyingi! meaning that there was still a lot of malaria in their respective villages. Elaborating on their response, more than 75% of the mothers attributed the persistence of malaria in their respective villages to poverty and poor environmental conditions (mazingira machafu). A 40-year-old mother of four children explained:While all mothers who participated in the FGDs and nearly 90% of those who were interviewed said that they were satisfied with ALu's efficacy, they frequently exclaimed We are poor so we don't have enough nets for everyone in the house. When our relatives come to visit us, we'll say, Ah! Alright, let the guests sleep there under the net, and I'll sleep on this side with my children without a net. Naturally the mosquitoes bite us and we get malaria. What can we do? We are poor. There are many large families in this village who have only one net, and many people sleep in places where there are no nets at all.Mothers gave multiple responses to describe their efforts to minimize the impact of malaria on their lives -- use of ITNs to prevent mosquito bites (76.5%) and covering the bed with an ITN early during the evening; keeping surroundings clean (76%) and making sure that children wear a sweater in the evenings before going to bed to prevent mosquitoes from biting them (27.5%)The Tanzanian government's decision to deploy ALu as the first-line anti-malarial on a large scale, mainly through public health facilities, is laudable from a public health point of view. This decision, however, also invites more attention to how communities that are affected by malaria interpret the efficacy and side effects of newer and older anti-malarials. Monitoring how the introduction of new anti-malarials affects people's treatment expectations, the cultural meanings they attribute to old and new drugs, their reckoning of the cost factor in their search for therapy, and their responses to uncertainty in the context of poverty is critical for the successful deployment of new anti-malarial regimens.The data from this study suggest that even though mothers are aware that they have access to a highly effective anti-malarial free of cost, the majority (88%) of them do not rush their child to a health facility for diagnosis and treatment within 24 hours of the onset of fever. Instead, they first treat their febrile child with a store-bought antipyretic to see if the fever subsides. They continuously monitor and evaluate their child's fever for up to three days, and in some cases for up to six days, before deciding to take him/her to a health facility. The data also suggest that self-treatment of febrile children with a store-bought anti-malarial in the Dar es Salaam region is uncommon. This observation is consistent with findings of recent studies in the Tanzanian context, which have reported that unlike during the chloroquine era when self-medication was the norm, there is a noticeable reluctance among the people of Tanzania to use a store-bought anti-malarial to treat childhood malaria as a first resort ,18-20.vipimo) on their children to confirm that they have malaria before prescribing ALu. While this often leads to longer waiting periods, not one mother in the study complained about the extended waiting periods at the dispensary. Thus, improved perception of ALu is related to improved perception of the quality of care exemplified by blood tests for malaria. These data are striking when compared to mothers' responses regarding sick children to a similar question pertaining to treatment with SP in a previous study in the same research setting [The data also suggest that mothers who participated in FGDs and those who were interviewed for this study were satisfied with ALu's therapeutic efficacy as well as what it costs them to access the drug; they were equally pleased with the fact that the personnel at the local dispensary perform a blood test ( setting . In the homa ya kawaida) or teething fever (mtoto anaota meno). Most of the mothers had decided to "wait and see" (unasubiri ukimtazamia) if the fever would go away following treatment with a store-bought antipyretic. These observations are significant in the context of recent discussions and debates surrounding the accuracy of malaria diagnosis, misdiagnosis, over-diagnosis, and the question whether to treat all fever cases presumptively with an anti-malarial or to rely on laboratory-confirmed diagnosis and treatment [For the majority of the mothers interviewed for this study, the blood test marks a significant departure from the SP era when children were routinely/clinically diagnosed with malaria and presumptively prescribed SP, even as the majority of the mothers deemed it a useless and/or a dangerous drug. In addition to efficacy, cost entered the evaluation of anti-malarials. During the SP era, dealing with childhood malaria was usually an expensive undertaking. In the previous study up to 59% of the mothers had consulted more than one health facility in search of an alternative therapy for their sick child. In the process they had incurred additional expenses and lost precious time . The finreatment ,22. On tWhile plans are being implemented to provide the public with better access to ACT, there is an urgent need to implement socio-cultural and behavioural interventions that would persuade mothers to bring their sick children to a health facility for diagnosis and treatment within 24 hours of the onset of symptoms, and not wait for three or more days to see if the fever would subside with an antipyretic. This would minimize childhood mortality resulting from other severe febrile illnesses, such as pneumonia or meningitis, which cannot be easily managed at home . Concurr® blister package meant for adults to mothers, asking them to break up the tablets into two or four parts, and give them to their febrile children. In the present case, the discrepancy between the recommended dosage and schedule for ALu and the mothers' reporting of the dosage and schedule they adhered to (see Table This is especially true in situations where, due to shortage of drugs, health care providers may give CoartemThis study has some limitations that should be considered. First, the study was conducted in a region and among a population that is relatively well served by the health care system, and where people have access to ACT. Caution must be exercised in extrapolating the findings of the study to other regions of Tanzania where there are remarkable differences in population configurations, health infrastructure, and people's access to ACT. Second, the sample size is relatively small to make major statistical inferences. Third, a bulk of the data analysed for this study is derived from narrative interviews with mothers. It was beyond the scope of the study to verify their reports about the drug's efficacy through other measures, including ascertaining drug levels in blood samples. The findings of this study are, therefore, context-based and limited in their generalizability. However, the findings of this study provide valuable insights into how community level interpretations of newly introduced anti-malarials can inform further research and policy decisions aimed at improving the coverage and delivery of ACT among economically vulnerable populations.in vivo clinical or pharmacological efficacy of various anti-malarials in controlled environments, and quite another to ensure the effectiveness of the drugs in "real-life" situations [The deployment of ALu in public health facilities for treatment of uncomplicated malaria has significantly altered people's perceptions of anti-malarial treatment. In the present study, the majority of the mothers not only regarded ALu as an effective anti-malarial, they also found that it significantly reduced their expenditure on dealing with a malarial episode because it did not require them to go through multiple treatment stages as was common during the SP era. However, the majority of the mothers delayed before accessing ALu, limiting the impact of the subsidized roll out of these drugs on health outcomes. Current efforts to make highly subsidized ACT more readily available through private retail pharmacies may result in patients being promptly treated with a highly effective anti-malarial. However, this complex intervention may also result in people's overdependence on the commercial sector for treatment of childhood fevers, and lead to additional financial burden on poor households ,28. Furttuations . In othetuations . Successtuations . Thus, atuations .The authors declare that they have no competing interests.DJN supervised data collection, translated the interviews, contributed to data analysis and write-up of the manuscript. VRK conceived of the study, participated in its design and coordination, carried out fieldwork, and drafted the manuscript. All authors read and approved the final manuscript.


