Malaria is a serious illness among children aged 5 years and below in Zambia, which carries with it many adverse effects including anemia and high parasites exposure that lead to infant and childhood mortality. Due to poor accessibility to modern health facilities, malaria is normally managed at home using indigenous and cosmopolitan medicines. In view of problems and implications associated with management of severe malaria at home, rectal artesunate is being proposed as a first aid drug to slow down multiplication of parasites in children before accessing appropriate treatment.A qualitative study using standardised in-depth and Focuss Group Discussions (FGDs) guides to collect information from four (4) villages in Nakonde district, was conducted between February and March 2004. The guides were administered on 29 key informants living in the community and those whose children were admitted in the health facility. Participants in the 12 FGDs came from the 4 participating villages. Participants and key informants were fathers, younger and older mothers including grandmothers and other influential people at household level. Others were traditional healers, headmen, village secretaries, tradtional birth attendants, church leaders and black smiths. FGDs and interview transcriptions were coded to identify common themes that were related to recognition, classification and naming of malaria illness, care-seeking behaviour and community treatment practices for severe malaria.Parental prior knowledge of the disease was important as the majority of informants (23 out of 29) and participants (69 out of 97) mentioned four combined symptoms that were used to recognise severe malaria. The symptoms were excessive body hotness, convulsions, vomiting yellow things and bulging of the fontanelle. On the other hand, all informants mentioned two or more of symptoms associated with severe malaria. In all 12 FGDs, participants reported that treatment of severe malaria commenced with the family and moved into the community as the illness progressed. Although treatment of severe diarrheal effects, were common among the winamwanga, no rectal medicines to treat severe malaria were identified. Apart from the anti-malarial fansidar, which was mentioned by 23 in IDIs and 40 in FGDs, participants and informants also frequently mentioned indigenous medicines provided by healers and other respectable herbalists for repelling evil spirits, once a child had severe malaria. Mothers were the important arms for administration of ant-malarial drugs in the villages. Referrals began with healers to CHWs, where no CHWs existed healers directly referred sick children to the health facility.Our findings showed that there is a precedent for rectal application of traditional medicine for childhood illness. Therefore rectal artesunate may be a well-received intervention in Nakonde District, provided effective sensitisation, to mothers and CHWs is given which will strengthen the health care delivery system at community level. The problem of malaria infection in the under five years children has been widely described -5. It isIn Zambia malaria continues to be the leading cause of deaths and illnesses, responsible for 47% of all deaths . The incWhere new drugs such as rectal artesunate have been developed and used for the emergency management of acute malaria in patients who cannot take drugs by mouth and for whom parenteral treatment is indicated but not available, positive results have been recorded ,12. The A lack of knowledge at family level on transmission of malaria hampers prevention of the disease. In the same study Simoes and others have shown that malaria case fatality rates were related to environmental and socio-economic status rather than poor knowledge of the disease 'malaria' . In ruraIn a clinical trial the rectal administration of artesunate has resulted in adequate drug absorbtion in all subjects and 92% children had achieved a parasite density lower than 60% of baseline after 12 hours compared with 14% of those administerd parenteral Quinine. Results were shown to be highly significant , at the helm, controls all government activities in health care provision including one District hospital for referred patients. Of twelve administrative Communication between families and the health centres is through the Neighbourhood Health Committees, established per 'zone' covering a catchment of 500 populations or 5 to 10 km radius. Community Health Worker (CHWs), trained Traditional Birth Attendants (TBAs) and Village Health Motivators (VHMs), who are trained by the District Health Management Board, also report directly to the Rural Health Centres. Community Health Workers cover between three and five villages in their catchment areas. The presence of local healers and untrained Traditional Birth Attendants (TBAs) providing indigenous health care has a direct effect on the health seeking behaviour of the families in the villages. The villages in Nakonde are spread over a vast area and are sparsely populated, making it difficult for the government health structures to be easily accessible and effective.The study was conducted in Nakonde District between February and March 2004. Data collected was qualitative in nature, primarily using key informant interviews. Focus group discussions provided a means of triangulation.Primarily interviews were conducted with caretakers and influential relatives at household level. Caretakers comprised of fathers of the under five years children, younger and older mothers and grandmothers. In view of the important role of traditional healers and other community members, village headmen, village secretaries, Traditional Birth Attendants, church leaders and blacksmiths were interviewed. The Focus Group Discussions (FGDs) were conducted to provide more responses and accommodated the emergence of hidden issues from the respondents. Finally, interviews with health care providers working at the closest Rural Health Centre (at Mwenzo) and the next referral level, the District Hospital (at Nakonde) were conducted.To provide a means of triangulating the information, key informant interviews and focus group discussions were conducted. Key informants consisted of 12 community informants, 11 mothers of children admitted at either Mwenzo Rural Health Centre or Nakonde District Hospital for severe malaria and 6 health care providers working either at Mwenzo Rural Health Centre or Nakonde Rural Health Centre. A total of 12 Focus Group Discussions (FGD), distributed equally in the four villages, were conducted. They lasted for between forty minutes and one hour. Separate groups of younger mothers, fathers and older mothers were organised and lastly a discussion with opinion leaders  was conducted (Table The number of people participating in each FGD varied between six and ten. The major criteria for participating in the FGD was availability of an under five child in the care of the mother or caretaker. Only younger mothers were categorized according to their age groups (15–24 years). However, no age restrictions were imposed on older mothers, fathers of under five or opinion leaders who participated in the FGDs.The scene was set by the research team who visited the study site prior to the interviews, explaining the purpose of the study. All those who participated in the study were identified by the research team. The village headmen mobilised participants for FGDs through the criteria provided by the investigators.The