However malaria was not identified as an independent risk factor for diazepam's failure to stop seizures in children.Malaria infection reduces the binding capacity of benzodiazepine receptors in mice. We studied the efficacy of diazepam terminating seizures in children with falciparum malaria. Diazepam stopped seizures in fewer patients with malaria parasitaemia ( Acute seizures are common in many children presenting to sub-Saharan African health facilities . In thesPlasmodium berghei infection in mice significantly decreases the binding capacity of Gamma-aminobutyric acid-A (GABA-A) receptors to which benzodiazepines bind  were excluded.Either intravenous (IV) diazepam (0.3 mg/kg) or intramuscular (IM) paraldehyde (0.4 ml/kg) was used as first line treatment, usually dependent upon the availability of venous access. In the event of refractory seizures, a repeat dose of either drug could be given. If the seizure did not stop, then second line IV phenobarbital (15 mg/kg) or IV phenytoin (18 mg/kg) and eventually the third line  were given, according to the hospital protocol  based onDiazepam was considered to be effective if it stopped the convulsion in less than 15 min with no recurrence . The poiMicrosoft Visual Fox-Pro 6.0 (Microsoft Corporation) and analysed with Stata . Measures of association were done using Pearson's Chi-square test. We performed a univariate analysis of the various risk factors  and stratified for possible confounders such as age and sex. A multiple logistic regression was performed adjusting for history of seizures, age, sex, seizure duration and malaria as an underlying condition.The data were entered and tabulated in A total of 1653 children aged 0–12.9 years, admitted during the 7-year period were given AED to stop acute seizures, of whom 1189 (72%) had adequate information recorded in the notes and proforma. Malaria was the most common underlying condition, occurring in 566 (48%) of children of whom 239 (42%) had cerebral malaria.P. falciparum parasitaemia compared to those without parasitemia  . However the level of parasitaemia did not influence the outcome and malaria was not an independent risk factor for the lack of termination of seizures (First line AEDs stopped seizures in 298 (25%) of all patients. Diazepam terminated seizures in fewer patients with = 0.047) . Diazepaseizures . FurtherThis study showed that diazepam is less efficacious in stopping seizures in children with falciaprum malaria. However, after adjusting for risk factors, the effect of malaria was not statistically significant . In a stPatients with cerebral malaria have reduced plasma and CSF concentrations of histidine an amino acid which is an important constituent of the GABA-A receptor , and thiThe effect of diazepam on respiration also depends upon the integrity of the benzodiazepine receptors . The incThe difference in diazepam efficacy is not only seen in the comparison between the malaria group versus the non-malaria group but those patients with and without parasitaemia as a whole. Thus diazepam may be less efficacious in controlling seizures in children with malaria. However a randomized trial is needed to clarify these findings and to elucidate the effect of malaria on benzodiazepine receptors in humans.


Transmission of malaria in West African urban areas is low and healthcare facilities are well organized. However, malaria mortality remains high. We conducted a survey in Dakar with the general objective to establish who died from severe malaria (SM) in urban areas (particularly looking at the age-groups) and to compare parasite isolates associated with mild or severe malaria.Pfdhfr/Pfcrt-exon2 and nine microsatellite loci in 102 matched cases of SM and MM. Half of the malaria cases recorded at the dispensaries and 87% of SM cases referred to hospitals, occurred in adults, although adults only accounted for 26% of all dispensary consultations. This suggests that, in urban settings, whatever the reason for this adult over-representation, health-workers are forced to take care of increasing numbers of malaria cases among adults. Inappropriate self treatment and mutations in genes associated with drug resistance were found associated with SM in adults. SM was also associated with a specific pool of isolates highly polymorphic and different from those associated with MM.The current study included mild- (MM) and severe malaria (SM) cases, treated in dispensaries (n = 2977) and hospitals (n = 104), We analysed In this urban setting, adults currently represent one of the major groups of patients attending dispensaries for malaria treatment. For these patients, despite the low level of transmission, SM was associated with a specific and highly polymorphic pool of parasites which may have been selected by inappropriate treatment. The UNESCO estimates that half of the African population will be living in an urban setting by 2030 Urbanization profoundly changes the epidemiology of malaria, causing a decrease in risk, when compared to rural areas To better document severe malaria cases in this urban setting, we conducted a survey in one of the biggest dispensaries and in two main hospitals. In this epidemiological setting, young adults were the major group at risk from malaria and severe malaria (SM) in both, dispensaries and hospitals. Self treatment and a delay in consultation appeared to be the major factors associated with SM. However, genetic analyses of the parasite isolates from SM and MM, using molecular markers, of drug resistance to chloroquine and pyrimethamine as well as parasite polymorphic  de Fann, and Hôpital Principal de Dakar (HPD), over the same period of time. In the hospitals, patients with fever and clinical signs of cerebral impairment underwent a lumbar puncture (to rule out meningitis) and twice rapid malaria diagnosis tests plus a thick blood smear, over a 48 hour period. The same protocol of examination and questioning (of the family) was followed as for MM. Assignment of patients to a cerebral malaria (CM) group was carried out following the WHO criteria. However, P.falciparum-positive patients with impaired consciousness but not fulfilling WHO definition, were called “hospitalized malaria” (HM). Most of them experienced a rapid recovery of consciousness during the time of presentation. During statistical analysis when no difference was found between the CM and HM groups, patients of the two groups were pooled into the severe malaria (SM) group for further analysis.In Senegal, mild malaria cases are managed at the dispensary and severe malaria cases are referred to a hospital. Patients with mild malaria, were registered during the rainy season (from September to December) at the Dispensaire St-Martin in Dakar. Routine registration and questioning of patients, medical examination, biological diagnosis and treatment were conducted by eight nurses and two medical doctors, in agreement with the Senegalese national recommendations. Medical consultations of adults, children and pregnant women were conducted in separate areas. All patients with fever and without obvious etiology underwent a thick blood smear examination prior to antimalarial treatment. Mild malaria was defined as fever associated with a positive thick smear and without other obvious causes of fever. Severe malaria cases were assessed by the local team, according to the national recommendation  and referred to a hospital. All biological examinations were confirmed at the Institut Pasteur de Dakar. Malaria cases were recruited by our team from two hospitals in Dakar, namely During this survey patients with MM were matched with SM for the genotyping study. This group of MM matched patients was submitted again to a careful interrogation  and physical examination. Immediately at presentation of a patient with SM, two MM cases were matched based on their area of residence, age group and gender. 144 MM were matched before the assignment of hospitalized patients to CM or HM groups. Only 102 control patients were kept as matched controls for the genotyping study after confirmation of the MM and SM status .Parasiteamia was measured by examination of Giemsa stained blood smears using a magnification ×1000 and by establishing the parasite count per 1000 leukocytes. The count per microliter was calculated according to WHO recommendations.For each patient enrolled in the genotyping study, 5 mL of peripheral venous blood was collected on EDTA.Written consent was obtained from the patients, or their relatives, after verbal information in the vernacular language, and the study was approved by the National Ethics Committee of the Ministry of Health of Senegal.Pfcrt exon_2, and Pfdhfr were sequenced as described Parasitic DNA was extracted using the standard Phenol-Chloroform procedure Differences between groups were compared using the Mann-Whitney (MW), Chi2 or Kruskal-Wallis (KW) tests. Correlations were calculated using a Spearman test (SP).A), and ii) expected heterozygosity under panmixia (HE) Polymorphism of the parasite populations was determined by the complexity of the isolates, i.e. the average number of parasite genotypes per patient, in the population  and for each microsatellite by i) the mean number of alleles per patient and ii) the number of alleles needed to describe 50% or 75% of the patients in the group. For population genetic analyses, individual haplotypes were reconstructed for the nine microsatellites, when possible . Genetic variability was quantified using FSTAT as i) mean number of alleles . Overall, 46% of the consultations were for children less than 5 years old . Older patients (>15 y) represented 26% of the consultations and their number increased during November and December. Malaria attacks accounted for 13.7% of all the consultations (n = 2977)   and incr = 2977) . InfantsP.falciparum was reported.Cases referred by the dispensary to hospitals were more frequent in November  than in any other month , but not in the use of prophylaxis (16%) . Severe cases (CM+HM) had experienced a longer delay before consultation than MM cases (CHI2 p = 0.0005), especially among the patients using self-treatment (Chi2 p = 0.00015). Geometric mean parasitaemia was higher in MM than in severe cases  used self-treatment before consultation, with a significant difference between groups   . 