interviews were conducted by local residents following a training session by the research team at Mwengu Social and Health Research Center. Despite the permission that was granted by the community leadership, individual participants were requested to consent participating in the study. This arrangement ensured that the quality of data was not compromised, as enough time was given to allow for respondents to gain confidence in the research assistants. The interviews with caretakers lasted about one hour and about two hours with traditional healers and other opinion leaders. At every stage, interviewees were encouraged to talk freely. Transcription was completed following each interview before moving on to the next one. The authors closely supervised both data collection and transcriptions of materials.All the interviews were tape recorded, transcribed and translated from Chinamwanga to English. The authors listened to recordings and checked the accuracy of the transcripts.During the course of the research, investigators took turns to witness an in-depth being conducted by the research assistants and, in some cases, assisted in manual recording of responses. At every interview, the presence of the investigator was critical. During focus group discussions the investigators actively participated as facilitators.A field guide was developed for use by interviewers and piloted in a community with the same socio-economic characteristics as the study site. The guide provided an opportunity to explore various community treatment behaviours of severe malaria, questions were related to serious illnesses in the community; information about health care; local terminologies of malaria; symptoms suggestive of simple and severe malaria; causes and treatment of different symptoms for simple and severe malaria; access to health facilities; dispensing practices at community and health facility levels; health information and communication channels regarding malaria and finally knowledge and practices regarding administration of rectal medicines.Criteria for selection of research assistants were completion of 12 years of schooling, previous experience as a research assistant, ability to speak the local language, Chinamwanga, fluently, resident in Nakonde, mature and respected by the people in the community. The four research assistants selected were all males aged between 27–35 years. The research assistants had not had any medical training; this assisted in minimizing the likely interviewer bias.The research team conducted an eight-hour training session for ten days familiarizing research assistants with research tools and practicing interviews. They were also taught basic techniques of recording responses and probing, identifying informants and note-taking for easy transcription. The fundamentals of organizing and conducting focus groups discussions were also included in the training. Investigators demonstrated in-depth interviewing.The authors read and re-read the transcripts and developed codes to identify important themes. All transcriptions were typed in MS Word and converted into an Atlas.ti readable text file. Each interview and focus group discussion was entered as a single file. There were 41 single files for analysis. The files were then coded and independently prepared matrices for interpretation of substantive themes were developed. The purpose of the texture analyses was to interpret the data and findings by interrelating codes and data, in order to create common concepts.To achieve this, data was segmented into variables, coded and then frequencies of codes were electronically assigned. Focus group discussion and in-depth interview transcripts were coded to identify common themes related to recognition, classification and naming of malaria illness, care seeking behaviour and traditional treatment practices for severe malaria. Codes were used to find specific occurrences of common responses in the data, which could not be searched by simply applying text based search techniques. They were also used to classify different levels of summaries that fell under different variables, for purposes of comparison between FGDs and in-depth interviews, as well as between categories of informants and participants. The process was followed to ensure a systematic basis for frequencies of the views expressed by participants during discussions and interviews.Codes were standardized in all files according to variable under analysis, in order to ensure inter-code reliability. Assessment and validity of information was made by re-reading the transcripts.The results are presented in themes previously selected to meet the objectives of the study; identification of malaria , causes of malaria, use of rectally administerd medicines, health seeking behaviour and decision-making processes. Information from twenty-nine (29) interviews, the point at which no new information was forthcoming, and 12 focus group discussions are presented.Malaria was recognized as being a common problem in the area. At least 24 out of 29 key informants and 78 among the 97 FGD participants continuously mentioned this. An in-depth interview with a grandmother caring for under five child reported this:There is this disease number one, malaria. The children have finished [meaning many children have died], when diarrhea starts, at the same time with vomiting, you haven't arrived in time and the child.. we have buried many children, and the child is dead. Eee, this is the disease, which has defeated us very, very much.The biggest problem, which we face in this area, especially for young children, is malaria [said it in English], inzekema is common here.In one of the focus group discussions with opinion leaders, an old man aged over 60 years expressing himself on the prevalence of malaria in the district said: Inzekema' to describe the syndrome of fever, chills, body pains and headache. The term fever was used synonymously with the English word malaria. The categories of variables varied as shown in table The Winamwanga at times considered fever as an illness on its own in the under five children. They used the term 'Two types of malaria were recognized, simple and severe, the latter being associated with convulsions. The Winamwanga use 'Umuwili ukulungua' as a general term to describe the symptom of fever. 'Impepo' which is translated as cold, includes body hotness, shivering, headache and general body pains. This is considered as less serious, as the 46-year-old traditional healer put it:There are two types of malaria. There is one where a child is shivering and the body is hot, that is one type of malaria, we call it impepo.The two symptoms associated with an episode of simple malaria, that were commonly mentioned by both informants and participants included shivering and body hotness.sinsa' [convulsion], was one of the key symptoms used by FGD participants to describe the progression of an illness, 'it is big malaria' they could say. Making a statement on severe malaria, the 46 year old traditional healer continued and said:The local term 'The other type of malaria comes with convulsions [sinsa], this is the other type. There are 2 different types of convulsions [sinsa].Describing severe malaria a 40 year old key informant said:Vomiting [ukuluka] yellow things, when he starts vomiting yellow things, shivering too much [ukuzakaza], coldness/hotness [impepo], this is how we know that this is big malaria.E malelya mukulu', meaning . A 54-year-old opinion leader in one of the FGDs said:In focus group discussions 78 out of 97 participants recognized that vomiting yellow things, convulsions, bulging of the fontanelle and excessive body hotness were some of the symptoms associated with severe malaria. These participants included those who had at one time taken their children to either Nakonde district hospital or Mwenzo rural health Centre, Convulsions were associated with severe malaria. At this point the reference to fever changes and they call 'we see when a child starts to say things we cannot understand; in the end you see the child prostate/stretching .breathing [meaning was relieved]. During the FGDs, it was common for participants to interpret vomiting yellow things as a sign of relief and beginning of healing of the illness. One participant in the FGD with younger mothers reported:Among the Winamwanga, vomiting yellow things was a clear sign that the malaria was coming out and that the child was at least That is when he is breathing; it means the illness is coming outA mother aged 39 years old, whose under five child was admitted at Nakonde hospital, reported that though most children with severe malaria vomited, not all vomiting was malaria. To show how vomiting due to severe malaria could be identified, she went on to say:They look yellow and they are slippery, if it is severe malaria [Inzekema]. But if it is another disease, because illnesses come different, he/she may just be vomiting [ukuluka] yellow things only and we say maybe it is severe malaria [Inzekema]. Ukupolomya]. A mother whose child was admitted at the hospital stressed on 'diarrhea' that was caused by witchcraft and said,'Caretakers were capable of identifying different causes of some symptoms, which included diarrhea , it is not cured easily. Child will keep on crying, keep on crying. That is how it is.It was common to have mothers associating symptoms such as diarrhea with witchcraft, not with the malaria episodes.When analyses were made on symptoms of convulsion and bulging fontanelle, caretakers knew differences between convulsions that occurred because of epilepsy and convulsions due to severe malaria attacks. They were able to associate a bulging fontanelle due malaria and that resulting from other illnesses. A father of the under five, who was 52 years old, said,' there are two. There is that one suffered by young children... an illness, which they say [akatuwi] sinking of fontanel, it causes the fontanel not closing quickly, this one is [ichapamutwe]. Now those... those illnesses are which differentiate them from malaria. And that one...that... [akatuwi], when you use a malaria drug, you find a child still sick. It closes without working. We... oh, this person needs African medicines which the people have been using long ago for cure. That is when we go to those who know the medicines.Reported malaria infection ranged from 50 to 80% in all villages. The prevalent conditions were convulsions, vomiting, high fever and loss of appetite.Rectally administered medications are already being used in this community.During an FGD with older women, one participant described how the medicine for Ilonda was administered, she reported:Yes, turning the child upside down Sikaona [referring to the interviewer], sprinkling him/her just there where the dirt comes out [anus], that is where I sprinkle... there since it is Ilonda.It was common for most informants and participants, to explain in detail how medicines given through the rectum were administered. In an interview with a male key informant, regarding administration of medicine for Ilonda, he said:We just apply it on the sores on the anus, bath in medicine in the water and put the powder inside the anus, and then some you drink it. [Meaning medicine is applied on the rectum]. But...but some they buy those things from Tanzania, which they put medicine and inject people .me... I would not care even if the medicine was inserted in the ears or nose, for as long as the child survived that is alrightWhen asked whether they would be willing to let their children have the drug inserted into the rectum, almost all the participants said it was alright as long as it made their children reach a health facility where they would continue seeking further treatment. As one member of the FGD said, ': We mama [the mother].The results from this study showed that the decision to use a facility when a child had severe malaria lay with the mother. Most informants and participants stressed on this point. During a Focus Group Discussion with opinion leaders, when asked who takes the decision for the child to go to the health facility for treatment, all participants shouted In another FGD with older mothers, a participant aged 25 years, with seven years of schooling and having three children reported;Just myself the brains come to decide that here it is not looking well. Me... when I tell the father [Father here meaning husband], you father this child is sick, aah.... It is just malaria you are just troubling let us just buy 'to' panadol that's how sometimes he will go and buy panadol, but we who are taking care of a child [meaning one sleeping closer to the child]. I am the one who is knowing how the child is becoming, so when I see that the child has not slept, I tell him my dear this child has not slept its better we take him/her where...? To the hospital.During discussions with opinion leaders, the responsibility for the mother to take the first decision was strongly stressed, as was evident in the following discussion:It's the mother of the child. Inf.3, 5 and 7 almost at the same time: Its the mother Inf.4: The very mother of a child, is she not the very one who can tell the father? That lets take the child to the hospital. Inf.5: She has the right Inf.4: Because she is the first one to know the disease, so it means she has the right to decide.Inf.6: According to this discussion, a father was only consulted in order to provide financial and material support, which enabled a mother to use services from specialised health facilities or service.The majority of the participants believed that once a child is sick, unless treated, it was inevitable that he/she would die. Households started using structures and medicines that they were familiar with, before they could start looking for help outside what they knew. The chart below demonstrates caretakers' movements. CHWs  Admitting Health CentreIndigenous Healer During an in-depth interview with a mother admitted at the hospital, she reported:It is just like when you meet the disease... some days you can waste even three days. Now if you have not met the illness, some days they fail like that they come and change and say go and buy this such you change again it is just like when you meet the illness like when it meets [referring to drugs bought and administered at home] with the illness it can even take two days [says it in English], and the child is better.While attempting to treat symptoms associated with severe malaria, local individuals, such as vendors and other people who knew some indigenous medicines were consulted. During an FGD, one informant aged 34 years stressed on this point when she reported:and those which you find here [respondent touching on her top of the head showing where it sinks] it is getting in side, even those they treat in the village, some people even here [referring to the group], they treat it.The informants emphasizing treatment-seeking patterns reported