36% of Despite the low transmission rate of malaria in Dakar, parasite isolates from the area contained highly polymorphic microsatellite loci, with 9 to 16 alleles observed per locus . The totPfcrt or Pfdhfr haplotype was found .In contrast, self-treatment was associated with an increased frequency of some alleles, which could indicate a selection of parasites (DNS). However, no significant association between a specific allele and a By taking the nine microsatellites together, it was found that the multiplicity of infection was high with a maximum of three distinct parasite populations detected per sample . It incrA high mean heterozygosity was found for the 235 haplotypes reconstructed (He = 0.81±0.07, Overall, no significant linkage between SM and a specific parasite genotype/allele was detected by single microsatellite analysis. However, global genetic analyses outline a larger diversity of parasite populations in SM and a clear genetic difference between parasites associated with either SM or MM.This study, conducted in Dakar during the peak of malaria transmission During this study, adult patients accounted for about 50% of all confirmed malaria cases at the dispensary. This high prevalence is unlikely to be due to under-representation of children, resulting e.g. from home treatment, as more than 70% of the consultations were for children <15 y. Adults were also highly susceptible to severe malaria, as they accounted for 87% of the 8% of the malaria cases referred to hospital, in agreement with other surveys at CHN-Fann Pfcrt, haplotype was found in all clinical groups. Furthermore, a higher prevalence of mutated Pf-dhfr isolates was found in severe cases. These findings are compatible with other studies in vitro cycloguanil resistance in isolates collected at hospital. Self-treatment was reported by 16.8% of the MM patients attending the dispensary. This rate is lower than that previously reported by Ndiaye et al Pfdhfr mutants in SM may indeed reflect poor efficacy of this self-treatment. Additionally we found that chloroquine-prophylaxis tended to be associated with a higher frequency of a Pfcrt mutated genotype. Chloroquine and SP are thus poorly efficient treatments. As was reported recently, they can, however, reduce parasite densities and result in delay in malaria presentation, which can contribute to asthenia and anaemia and thus to referral to hospital For the genotyping study, three groups of patients were compared: mild malaria, hospitalized malaria and cerebral malaria. A very high rate of chloroquine resistance-associated CVIET Microsatellite genotyping of the isolates showed a high prevalence of multiple infections with a higher infection complexity and a larger genetic diversity in parasite isolates from patients with CM and HM. These data are in agreement with Nzila Pfcrt or Pfdhfr haplotypes. However, Fst analysis and linkage disequilibrium confirmed differences between MM isolates collected from dispensaries and those from severe malaria, collected from referral hospitals. They suggest a non-panmictic structure in P. falciparum populations. As patients were matched on their household location and recruitment period, this cannot be due to different spatial origins of the samples, and isolation between MM and severe malaria is also unlikely to have occurred. The results indicate that severe malaria is not associated with a small subset of highly virulent genotypes/isolates, but rather demonstrates that it is associated with a highly polymorphic group of isolates presenting population characteristics different from parasites associated with mild malaria. These differences could be related either to drug resistance or to antigenic specificity. As self-medication and treatment delay were found to be major factors associated with SM, a strong involvement of drug resistance in the selection of a pool of isolates is suggested. However, this does not explain genetic isolation of these isolates from those inducing MM. This could be explained by their newly introduction into the urban biotope from other parts of the country, followed by their selection due to inappropriate treatment. However, the role of specific antigenic properties of these isolates in their pathogenicity cannot be ruled out; as the immune response developed by inhabitants of this low transmission area could be mostly strain-specific.Microsatellite genotyping showed roughly similar allele frequencies in CM, HM and MM. There was no association of any specific allele with a clinical group or fatal outcome, or between a specific microsatellite allele and Pf-dhfr parasites were associated with severe malaria, but parasite isolates collected from severe malaria patients were clearly identified as a separate pool of parasites from those detected in MM. This supports the need for full sequence analysis of the genome of these parasites in order to understand their association with patho-physiology.Whatever the origin of these isolates, this study highlights that young adults are the major group of patients attending dispensaries with mild malaria and hospitals with severe malaria in urban settings. According to the new strategy for the elimination of malaria proposed by WHO in countries with low transmission, this clinical profile could be the most prominent in the next 5 or 10 years in most of the African urban areas. National Malaria teams must be aware of this evolution to adapt their strategies. Multiple infections and highly mutated


It is important to establish whether or not the presence of malaria parasites in peripheral blood of asymptomatic individuals is a predictor of future clinical mild malaria attacks (MMA). The aim of this study was to determine how an asymptomatic positive thick blood smear could be related to the occurrence of a MMA during the nine following days.Plasmodium falciparum asymptomatic carriage at two different times of the transmission season, the beginning (September) and the end (November). The occurrence of MMA was investigated in asymptomatic carriers and non-carriers, every three days for nine consecutive days. Survival analysis was performed and risk estimates were calculated by Cox proportional hazards model.The study was conducted in a cohort of 569 Senegalese children, who were investigated for At the beginning of the transmission season, 27.8% (147/529) of the children were asymptomatic carriers (ACs) and 5.4% (8/147) of MMA occurred among these, versus 1% (4/382) among non-carriers . At the end of the transmission season, the frequency of asymptomatic carriers was similar to that observed at the beginning of the season , but no MMA was detected during this period.P. falciparum asymptomatic carriage and the occurrence of MMA at the beginning of the transmission season was demonstrated, with a five-fold increase in the risk of developing a MMA in ACs. In the context of a possible distribution of IPTc in the future, drug strategies may have dramatic consequences due to the existence of ACs (both long term and short term), as they seem to play an important role in the individual protection to malaria, in the most exposed age groups.A significant association between  Plasmodium falciparum asymptomatic carriers (ACs), i.e., individuals harbouring parasites without clinical signs, are numerous in areas of high transmission. The consequences and significance of such asymptomatic infections have both been studied in diverse situations and from complementary approaches, but these studies led to contradictory results [ results -4. Accor results -3. Conve results .Plasmodium falciparum infection and MMA occurrence needed to be investigated [It is important to understand the process which leads some of these children to suddenly develop a MMA. The time course of the relation between stigated . If the stigated ,6,7. Invstigated -4.Treatment of asymptomatic individuals, regardless of their malaria infection status, with regularly spaced therapeutic doses of antimalarial drugs has been proposed as a method to reduce malaria morbidity and mortality . This stTo evaluate how asymptomatic carriage could be related to the occurrence of uncomplicated malaria attacks, a follow-up of a cohort of Senegalese children was carried on in an area of marked seasonal transmission. To determine the variations of the balance between clinical signs and the absence of symptoms during the transmission season, a survey on the same population at the beginning and at the end of the transmission season was performed (i.e. September and November 2002).Anopheles gambiae s.l. complex, mainly by Anopheles arabiensis [This study was carried out during the 2002 malaria transmission season, in two villages of the Niakhar area (Diohine and Toucar), located 150 km south-east of Dakar . The cliabiensis ,11. PluvP. falciparum parasitaemia without fever or clinical signs were classified as asymptomatic carriers (ACs); (2) children with a negative thick blood smear were classified as non-carriers, and (3) children presenting with a clinical malaria attack (see below) were treated and further excluded from the analysis.566 children and adolescents, from two to 17 years of age, were investigated twice during the study: at the beginning (September 2002) and at the end (November 2002) of the transmission season. All eligible children in the area were asked, at each period, to attend the health centre where a systematic thick blood smear was performed. A urine sample was collected in order to check for the presence of chloroquine metabolites. Children were classified according to their clinical and parasitological status: (1) children harbouring a P. falciparum, in order to minimize the probability to deal with reinfections [An active survey of clinical malaria attacks was set up. Children were visited at home every three days by investigators recording axillary temperature. In case of temperature exceeding 37.5°C or fever reported by the parents since the last visit , a thick blood smear was performed. Parents were urged to bring their child to the health centre between the scheduled visits if any kind of symptom appeared. A MMA was defined as the association of a temperature over 37.5°C (or reported fever between two visits) and a parasite density over 2,500 trophozoites/μl of blood. The same threshold had been previously used in various studies performed among populations living in the Niakhar area -14. At efections .When a clinical malaria attack was suspected, a presumptive treatment was administered according to the "Programme National de Lutte contre le Paludisme" (PNLP) recommendations. This protocol was approved by the ethics committee of the Health Ministry of the Republic of Senegal (N°000526/MS/DERF/DER).Plasmodium falciparum asexual blood forms were counted on Giemsa-stained smears, using a previously described method [d method . A bloodd method .For each child the following covariates were collected: (1) sex; (2) age; (3) village of residence and (4) presence of chloroquine metabolites in urine (≥ 3 μg/ml).The same statistical strategy was applied during the two periods. We first performed an univariate analysis to test for associations between the occurrence of MMA (yes/no), asymptomatic carriage (yes/no), and measured covariates. Pearson's chi-square and one-way analysis of variance were used for qualitative and quantitative variables respectively. When non parametric methods were required, Fisher's exact test or Kruskall-Wallis test were applied. Factors associated with asymptomatic carriage and MMA occurrence were selected at a p value < 0.15.The delay between the systematic parasitological measurement and the appearance (or not) of a MMA during the nine following days was analysed, using survival time data procedures.- The Kaplan-Meier survival analysis and the Log-Rank test were used to compare incidence rates of MMA between ACs and non-carriers.- The Cox proportional hazards model was used to analyse the time to development of a MMA in each group, adjusted on the covariates found significant (p < 0.15) in the univariate analysis.Survival times were estimated as follows Figure : the basA total of 529 and 527 children from the original 566 were enrolled respectively at the beginning and at the end of the transmission season. The mean age (SD) of the population was 8.6 (0.15) years and the sex-ratio  was 1.35. Fifty seven per cent of children belonged to the village of Diohine and forty three percent to the village of Toucar. Children who did not attend the health centre in September (n = 37) and in November (n = 39) were significantly older than children who did. Activities such as farm work or looking after livestock may explain this difference.-3), bur without any particular trend of increase. The proportion of AC did not differ (p > 0.70) between children with CQ metabolites in their urine  and others . No child was lost to follow-up. During the nine following days, 2.3% (12/529) of the whole group presented a MMA, i.e. 5.4% (8/147) of the ACs and 1.0% (4/382) of the non carriers. The mean parasite density was 34,243.3 parasites/μl of blood , extremes values going from 4,040 to 108,800 parasites/μl of blood. The information concerning the use of impregnated bednets was collected. It appears that 7.52% of asymptomatic carriers reported the use of these bednets, vs 5.38% among non carriers, p = 0.624. All children who presented a mild malaria attack were not sleeping under impregnated bednets (unknown data for 2 children in this group). Information concerning the treatment delivered in the area by the dispensaries during the study was collected by our team and we didn't notice differences among villages.In September, 27.8% 147/529) of the children were asymptomatic carriers  and, in a lesser extent, sex (p = 0.06) were associated with asymptomatic carriage. Age was neither associated with MMA nor asymptomatic carriage. However because of its importance, it was included in multivariate analysis as well.Associations between MMA, asymptomatic carriage, and measured covariates  in univariate analysis are shown in Table The rate of MMA occurrence was significantly higher over the nine days of follow-up in ACs compared to non carriers   . The parasite density was homogenous during the 10 days of enrolment (p = 0.13), but not the plasmodial index (p = 0.005), even if there was no particular trend of increase. The same pattern of results concerning the association of AC and measured covariates as observed in September, was observed at this period. No MMA occurred during the nine following days.527 children out of 566 were present at the end of the malaria transmission season Figure . Three cP. falciparum asymptomatic carriage and the occurrence of MMA. The relation was true at the beginning (September), but not at the end of the transmission season (November). In the first period, children harbouring P. falciparum had a five-fold increased risk to develop a MMA during the nine following days, independently of their age.This study demonstrated that, in a cohort of Senegalese children, there was a significant association between Plasmodium falciparum asymptomatic carriage concerns a very important proportion of exposed populations in endemic areas. However, the accurate definition of asymptomatic carriage relative to its duration (i.e. long term or short term) differs from one study to another. In the present work it was intended to minimize the probability to deal with reinfections and a short term follow-up of nine days was chosen, as done by Bouvier et al in a previous study [us study . The defus study .To see if there was a similarity with the results of Bouvier, the survival analysis was performed using different diagnosis criteria for MMA (temperature over 37.5°C associated with a positive blood smear regardless of parasite density). The main results of the study were confirmed with these different criteria, both at the beginning and at the end of the transmission season.P. falciparum infections induced a protective response in older children regularly exposed with less subsequent morbidity. Conversely, in young children with low previous exposure, recent malaria infection represented a higher risk of clinical attacks [The results of the present study show the absence of effect of age on children susceptibility to present a MMA, which is an important individual parameter concerning the acquisition of immunity. They differ from a recent study performed in Tanzania, which evidenced an age-dependent risk of clinical malaria associated with asymptomatic carriage . In this attacks ,18.Differences in the levels of malaria transmission between the two study areas  can be an explanation to the observed discrepancies. Transmission is intense and perennial in Tanzania, while in Niakhar it is low and strongly seasonal. Moreover, in the study period (2002), the overall rainfall level in Niakhar was notably lower than the mean rainfall over the past 16 years (1984–2000) . It can Differences observed between the beginning and the end of transmission season could be explained by the hypothesis that immunity to uncomplicated malaria symptoms, which requires an important number of infections over many years to become established, can be partitioned in two components: an immunity refraining the outbreak of symptoms  and an immunity allowing to control parasite density (infection immunity) (reviewed in ). The abet al have shown that multiple infections are associated with an increased number of clinical episodes in very low transmission areas [msp2 genotyping, was performed in June 2002 (before the transmission season) and January 2003 (at the end of the transmission season) in a cohort of 400 children living in the same villages as the children enrolled in the present study. A significant increase of the mean value of MOI was found between June 2002 and January 2003, i.e. 2.5 and 4.1 respectively (p < 10-4) [Plasmodium falciparum genotypes during the transmission peak in September can be associated with an increased incidence of MMA. Later in the transmission season, children may have recovered their clinical immunity quickly as they have been accumulating infections by various parasite strains.Moreover, the multiplicity of infections (MOI) by different plasmodial strains can bring a complementary explanation to the differences found in the study between the beginning and the end of the transmission season. The role of MOI on malaria morbidity has recently drawn a lot of attention but several