thus:We get ourmedicine from wa sing'anga, here . If things do not work, then we go to the hospital where doctors try. (FGDs).we just use traditional medicines here. If we all fail, then we send them to the hospital. [Q: Do you have someone in this village that gives drugs from the hospital?] A: Yes, we have one, Mr. Sikaona, if he has medicine, that is why I am saying, when we fail we send to Mr. Sikaonga [referring to a CHW], then he knows what to do next.One informant reported: Normally, a healer would take between two and four days before a referral was made to a community health worker. It was common for both informants and participants to report this number of days. One key informant in the village said:It takes 2 days...3 days to 4 days and when you give a child treatment, you now see a child starts playing, now you know he/she is okay.In an interview with mothers whose children were admitted in hospital, it was common for them to mention this pattern. One informant aged 37 years old reported:We take him/her to the village doctor [referring to the CHW]... the reason why it takes days because we first go to the village doctor, that is why it takes days because he gives us a dose and then we go back to him, or go to the hospital, when there is no change... If it is Fansidar, we wait for four days, if she/he has not changed, we go back to the village doctor and the village doctor says, to go to the hospital. Then we lift the child to the hospital, that's all.The study showed that severe malaria was well described by the Nakonde residents and was consistent across all the age groups of participants and key informants. They were able to correctly define and differentiate simple from severe malaria .' Both participants and informants expressed high level of knowledge of symptoms of severe malaria 'ichinzekema', which was directly reflected in their definition of 'big' malaria and were able to report symptoms in pairs or in groups, which might appear one at a time, accompanied with high temperature. The studies among the Tumbuka of Lundazi in the Eastern part of Zambia ,18 preseThe study showed that caretakers recognise fever as being associated with malaria, they can determine severe and simple malaria and can differentiate between convulsions due to malaria and those due to epilepsy. In respect of rectal administration of drugs, it was found that local practices included rectal administration of local medications already exist. To determine whether caretakers then proceed to take the sick child to the health facility for follow up oral treatment, several factors were apparent. Firstly it is the mothers who would make this decision, secondly that health seeking behaviour follows a pattern of using the local traditional healers/CHWs and then, if no success is found then taking the child to the health facility.Mothers and other caretakers in Nakonde could also identify severe malaria with many other complications and distinguish between convulsions due to other conditions. For example, only convulsions that were accompanied by high fever were regarded as 'big malaria' or severe malaria. Tchinda and others  in theirWhile the majority of participants and informants perceived malaria to be an inevitable part of life in Nakonde others believed the causes for symptoms included evil spirits. This has implications for the introduction of home-based management of malaria since the casting out of evil spirits will delay seeking of biomedical treatment.In this study, investigations were made on whether there were any local medicines that were administered rectally to treat acute illness. During the in-depth interviews, it was shown that, although rectally administered medicines were not used for treatment of severe malaria in the Nakonde community; it was common for rectal medicines to be used for treating sores/wounds resulting from episode/s of severe diarrhea. The rectal medicines were (Inkula), local herbs and some backs of trees, like ]. Inkula was widely used in the Northern and most of the Eastern part of Zambia. Inkula is sometimes called cassia in English.Treating 'Ilonda', sometimes a child was made to sit in cold or warm water in which the medicine had been soaking. It was believed medicines soaked in water would go in the wound and treat it. The other interesting finding from this study was that healers were able to administer rectal medicines using syringes that were bought from chemist shops in Tanzania. Medicines that were in powdered form could be injected in the child's rectum. Healers, who could not afford syringes, used local materials such as straws to inject the rectal medicines into the children's rectum. With this background, introducing rectal artesunate in the Nakonde community might not face any resistance, as people were already administering some of their medicines in the same way as the modern rectal artesunate.We believe that the most important element in the intervention would be to provide accurate and correct information to caretakers. Other studies ,27, had CHWs have been identified as the first port of call, along with the indigenous healers, once home treatment had been given. However the lack of essential drugs available to these workers contributes to delay in providing appropriate treatment. This study suggests that the provision of artesunate rectal suppositories to these health workers for the initial management of malaria patients who were not able to take medication by mouth would also reduce case fatality.In this study, results revealed that a woman had a right to decide when to take a sick child to the health facility. According to these findings, a father was only consulted in order to provide financial and material support, which enabled a mother to use services from specialised health facilities. This finding is consistent with a study by Kaona et al , which iYoung women in particular, felt very strongly that a severely sick child should never be kept at home or would certainly die. They appreciated the fact that early treatment would help the family avoid expending the family's' meager resources. Moreover, participants and informants categorically stated that children affected by severe malaria should be quickly referred to the health centres. Residents were deeply concerned that even the health facilities run without the qualified staff. If emergency treatment has been given prior to arrival at the health facility then unqualified staff can provide the oral treatment required to complete the cure.The people of Nakonde commonly manage malaria episodes at household level initially. This is consistent with similar studies in other parts of Africa ,26,25. BIt was established in this study, that the family was able to use various methods including sponging which was most common. Applications of these medicines varied though most of the respondents mentioned preparations through mixing with the child's porridge and drinks. Situations like this, made mothers and other caretakers to rely heavily on the indigenous medicines from healers, which were not anti-malarial drugs. There is no doubt, therefore, that caretakers can be trusted to identify when they would need to give rectal artesunate. However, there will be need for some extra sensitisation for mothers.nankololwe, utunvumbe, imbozyo, isyoyo, inkowamatete and umupene wambuzi, as repellants for evil spirits. Sensitization messages must therefore address the need for the rectal artesunate to be administerd before evil spirits are expelled.The study by Savingry and colleagues , found t1. Lack of knowledge of the local language by the PI, could have made the research assistants fail to stress important issues during interviews. However, the Co-PI who comes from the area, managed to complement this.2. The study recruited younger men as research assistants, to interview elderly men and women. This could have affected the responses from some of the participants.3. The sampled individuals were purposively selected; these have led the study to generalize the findings.4. The nature of the qualitative study, does not allow a researcher to measure the significance of the occurrence of an event.Our findings showed that there is a precedent for rectal application of traditional medicine for childhood illness. Therefore rectal artesunate may be a well-received intervention in Nakonde District, provided effective sensitisation, to mothers and CHWs is given which will strengthen the health care delivery system at community level.The author(s) delclare that they have no competing interests.FADK the Principal author of this paper, assisted in the design of the project to be adapted to the country's situation. He was instrumental in data collection, supervision during data collection, analysis and the production of this manuscript. MT was PI to the project. She contributed to the design of the project, supervised all field activities including data collection, transcription and editing of typed manuscripts. She was instrumental in data analysis. She has worked closely with the Principal author in the production of the manuscript. Authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


P. falciparum.There is a need for improved targeting of antimalarial treatment if artemisinin combination therapy is to be successfully introduced in Africa. This study aimed to explore why malaria slides are requested and how their results guide treatment decisions in an area of low transmission of Outpatients attending a district hospital in a highland area of Tanzania were studied over a 3-week period. Clinical and social data were collected from patients who had been prescribed an antimalarial or sent for a malaria slide. Hospital slides were re-read later by research methods.P. falciparum but 96(48%) of the 201 slide-negative cases were treated for malaria anyway. In a logistic regression model, adults , a history of fever  and a longer travel time to the clinic  independently predicted the request for a malaria slide. Only a history of a cough predicted (negatively) the prescription of an antimalarial with a negative slide result . The sensitivity and specificity of hospital slide results were 50% and 96% respectively.Of 1,273 consultations 132(10%) were treated presumptively for malaria and 214(17%) were sent for a malaria slide; only 13(6%) of these were reported positive for Progress in targeting of antimalarials in low malaria transmission settings is likely to depend on consistent use of malaria microscopy and on the willingness of health workers to be guided by negative slide results. Further studies are needed to identify how this can be achieved. In Tanzania, as in a number of African countries, existing antimalarial treatment  is being replaced by artemesinin combination treatment (ACT) as first line treatment for non-severe malaria. However, at 5–10 times the cost of SP, its introduction creates an urgent need to review the diagnostic and treatment practices that have evolved over many years of cheap and safe antimalarials .Where there is no access to microscopy presumptive diagnosis of malaria  is widely practiced and results in a large degree of unnecessary use of antimalarials, especially at low transmission and in older children and adults in endemic areas . Where aNjombe town  is situated in the southern highlands of Tanzania at an altitude of between 1,800 and 1,920 metres and with a rainy season between November and May. Economic activity in the area consists of subsistence agriculture with some commercial farming of pyrethrum and coffee.P. falciparum transmission in the Njombe area as 'unstable or absent' [P. falciparum [The 'Malaria Assessment of Risk in Africa' initiative  classifies the level of  absent' . A survelciparum .Njombe District hospital serves the population of the town and surrounding villages, most of which are served by a dispensary without malaria microscopy. According to routinely collected data in 2003 there were 53,546 outpatient consultations, 19,812(37%) of which resulted in a diagnosis of malaria; 10,165 blood slides were examined in the hospital laboratory by 4 trained staff supported by 2 unqualified assistants. The mean number of slides/working day/slide reader was 10.6(SD 2.0). Malaria slides were charged at approximately $0.2 (U.S.) each for patients over the age of 5 years.All patients exiting from a medical out-patient consultation during a 3-week period and who had been prescribed an antimalarial or who had a request for a malaria slide were identified. Following consenting procedures, data were collected on age, sex, village of residence, distance and travel time to the clinic and the presence within the previous 2 days of cough, fever, vomiting or diarrhoea, 'body pain', or any other symptom. Possible indicators of socio-economic status were identified from local interviews and from opinions of researchers in Tanzania. Data on 8 items  were analysed for correlation; household possession of a bicycle or bed net were dropped due to poor correlation and the remaining factors were found to highly correlate  and added to produce a socio-economic score.Patients were identified on exit from the review consultation and any changes to their medication were noted. Any patient who appeared too ill to participate was referred back to the clinic medical staff and not included in the study.Data were collected by locally recruited research assistants who were not part of the normal hospital staff.P. falciparum asexual parasites per 200 leucocytes was counted on Giemsa-stained thick blood films. A slide was considered negative only after scanning 100 high power fields. All slides were read twice independently with a third reading if there was discordance, the majority result was accepted as final.Blood slide results were tracked at the end of each day and the results were recorded from the laboratory results register, although it was not possible to determine if there had been an error in transcribing results to the register. Blood slides in the hospital laboratory were stained with Giemsa and 50 high power thick film fields were examined before declaring a slide to be negative, with no system of reporting parasite densities. All clinic blood slides were marked with the patient study number and retained for later research-quality reading where the number of Verbal consent of outpatient staff and written consent from patients was obtained in all cases. Ethical approval for the study was obtained from the review committees of Kilimanjaro Christian Medical Centre, Tanzania and the London School of Tropical Medicine and Hygiene.Data were double-entered in Access  and statistical analysis was performed using STATA 8 .During the 3-week period of the study there were there were 1,273 outpatient attendances, 240(19%) of which resulted in treatment for malaria.  