contradictory results have been published. Mayor on areas . Converson areas ,3 as welon areas . In the  < 10-4) . These ret al to determine the efficacy of IPTc to protect children from presenting a MMA. IPTc was distributed to children from two to 59 months living in villages surrounding the two villages of the present study, at three periods of the transmission season. Doses where delivered in September (beginning of the transmission season), October (four weeks after the first dose) and November (end of the transmission season) and they obtained high degrees of protection against clinical malaria, with a 90% decrease in MMA incidence [Treatment of asymptomatic individuals with regularly spaced therapeutic doses of antimalarial drugs, called IPT, has been proposed as a method to reduce malaria morbidity and mortality. At the same period and in the same area, a clinical trial was undertaken by Cisse ncidence . Such hincidence  or Ghanancidence .The results of the present study lead to interrogations. In the case of a systematic distribution of IPTc by health workers at the beginning of the transmission season, all children would be treated independently of their parasite carriage, i.e. children at risk at the beginning and protected at the end of the transmission season (ACs) as well as non carriers, protected at the end of the season. Immunological protection against malaria would be somehow replaced by chemical protection. The consequence of an interruption of IPT distribution, due to low availability of the drugs or dysfunction of the distribution process would be an incomplete coverage of chemical protection during the transmission season. Supposing the second dose, given in October, was not distributed, the situation would be similar to the beginning of the transmission season as described in the present study. Children could present a MMA at the end of the transmission season, with no noticeable benefit due to IPT.A recently published study on the same population has focused on long term asymptomatic carriage . ACs werP. falciparum carriage would be suppressed, as well as short term carriage, in the event of IPT distribution. Thus, the consequences of a shortage of drugs would be dramatic, as potentially increasing the population of susceptible children. The existence of ACs, both short term and long term, stresses the atmost importance of a perfect coverage of the transmission season by IPT, with sufficient drug supplies and well motivated health workers, as explained by Greenwood [The long term reenwood .In a seasonal marked malaria transmission area, asymptomatic carriers are at risk of presenting a MMA at the beginning of the transmission season, and not at the end. Such differences observed between the two periods within the transmission season can be related both to the acquisition of immunity and the evolution of the genetic diversity of parasite strains in the course of the transmission season. In the future, it may be important to assess epidemiological or interventional studies in transmission area, taking into account the period within the transmission season. The interest on asymptomatic carriage should not be abandoned, as it may be a form of protection at some moments of the year. The probable existence of several types of asymptomatic carriage (i.e. short term and long term) and their characteristics have to continue to be investigated, at the time when intermittent preventive treatment for infants (IPTi) and children (IPTc) is under evaluation and could be applied generally.AC: asymptomatic carrier; MMA: mild malaria attack; MOI: multiplicity of infection.The authors declare that they have no competing interests.ALP participated in the collection of data, performed the statistical analysis and drafted the manuscript. MC participated to in the design of the study, helped to draft the manuscript and revised the paper critically. FMN participated in the design and coordination of the study, the collection of data and made helpful comments on the manuscript. JFE and OG participated in design of the study and revised the paper. AG was the conceptor of the study, participated to its design and coordination, organized the collection of data, helped to draft the manuscript and revised it. All authors read and approved the final manuscript.


Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects.Susceptibility/resistance to TNF enhancer and the FCGR2A G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfo™ version 3.4.Allelic frequency distribution in populations across India was first determined by typing genetic variants of the TNF enhancer along with other reported variants. Five TNF enhancer SNPs and the FCGR2A R131H (G/A) SNP were analyzed for association with severity of P. falciparum malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. TNF -1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG  correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcγRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of P. falciparum severity/resistance in the Indian population.A novel single nucleotide polymorphism (SNP) at position -76 was identified in the TNF and FCGR2A SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.Association of specific  Plasmodium falciparum blood infection levels and fever episodes have been linked to chr 5q31–33 [The association of severity of malaria with several human genetic factors is well documented  and the  5q31–33  and chr1 5q31–33 -8.P. falciparum malaria in India is high with ~0.9 million cases reported annually in the last few years [P. falciparum with the infection accounting for more than 80% of all malaria cases in certain areas [Incidence of in areas ,11  in the 5' regulatory region of  malaria -17. Tumo malaria . The traegulated ,20. SNPsegulated ,22 with ulations -14,23-25FCGR2A  alters its function in vitro; the product of the G allele (131R) has preferential affinity for IgG1 and IgG3 while the A allele (131H) product binds efficiently to IgG2 while retaining its affinity for IgG1 and IgG3 [P. falciparum malaria in Africa, particularly in children in Kenya and Gambia [The human IgG receptor FcγRIIa (CD32) is an important link between the cellular and humoral arms of the immune system. It is a low affinity receptor for immunoglobulin subtypes IgG1–4 and also binds C-reactive protein (CRP) with high affinity. A polymorphism in exon 4 of and IgG3 . The higd Gambia ,16, but d Gambia . A recend Gambia  has implTNF gene for genetic variants in the ethnically diverse Indian population and correlation of two SNPs (-1031 and -863) with circulating plasma TNF levels in healthy individuals and P. falciparum malaria patients is reported here. Correlation of TNF levels and 5' regulatory region SNPs with severity of disease was investigated in a case-control study with patients and controls drawn from a P. falciparum endemic and a non-endemic region of the country. The FcγRIIa R131H polymorphism was also evaluated for correlation with susceptibility to severe malaria in these regions.Screening of the enhancer region of the TNF (RefSeq NT_007592.14) and FCGR2A (RefSeq NM_021642) SNPs was carried out in the existing Indian Genome Variation Consortium (IGVC) samples. This validation panel consisted of 1,871 samples drawn from 55 ethnically and linguistically diverse endogamous populations across India [Analysis of allele frequency distribution of the selected ss India .P. falciparum malaria. Diagnosis was carried out by rapid diagnostic test kits  followed by confirmation by examination of thick and thin film blood smears. Categorization of severe and non-severe cases was performed according to WHO guidelines [For the case-control study for an endemic  and a non-endemic (Lucknow and surrounding areas of Uttar Pradesh) region Figure , approvaidelines . Non-sevTNF and FCGR2A SNPs in the IGVC validation panel was done by Sequenom mass spectroscopy. For patient and control samples, genotyping of -1031, -863 and -857 TNF SNPs was done by direct sequencing of a 292 bp PCR product covering nt -782 to -1073. Typing of other TNF SNPs  and FCGR2A G/A (R131H) SNP was done by SNaPShot method (Applied Biosystems). All DNA sequencing and genotyping was performed on 3130xl automated DNA sequencer (Applied Biosystems). Approximately 10% genotypings carried out by SNaPshot were quality checked by DNA sequencing.Genomic DNA was extracted from peripheral blood leucocytes using salting-out procedure . For valA sandwich ELISA was performed to measure plasma TNF levels using capture monoclonal anti-human TNF antibody (Pierce) and a paired biotinylated anti-human TNF antibody (Pierce). The minimum detection limit of the assay was 2 pg/ml.TNF SNPs were generated by PHASE. Linkage disequilibrium (LD) plot for TNF SNPs was generated using Haploview. Odds ratio (OR) for risk assessment was determined using EpiInfo™ version 3.4 software with the P-value calculated by Fisher Exact or Mantel-Haenszel test.The chi-square test was performed to evaluate whether the allele frequencies of the populations are in Hardy-Weinberg equilibrium. Comparison of TNF levels among genotypes of the -1031 and -863 SNPs was carried out by non-parametric Kruskal-Wallis test [TNF 5' regulatory region polymorphisms as well as discovery of possible novel SNPs in TNF promoter/enhancer was carried out by sequencing in representative samples of 43 populations of India (discovery panel) under the Indian Genome Variation Consortium (IGVC) [TNF enhancer that has been reported to be highly conserved in humans and primates [TNF -76 T>A was among those exhibiting the lowest FST