Of the 346 patients who were recruited into the study approximately half  were under the age of 15 years. Females predominated in the 186 patients over the age of 15 years .P. falciparum asexual parasites and an additional 35(16%) were reported as positive for P. falciparum gametocytes. No other Plasmodium species was reported. Twelve (92%) of the cases reported as positive for asexual parasites were treated with an antimalarial. Thirty-three (94%) of the 35 patients with gametocytaemia reported by the hospital laboratory were also treated with an antimalarial and all were reported as negative for asexual parasites. Since gametocytes do not cause human illness these cases have been classified as 'slide negative'.A blood slide was requested for 214 patients and all of these had a hospital laboratory slide result recorded. Thirteen (6%) of these were reported by the hospital laboratory as positive for P. falciparum asexual parasite densities of the 8 positive research slide results were 2 at <2000/μl, 2 at 2000–4,999/μl, and 4 at >5,000/μl. The research slide results identified only 3 positive slides for gametocytes, 2 of which were reported as negative by the hospital laboratory.The sensitivity and specificity of hospital slide results for asexual parasitaemia (using the research slide result as the reference) were 50% (95% CI 43–57) and 96% (95% CI 93–98) respectively, and positive and negative predictive values were 31% (95% CI 26–37) and 98% (95% CI 96–100) respectively. Five slides were judged to be unreadable . In a logistic regression model the request for a malaria slide (as compared to presumptive treatment with no slide request) was independently predicted by being aged 15 years or over , reporting a history of fever in the previous 48 hours  and reporting a longer travel time to the clinic . The request for a malaria slide request was not significantly associated with the socio-economic score (P = 0.26). were treated with an antimalarial only, 52(26%) with an antimalarial and an antibiotic, 68 (34%) with an antibiotic only, and 37(18%) with neither an antimalarial nor an antibiotic. Thus overall, 96(48%) of patients whose slide was reported as negative were treated with an antimalarial and of these 29(30%) did not report a fever within the previous 48 hours. Similarly, 47(36%) of those treated presumptively for malaria also denied having a fever within the previous 48 hours.A logistic regression model using the same factors listed in Table In this study almost 20% of all outpatients were diagnosed and treated for malaria and routine data reported this proportion as almost 40% in the previous year, suggesting a possible moderating influence of the study itself. Blood slides were not collected from presumptively treated cases for fear of influencing clinical practice, but even if twice as many of these were positive compared to those who were sent for a malaria slide, well over 90% of treated cases did not have malaria, a finding consistent with other studies  and, altHealth workers demonstrated unrealistic confidence in their ability to make a clinical diagnosis of malaria. Thus, as would be reasonable in a high transmission area, children tended to be treated presumptively. The failure to request a blood slide did not seem to be justified by excessive workload  and data were not collected on the influence of waiting times on the request for a slide. However, if a blood slide is requested it is logical to expect the result to guide treatment decisions but almost half of those with a negative result were treated with an antimalarial anyway. If the clinical suspicion of malaria is strong then this might override the slide result  but 30% of slide-negative cases treated for malaria did not report a recent history of a fever. When requested, it was not clear if slide results were being used to confirm or exclude malaria; while children were almost 4 times less likely than adults to be slide-tested, those sent for a slide were more likely to have reported a recent history of fever than those treated presumptively.Our results suggest that malaria slides in clinics may fulfil a social or ritual function. In this study, a slide request was more likely if patients had travelled further (suggesting patient motivation to attend a facility where microscopy was available) although there was no association with socio-economic status suggesting that the cost of the slide does not play a major role. Clearly, for health workers, malaria is a convenient and acceptable label for non-specific illness but little is known about the understanding of malaria that leads to such practice and whether the phenomenon is driven more by patients or health workers.Presumptive diagnosis of malaria is an effective strategy to increase coverage of antimalarials in high transmission areas where malaria is common, the risk of progression to severe malaria is significant, and diagnostic facilities are lacking. In low transmission areas the justification for presumptive diagnosis is much less clear. Current Malaria Guidelines in Tanzania do not vThe accuracy of slide reading in this study was low although the negative predictive value  was still in excess of 95%, providing a high likelihood that a decision to withhold an antimalarial on the basis of a negative slide would be correct. In spite of this there is clearly an urgent need to improve laboratory standards; Bates et al. have shoReducing the overuse of antimalarials is an important and complex challenge. The criteria that are used to request a malaria slide are inconsistent and, where requested, negative results are often disregarded. If RDTs are introduced on a wide scale in Africa similar questions regarding consistent use, quality control and use of the results will need to be addressed.HR and CD conceived of the study and its design and drafted the manuscript. JR coordinated the data collection and OM analysed the data; both JR and OM made critical comments to the manuscript.


Malaria is one of the strongest recent selective pressures on the human genome, as evidenced by the high levels of varying haemoglobinopathies in human populations–despite the increased risk of mortality in the homozygous states. Previously, functional polymorphisms of Hp, coded by the co-dominant alleles Hp1 and Hp2, have been variously associated with several infectious diseases, including malaria susceptibility.Risk of a clinical malarial episode over the course of a malarial transmission season was assessed using active surveillance in a cohort of Gambian children aged 10–72 months. We report for the first time that the major haplotype for the A-61C mutant allele in the promoter of haptoglobin (Hp)–an acute phase protein that clears haemoglobin released from haemolysis of red cells–is associated with protection from malarial infection in older children,  . Protection was also observed using two other definitions, including temperature >37.5°C, dipstick positive, plus clinical judgement of malaria blinded to dipstick result . A similar level of protection was observed for the known protective genetic variant, sickle cell trait (HbAS).in vitro studies. Decreased Hp expression may lead to increased oxidant stress and increased red cell turnover, and facilitate the development of acquired immunity, similar to a mechanism suggested for sickle cell trait.We propose that previous conflicting results between Hp phenotypes/genotypes and malaria susceptibility may be explained by differing prevalence of the A-61C SNP in the populations studied, which we found to be highly associated with the Hp2 allele. We