and heterozygosity (Ho) values  suggesting negative selection. The distribution of TNF SNPs at positions -1031, -863, -308 and -238 across Indian populations is shown in Figure FCGR2A exon 4 G/A R131H polymorphism across the 55 populations varied from 0.38 to 0.83. The distribution of the AA genotype is shown in Figure Validation of reported m (IGVC) . A novelprimates  and is pprimates . Of the TNF and FCGR2A SNPs with P. falciparum malaria in India. Patient samples were collected from a P. falciparum-endemic area (Chattisgarh and Orissa) that has perennial and high malaria transmission [TNF -1031 and -863 SNPs. The frequency of the FCGR2A (R131H) A allele was also higher in the endemic region although the P value was 0.07. Thus, the healthy control panel constituted for this study comprised ethnically-matched individuals from both regions. The combined control panel comprised equivalent numbers from both these groups and included populations represented in the patient pool.This SNP frequency data was used to plan a case-control study to analyze the association of smission  and a noTNF regulatory region SNPs described above as well as the -857 SNP previously correlated with cerebral malaria [The five  malaria  were typWithin the patient and control groups, significant differences in TNF levels were seen between genotypes of both -1031 and -863 (Table 2 = 0.494) while no linkage was observed with -308 and -238  or with patients with non-severe malaria (P = 0.0009)  Figure . The difTNF -1031 and -863 SNPs and cytokine levels in individuals, association of high TNF level with severe malaria as well as correlation of -1031C and -863A polymorphisms with disease severity.The -1031C allele was associated with disease severity with higher percentage of the mutant genotypes observed in severe patients compared with controls  (Table FCGR2A G/A (R131H) polymorphism with P. falciparum malaria in different populations, we analyzed its distribution in patient and control groups. Combined analysis of endemic and non-endemic region samples indicated that the high affinity IgG2-binding 131H allele associates with protection from disease manifestation; the GG homozygote for 131R correlated with susceptibility to severe malaria   Figure . Heteroz) Figure . No signP. falciparum malaria infection has been described as both protective and pathogenic [TNF enhancer SNPs on transcription levels of the cytokine remains controversial [P. falciparum malaria [The role of TNF during thogenic ,38. At lthogenic . Individoversial ,41, seve malaria ,43. Whil malaria , a study malaria  reported malaria . In our TNF SNPs with increased risk of severe malaria. A recent study on Thailand/Myanmar border populations reported association of the TNF -1031C, -863C, -857C allele with severe malaria [Our results indicated association of the -1031 and -863  malaria . This alP. falciparum malaria with the high-affinity IgG2 binding 131H/H genotype being associated with protection from disease. Our results are in agreement with a recent study on subjects from Sudan that demonstrated that the 131R/R genotype and the 131R allele were significantly associated with the odds of severe malarial disease [P. falciparum malaria in Burkina Faso [P. falciparum-infected erythrocytes by cells expressing the 131H receptor [In contrast to previous reports ,16,27, o disease . The 131 disease ,48. Cyto disease -51. Highina Faso . IgG2 anina Faso  showed treceptor  or by Fcreceptor .P. falciparum malaria observed in the Burkina Faso study [P. falciparum malaria transmission. As evident from the map of P. falciparum incidence in India  comprises scientists from six institutes of the Council for Scientific and Industrial Research with Dr. S.K. Brahmachari, Institute of Genomics and Integrative Biology, New Delhi as coordinator. Collection of endogamous population samples across India and Sequenom genotyping data analyzed here was coordinated under IGVC. SH is a member of the consortium. All authors have read and approved the final manuscript.


Whiles awaiting for the arrival of an effective and affordable malaria vaccine, there is a need to make use of the available control tools to reduce malaria risk, especially in children under five years and pregnant women. Intermittent preventive treatment (IPT) has recently been accepted as an important component of the malaria control strategy. This study explored the potential of a strategy of intermittent preventive treatment for children (IPTC) and timely treatment of malaria-related febrile illness in the home in reducing the parasite prevalence and malaria morbidity in young children in a coastal village in Ghana.The study combined home-based delivery of IPTC among six to 60 months old and home treatment of suspected febrile malaria illness within 24 hours. All children between six and 60 months of age received intermittent preventive treatment using amodiaquine and artesunate, delivered by community assistants every four months (three times in 12 months). Malaria parasite prevalence surveys were conducted before the first and after the third dose of IPTC.Parasite prevalence was reduced from 25% to 3%  one year after the inception of the two interventions. At baseline, 13.8% of the children were febrile  compared to 2.2% at evaluation (post IPTC3 combined with timely home management of fever) .The evaluation result indicates that IPTC given three times in a year combined with timely treatment of febrile malaria illness, impacts significantly on the parasite prevalence. The marked reduction in the parasite prevalence with this strategy points to the potential for reducing malaria-related childhood morbidity and mortality, and this should be explored by control programme managers. Malaria is estimated to cause between 300 and 500 million clinical cases with about 700,000 to 1.6 million deaths every year. About 94% of these deaths are believed to occur in sub-Saharan Africa, which is also the most resource-limited continent on the globe . MalariaPlasmodium falciparum resistance to these anti-malarial drugs, as with chloroquine and sulphadoxine/pyrimethamine.Malaria control in Ghana relies on early diagnosis and prompt treatment of suspected cases (fevers) and the home is where early recognition and, in most cases, prompt treatment is initiated ,8. HowevPrompt and efficacious treatment of malaria cases is an effective strategy for malaria control. It is especially important for vulnerable groups including children under five years of age and pregnant women. IPT has also recently been accepted as an important component of the malaria control strategy following the demonstration, in perennial malaria transmission areas, that IPT given with childhood vaccinations reduced incidence of first episode of malaria. It also reduced severe anaemia by more than 50% during the first year of life -11. IPTCAlthough the Abuja target of treating 60% of under-five suspected malaria cases within 24 hours of symptom onset by 2005 was reduced by the Ghana Health Services to 55% in 2007, this goal has not been achieved, and the question remains as to how this will be achieved. Improved access to IPTC especially in communities with no health facilities is one way to reduce the prevalence of infection and associated morbidity. IPTC studies were carried out in areas with seasonal malaria transmission and were found to be effective ,12, but The goal of this study was to demonstrate the feasibility of controlling febrile malaria illness at the community level through combined interventions of intermittent preventive treatment and timely febrile malaria illness management in children between six and 60 months of age in southern Ghana. Specifically, the study was designed to: deliver intermittent preventive treatment to children (IPTC) at home and to provide timely treatment for febrile malaria illness in children within 24 hours of symptom onset.The study was conducted at Shime sub-district of the Keta district in the Volta region of Ghana where malaria accounts for over 40% outpatient clinic attendance in the district . The malaria situation in the district is perennial with higher transmission occurring in March (the beginning of the raining season) to November (the end of the raining season). The Shime sub-district is one of the four administrative sub-districts recognized by the Ghana Health Service in the Keta District. It is the most deprived area in the Keta district. Electricity supply is limited; it has no secondary school and a very poor road network. During the rainy season most parts of the area are cut off from the rest of the district. The land is marshy and covered with streams, lagoons and creeks. The entire sub-district has a population of 19,972. The study took place in an area with a population of 8,000. About 99% are Anlo-ewe-speaking people. Their main occupation is farming and fishing.The sub-district is served by two health centers and one of them is located within the study area. Almost all the communities in the area have pipe-borne water, but because of the 'cash and carry system' where residents pay for every bucket of water fetched at the point of fetching, the people continue to use unsafe water from the many water bodies around to complement the safe one. Four out of the 36 communities in the sub-district participated in the study, mainly because of geographical accessibility.Community entry activities, involving meetings and durbars of chiefs and people in the study communities, were done at the onset of the project. After gaining the support and willingness of the chiefs and people to participate in the study, the community registers of eligible children aged ≤60 months were updated. After obtaining consent from caretakers/parents, children aged six to 60 months were enrolled in the study. No caretaker refused to participate. All children (six to 60 months old), including those on antiretroviral treatment, were included in the study.All caretakers of children selected to participate in the baseline parasite survey were selected as respondents in a semi-structured questionnaire interview. The questionnaire was designed to solicit information on the demography of caretakers, all cause mortality, morbidity, bed net usage among others. This short interview guide was translated into the local language and back translated into English to ensure that they convey the meanings they were intended to. The questionnaire was pre-tested in a field situation in a community with similar characteristics as the study community. The interview was conducted in the local Ewe language by the first author.IPTC was delivered every four months beginning in July 2007 for one year  and the strategy was evaluated in July, four months after the last round of IPTC and just before the fifth round of IPTC. Children received 10 mg/kg body weight of amodiaquine (AQ) daily and 4 mg/kg of artesunate (AS) daily (given as a single dose) over three days. All treatments were given under direct observation and children were observed for five minutes after drug administration to ensure that they retained the medication. Children who vomited within the five-minute observation period received a repeated full dose of the medication. Children were followed up on days 1, 2, 3 and 7 after treatment (the day of first dose (treatment) was counted as Day 0) to document adverse events reported by caretakers using the adverse report form.Any child with febrile illness suspected to be malaria by the caretaker was reported to the fieldworkers who then evaluated and treated those who met the protocol criteria for suspected febrile malaria illness. Those who did not meet the criteria were referred to the health post located within the sub-district for further evaluation and treatment. In short, any child with axillary temperature of ≥37.5 degree Celsius, whose caretaker suspected febrile malaria illness, was treated with the full three-day course of artesunate and amodiaquine.rd microscopist and the majority result was taken as the final result. Haemoglobin concentration was determined from finger pricks using a portable automated Hemocue® photometer  at the field site.A diagnosis of malaria infection was made by light microscopy of thick and thin blood smears. Finger print peripheral blood was taken by trained technologists from the Noguchi Memorial Institute for Medical Research for the preparation of the thick and thin blood smears. All specimens were taken from the field. The thick and thin blood smears were stained with 3% Giemsa for 30 minutes. Parasite density was determined by counting the number of asexual parasites per 200 white blood cells, and calculated per μL assuming a white blood cell count of 8,000 cells per μL. Sexual parasite count was done per 1,000 white blood cells. A smear was declared negative when the examination of 100 thick-film fields did not reveal the presence of asexual parasites. Quality control was done by allowing a second microscopist to read a random 10% sample of both negative and positive slides to confirm the absence or presence of parasites. Discrepant results were read by a 3Every qualified person living in the study community was enrolled with the consent of his or her caretaker, mostly parents. However, for the prevalence survey, a sample size of 174 was needed to determine malaria parasite prevalence estimated at 20% in the study population. Also, to be able to determine at least10% reduction in malaria parasite prevalence in the study population at evaluation, we needed to enrol about 360 children into the IPTC intervention programme . At evalData were analysed to compare pre-intervention findings with post-intervention findings. All quantitative analyses were done using EpiInfo version 3.4.1. Proportions of pre- and post-intervention clinical findings and parasite levels were compared using Mann-Whitney or chi-squared tests. Statistical significance was set at p ≥ 0.05.The mean (± SD) age of respondents was 28.2 (± 7.5) years. Most, (71.0%) of respondents were married. The mean (± SD) years of formal education was 3.9 (± 3.6) ranging from 0 to 12 years. However, 37.1% of respondents had no formal education.Respondents  reported that they and their families usually sleep under bed net. Comparatively, the number of insecticide-treated net usage has increased significantly from 38.5% to 60.0% in the study community within one year of project implementation. This was as a result of continual information provided by community assistants to caretakers that treated bed net was an effective way to reduce febrile malaria illness, and also participants were encouraged to take advantage of a highly subsidized treated bed net provision programme available at the health facility for children under five years of age and pregnant women. All respondents  said it was important for children under five years to sleep under bed nets.Asra - febrile malaria (2), convulsion (1), swelled up (1) and could not tell (1). Also, 35 (20.1%) respondents said they knew a close relative who had lost a child less than five years of age within the past 12 months at baseline. No caretaker, however, had lost a child or had a relative that had lost a child in the twelve months of project implementation.The mortality data generated was too small to make any meaningful analysis, but it is important to report that, at baseline, five (3.0%) caretakers reported that they had lost a child within the 12 months preceding the interview. The conditions that killed these children were reported as During the prevalence surveys  a number of febrile illnesses were reported for the children (within the seven days prior to data collection) Table . On the malaherb  and Kinaquine® . None of these five children were taken to the clinic/hospital. On the other hand, during the evaluation, only two caretakers reported that their children had been treated within the seven days prior to data collection and they were treated by the community assistants working with the project using amodiaquine and artesunate. However, community assistants' have referred a total of 29 children whose conditions they suspected not to be febrile malaria-related-mostly children having running nose/catarrh, stomach problems, ear infections, eye problems, watery diarrhoea and fast breathing among others, to the health post (clinic) for further investigations and treatment within the 12 months of study implementation.At baseline, five (2.9%) caretakers reported that the children were treated for febrile malaria within the seven days prior to data collection. These five were reportedly treated with anti-malarials, such as chloroquine, P. falciparum, the dominant species in Ghana. Detailed clinical and parasitological findings are presented in Table Out of 174 children tested for malaria parasites at baseline, 44 25%) of them were positive for malaria parasite compared with only 10 (3%) out of 357, a significant difference of over 80% . All the positive infections were 5% of theIn this study, anaemia was defined as haemoglobin < 10 g/dl. There was a significant drop of anaemia in the study population one year into the intervention , at baseline, 27.6% of the children were anaemic compared to 16.8% recorded during evaluation , but this was not the case at evaluation. Also, at baseline there was a significant relationship between parasitaemia and anaemia , but this was not the case at evaluation. There was a significant relationship between febrile status and anaemia at evaluation (P = 0.01 (Fisher exact test)), but this was not the case at baseline.There was no relationship between gender and anaemia in the study population. There was a significant relationship between reported fever in the seven days prior to baseline survey and anaemia ) - evaluation). There was no gender difference in parasitaemia in the study population.There was a significant relationship between febrile status and parasitaemia at both baseline and evaluation (XIPTC was delivered to children aged six to 60 months in the study community three times (July and November 2007 and March 2008). There have been marginal increases in the number of children at each subsequent treatment round   IPTC1, 4 (0.95%) IPTC2 and 7 (1.65%) IPTC3, caretakers reported that their children were weak for about two to four hours after taking the drugs. None of these cases warranted any medical intervention.Only five (1.2%) febrile malaria-related illnesses were treated in the study communities between IPTC1 and IPTC2, while 39 (9.3) were treated between IPTC2 and IPTC3 and 17 (3.9%) were treated between IPTC3 and evaluation.Throughout the 12-month intervention period, community assistants continued to informally advice community members on the importance of IPTC, the need for timely treatment of suspected febrile malaria illness in children within 24 hours of the onset of symptoms and the need to replace the predominantly untreated bed nets with treated ones. Community members were encouraged to take advantage of the highly subsidized treated bed nets available at the health centre for children under five years and pregnant women. Pregnant women were also encouraged and in some cases assisted to attend antenatal clinics.The