report the -61C allele to be associated with decreased Hp protein levels (independent of Hp phenotype), confirming  Haptoglobin (Hp) is an acute phase protein that binds haemoglobin released during the intravascular lysis of erythrocytes. Cell-free plasma haemoglobin is a potent pro-oxidant and haptoglobin is thought to be important in removing it from circulation and recycling the iron component for erythropoeisis via the reticulo-endothelial system and binding of CD163 on circulating monocytes and macrophages Haptoglobin phenotypes and genotypes have been variously reported to be associated with a range of disease conditions, reviewed by McDermid et al in vitroIn humans, Hp is polymorphic with two co-dominant alleles, Hp1 and Hp2 encoded by a single gene on chromosome 16, resulting in three phenotypes Hp11, Hp12 and Hp22. Unlike Hp11 and Hp12, Hp22 exists as large circular polymers, whilst Hp12 phenotypes form a variety of polymer formations. Hp22 is reported to have decreased binding affinity for free haemoglobin A fourth phenotype, Hp0 (i.e. no detectable circulating haptoglobin) has been reported in various African populations. In cross-sectional studies, the occurrence of ahaptoglobinaemia has been associated with malaria endemicity In the Japanese a genetic basis of ahaptoglobinaemia has been identified as the homozygous inheritance of a deletion spanning the entire Hp gene (Hp-del) Ethical permission for the study was granted by the Gambian Government and Medical Research Council Ethics Committee, and Gambian National DNA Collection Guidelines were followed regarding the handling of genetic material and information. Parental written informed consent was obtained for all study participants.The main study described consists of a longitudinal study with active surveillance for malaria and febrile episodes across the malaria transmission season in 2003, in children aged 10–72 months. This was conducted in eight rural villages in the West Kiang District of The Gambia, including the village of Keneba in which the Medical Research Council (MRC) clinic and laboratories are situated. Additionally two cross-sectional surveys of malarial parasitaemia prevalence were conducted, before (N = 873) and after (N = 801) the malaria transmission season in July and December 2003.At the cross-sectional surveys, children found to have anaemia (Hb<80 g/l) were treated with iron sulphate supplements (30 mg/day for 30 days), whilst children found to have malaria parasites of any density were treated with chloroquine and suphadoxine-pyrimethamine as per Gambian government treatment guidelines. The definition of malaria used for the cross-sectional survey was slide-confirmed parasitaemia of any density.The longitudinal surveillance study enrolled 889 children. Children were not eligible if they were known to have sickle cell disease or were found to have protein-energy malnutrition as determined by weight-for-height z-score of less than minus three (such children were referred to the Keneba supplement centre for treatment). DNA samples were available from 735 of the enrolled children. One child later found to be homozygous for HbS was excluded from the final analysis. Complete genetic and clinical follow up data were available for 599 children, whilst 1 further child was excluded from the final analysis as lost to follow up after only 1 week. Village assistants measured children's temperatures twice weekly and children with recorded temperature (>37.5°C) were seen by the study clinician at a twice weekly mobile clinic in each village. In addition, mothers were encouraged to bring their children to the mobile clinic if they felt their children had fever or was unwell. Those children with an increased temperature (>37.5°C axilla) or whose mothers reported a history of fever in the previous 48 hours were tested for malaria infection using a dipstick . Children with positive dipstick results had a 4 ml blood sample collected and parasitaemia confirmed by microscopy (12 out of the 271 positive dipstick tests were negative by slide result). Three different definitions were used to define malarial episodes: –(i) recorded temperature >37.5°C within the previous 3 days, plus a positive malarial dipstick, confirmed by microscopy to have asexual stage parasites >500/ul of blood (N = 255); (ii) as for (i) but including all those that were dipstick positive (N = 271) (iii) dipstick positive with a recorded temperature >37.5°C within the previous 3 days, plus clinical judgement of malaria by the study clinician, initially blinded to the dipstick result (N = 167). Malaria as a cause of subsequent febrile episodes was determined based on clinical judgement and few were confirmed by dipstick or microscopy. All children seen by the study clinician at these clinics received treatment as appropriate, either in the field or at MRC Keneba clinic and free of charge. Thus all first malaria episodes were detected and confirmed by microscopy.et al.et al.Blood films were stained with Field's and Giemsa, and examined for malaria parasites according to standard methods. Frozen plasma samples were analysed at the MRC Human Nutrition Research Laboratories Cambridge, UK. Plasma haptoglobin and CRP concentrations were measured by immunoturbidimetry, Tina-quant on a Hitachi 912 Bioanalyser . DNA was extracted from peripheral blood leukocytes using standard methods ® MassARRAY®  was used to genotype the following SNPs according to manufacturer's instructions: Haptoglobin promoter SNP's A-61C (rs5471) and C-101G (rs5470), HbS (rs334: haemoglobin–sickle), plus the A-376G (rs1050829) SNP in G6PD (glucose-6-phosphate dehydrogenase) resulting in mild enzyme deficiency, type A phenotype Sequenom® MassARRAY®. This confirmed that the assay was able to correctly identify the AA, AC and CC genotypes (data not shown).Due to the unexpected lack of homozygotes for the -61C allele of the Hp promoter SNP see . PositivHaplotypes for the two Hp promoter SNPs and the haptoglobin gene alleles–Hp1&Hp2–were constructed using SNPHAP 1.1 Analyses were conducted using STATA version 9 . Associations between genotypes were tested using Pearson's Chi-square test and binomial regression, which was also used to determine whether alleles were geographically clustered.Analysis of associations between Hp haplotypes, HbS and G6PD genotypes and the prevalence of malarial parasitaemia (any density) at the cross sectional surveys, was performed using logistic regression controlling for age, sex and village. Analysis of associations between Hp haplotypes, HbS and G6PD genotypes and risk of at least one malarial episode, assessed in the longitudinal cohort, was performed by both Cox-regression–time to first malarial event, and logistic regression, whereby children were classified as having had one or more malarial episodes or none. Children who did not have data for all of the genes tested were not included in this analysis. The mean follow up time of the 598 children included in the analysis was 99 days (SD 9.6) whilst the minimum and maximum follow up times were 21 and 102 days. No material differences were observed between models using Cox-regression time to first event or logistic regression. Hence, here we report results of the logistic regression models. Age, sex and village were included in all models a-priori. Hp haplotypes were fitted as a dose effect, coded by the number of copies . Thereafter if a haplotype was found to be associated, we attempted to test for a dominance