study clearly demonstrates that it is possible to train community assistants to deliver IPTC and timely home treatment to children aged six to 60 months. Visits were made to the community twice in a month to collect timely home treatment forms. However, during each IPTC rounds, the first author spent three days in the community to supervise the research assistants.This study looked at the effectiveness of intermittent preventive treatment for children (IPTC) combined with timely treatment at home for malaria control, targeting children aged six to 60 months old in an all year round malaria endemic area in Ghana. The main finding of the study was a reduction of about 88.0% in the prevalence of malaria parasite infections in the target population  within one year of the project implementation. It may be argued that the increase in the use of insecticide treated net (from 38.5% to 60.0%) could be implicated in the reduction of the malaria parasite prevalence in the study population. However, it is unlikely that all the reduction of over 80.0% was due to the 55.8% increase in ITN use alone. Several studies in IPT intervention measured clinical incidence rather than prevalence and found between 20% and 86.0% reduction with strong variations depending on transmission duration and intensity, target population and intervals between treatments -12,14,15Anaemia in the children, defined as haemoglobin <10 g/dl, improved from 27.6% to 16.8%, 12 months after the intervention was implemented. This compares well with a recent randomized trial in Tanzania, which showed that IPT given to infants at the time of childhood immunization reduced the incidence of the first episode of malaria and anaemia by more than 50.0% during the first year of life .There was a noticeable reduction in malaria-related morbidity in the study population as expressed by fever and other malaria-related signs and symptoms reported either in the seven days before prevalence surveys or IPTC administration. The reduction could also be seen from reported treatment sought for the children within the seven days prior to prevalence surveys or IPTC administration. Timely treatment of febrile malaria cases in the community did not follow any pattern as can be seen from the result. However, it could be argued that because the community assistants were on hand to deliver effective treatment in a timely fashion, this could have contributed to the marked reduction of parasitaemia seen at the evaluation survey. The community assistants also contributed to heightened health information in the study community through informal education on the use of treated bed net and timely treatment. This should encourage malaria control programmes to have confidence in community assistants to deliver timely treatment and IPTC to children at the community level, once they are well trained by the programme coupled with reference manual for easy and quick referencing when in doubt.et al [et al [Findings reported here present a challenge to the existing practice, especially in most sub-Saharan African countries, where malaria diagnosis is based on presumption without confirmation. As community intervention or treatment increases, this may lead to fewer malaria infections and this may lead to over diagnosis and treatment with expensive drugs for people who do not need them. When this happened, control programmes would have to invest in rapid diagnostic test kits where microscopy is not possible. As reported by Zikusooka et al , this mal [et al  also makThe sharp increases in the number of febrile malaria cases treated at home between IPTC2 and IPTC3 might have been largely due to increased confidence of caretakers in the ability of community assistants to treat their children with suspected febrile malaria effectively and the recognition of caretakers of the importance of timely treatment. The decrease in the numbers between IPTC3 and evaluation could be attributed to the reduction of malaria prevalence in the study community as a result of the interventions implemented.The use of IPTi which is similar to IPTC in principle was found to reduce malaria incidence in infants . AlthougThe results indicate that it is possible to deliver IPTC and timely home treatment to children between six and 60 months old. Since there were no timely treatment form to collect at some of the biweekly visits to the community, it should be possible to reduce the visit to once in a month to reduce supervision cost.This indicates that, it is possible to reduce malaria prevalence and this may reduce malaria-related childhood morbidity and mortality and this should be explored by control programme managers as one of the effective options available for the fight against malaria, especially in sub-Saharan Africa.The authors declare that they have no competing interests.CKA was involved in the conception, design and implementation of the study as well as data management and writing of this paper. KAK was involved in the conception, design and writing of this paper. AKS was involved in the design, implementation and writing of this paper. MGW was involved in the conception, design and writing of this paper. All the authors have read and approved the final version of the manuscript.The study was approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana and the Ethical Review Committee of WHO/TDR.


Bupropion is approved for the treatment of mood disorders and as an adjuvant medication for smoking cessation. Bupropion is generally well tolerated and considered safe. Two randomized controlled trials of bupropion therapy for smoking cessation did not report any hepatic adverse events. However, there are three reports of severe but non-fatal bupropion hepatotoxicity published in the literature.We present the case of a 55-year old man who presented with jaundice and severe hepatic injury approximately 6 months after starting bupropion for smoking cessation. Laboratory evaluation demonstrated a mixed picture of hepatocellular injury and cholestasis. Liver biopsy demonstrated findings consistent with severe hepatotoxic injury due to drug induced liver injury. Laboratory testing was also notable for positive autoimmune markers. The patient initially had clinical improvement with steroid therapy but eventually died of infectious complications.This report represents the first fatal report of bupropion related hepatotoxicity and the second case of bupropion related liver injury demonstrating autoimmune features. The common use of this medication for multiple indications makes it important for physicians to consider this medication as an etiologic agent in patients with otherwise unexplained hepatocellular jaundice. Bupropion is a weak inhibitor of the neuronal uptake of norepinephrine, serotonin, and dopamine but is chemically unrelated to other known antidepressant drugs including tricyclic and tetracyclic agents as well as selective serotonin re-uptake inhibitors. The mechanism by which bupropion enhances the ability of patients to abstain from smoking is unknown but may involve central noradrenergic and/or dopaminergic pathways.Bupropion  with a left shift, hemoglobin 15.5 (13.5–17.5 GM/DL), platelets 200 (140–450 K/UL), AST 1466 (20–57 IU/L), ALT 1459 (21–72 IU/L), total bilirubin 5.3 (0.0–1.5 mg/dl), direct bilirubin 4.9 (0.0–0.8 mg/dl), alkaline phosphatase 219 (30–136 IU/L), INR 13, and prothrombin time 145.8 (10.0–13.5 sec). Serum liver biochemistries were normal 4 months prior . An abdominopelvic CT scan without contrast was unremarkable.The patient had a history of mild depression, hypertension, and hyperlipidemia. He denied using intravenous drugs or recent travel or sick contacts. He had discontinued alcohol in 2002 but smoked a half-pack of cigarettes for the past 8 years. He was receiving warfarin for a prosthetic mitral valve since 2002 and had a previously stable and therapeutic INR. His other medications for the past 3 years included metoprolol XL, atorvastatin, and aspirin. Paroxetine had been started shortly after surgery and discontinued in May 2004 but restarted in October 2004 for recurrent depressive symptoms. Bupropion 150 mg bid was started for smoking cessation in July 2004 and was continued up until hospitalization (6 months of treatment). The patient reported never having received bupropion or other anti-depressants beyond the paroxetine previously. He also denied ingesting over the counter products such as acetaminophen or herbals. He had allergies to penicillin and sulfa drugs that caused hives.After receiving several units of fresh frozen plasma, he was temporarily placed on intravenous heparin. Diagnostic studies included a serum iron of 193 ug/dl, transferrin saturation of 55%, and ferritin of 974 mg/dl but subsequent hemochromatosis genotyping was negative. Serum ceruloplasmin was normal at 28 mg/dl. Serological studies for acute hepatitis A IgM, hepatitis B surface antigen and anti-HB core antibody, and hepatitis C RNA by PCR as well as CMV and EBV serologies were negative. However, an anti-nuclear antibody  and anti-smooth muscle antibody (ASMA titer = 1:40) were positive. A surface echocardiogram revealed an ejection fraction of 75–80%. Despite withdrawal of all outpatient medications, his serum aminotransferases and bilirubin continued to rise  declare that they have no competing interests.FH established diagnosis and medical treatment. TS participated in patient care and the drafting of the manuscript. JT read the original pathology. RJF participated in patient care and drafting of the manuscript. All authors read and approved the final manuscript.