effect (i.e. to test if the effect per copy was linear or differed in the heterozygote or homozygote condition). However, the small numbers of individuals with more than 1 copy of the relevant haplotype resulted in break-down of the assumptions of the model and the results were uninterpretable. Children who were found to be HbSS were excluded from analyses and therefore sickle genotype was fitted as a simple binary variable, whilst G6PD A376-G was fitted taking into account the phenotypic affects of sex-linkage by coding males with one copy of the mutant 376-G allele (G6PD AZ) and females with two copies of the 376G allele (G6PD AA), similarly.Malarial parasitaemia at the pre-season cross-sectional survey was assessed but not found to be associated with risk of subsequent malaria during surveillance.Comparison of concentrations of plasma haptoglobin (log-transformed) measured at malaria episodes and at convalescence was assessed using a paired student's t-test with levels less than the minimum detectable level  assigned a value of 10 ng/dl. However, a considerable proportion of samples, especially at convalescence were below the MDL. Therefore further analyses were conducted of the proportions of samples above and below the MDL–at malaria and convalescence–and associations with Hp haplotypes and genotypes using multiple logistic regression.Genotypes were available for between 665 (haptoglobin) and 755 (G6PD–G376-A) children for each of the six genes tested. The overall prevalence of the genotypes for each gene did not vary between the total number and the 598 children included in the final analysis of the longitudinal study.The prevalence of the different genotypes for the 598 children included in the final analysis of the longitudinal study is presented in Haplotypes were constructed for the haptoglobin gene and the two haptoglobin promoter SNPs. Four common and two uncommon haplotypes were detected Table 2.Multiple binomial regression analysis of the Hp 2 allele revealed geographic clustering in some villages (2/9) plus independent associations with the number of -61C and -101G alleles  and also G6PD deficiency, whereby male hemizygotes (AZ) and female homozygotes (AA) were less likely to have the Hp2 allele . No associations between the haptoglobin promoter SNPs and HbS and G6PD deficiency alleles were detected.The prevalence of malarial parasitaemia was 8.6% at both the pre-season (50/585) and post-season surveys (49/568), limited to those with Hp haplotype data available. There were no differences in the prevalence of malaria between individuals with and without complete genotype data available (data not shown). At the pre-season cross-sectional survey, in multiple logistic regression, controlling for age (grouped in years), sex and village, the prevalence of malarial parasitaemia (any density) was significantly decreased in individuals with 1 copy of haplotype “D” . As no homozygotes for -61C were detected there were no individuals with two copies of this haplotype. No other genotype associations were detected at this time point. At the post-season survey there was no evidence for an association with malaria for any of the haplotypes or with HbAS or G6PD genotypes.Of children enrolled in the longitudinal study, 598 children had complete genotype data, of which 255 were detected as having at least one malarial episode as described previously, with a parasite density greater than 500 asexual parasites/ul. Of these children, only three were recorded as having a subsequent malarial episode–as determined by clinical judgement by the study clinician. Sex and age groups were evenly distributed amongst the 598 children included in the final analysis.Children with one copy of Hp haplotype “D”  had a significantly decreased risk of having a malarial episode (definition (i) >500 parasites/ul and history of or current temp >37.5°C) , results from the multivariate analysis were similar for haplotype “D”  and convalescence  in individuals with Hp haplotype data available. Alternatively, samples with concentrations less than this detection limit were coded as being “hypo-haptoglobinaemic”. Contrary to previous reports, the geometric mean concentration of haptoglobin was increased at malaria compared to convalescence . Additionally, the prevalence of hypo-haptoglobinaemia  was significantly increased at convalescence compared to malarial episodes 54.9% vs 30.0%, . As expected, the frequencies of haplotypes D and E differed between the group who had a malarial episode and were thus included in analyses of Hp concentrations and the group in analysis of malarial risk. Haplotype prevalence was 10.3% and 14.2% for D and E haplotypes in analyses of haptoglobin concentration compared to 13% for both haplotypes in analysis of malaria susceptibility.Interestingly, different haplotypes were associated with the risk of hypo-haptoglobinaemia at malaria and at convalescence. During malaria, the only haplotype to be associated was haplotype “A” , associated with a decreased risk  Table 4.There was no evidence of an association of HbAS or with G6PD deficiency with hypo-haptoglobinaemia at either time point.We report for the first time, that the A-61C Hp promoter SNP is associated with protection from clinical malaria and that this effect is limited to older children, in this analysis, children between 36 and 72 months. We also confirm the previous report that this SNP is highly associated with the Hp2 allele The A-61C SNP has been demonstrated to cause decreased promoter activity in human HepG2 cells and decreased responsiveness to IL-6 MODThe -61C allele has been found to be the cause of a “modified” phenotype of Hp as assessed in gel electrophoresis as Hp12It is not clear why, if there are significant benefits of the heterozygous state, we did not observe any homozygotes for the base substitution. In order to rule out methodological error, we retested the ability of the Sequenom method to detect the -61CC genotype using cloned -61C or -61A DNA. In all of the samples tested, the results of the Sequenome method matched those from restriction digests, suggesting that the lack of detection of -61CC homozygotes was not due to methodological error (data not shown). Based on the current results, Hardy Weinberg would predict 12 individuals homozygote for -61C out of the 750 tested, suggesting a balanced polymorphism similar to HbS in which the homozygous inheritance of the -61CC is potentially lethal. In support of this supposition we have not detected any -61CC homozygotes in a large cohort of Tanzanian women and in another cohort of Gambian children (unpublished data). We plan to test a set of umbilical samples to determine if this genotype can be detected in a birth cohort. However, one female homozygote for -61CC out of 17 individuals found to be ahaptoglobinaemic has been reported from 123 tested In conclusion, we report an age-dependent protective effect of the -61C allele-associated Hp haplotype against symptomatic malaria in Gambian children. We suggest that the mechanism of protection may be similar to that recently proposed for sickle cell trait and are currently investigating if this haplotype is associated with increased oxidant stress, as a critical component of the proposed mechanism of action.Table S1Haptoglobin Hp1/Hp2-PCR conditions and primer sequences-method adapted from Koch et al. 2003(0.02 MB XLS)Click here for additional data file.Table S2Haptoglobin deletion assay-PCR conditions and primer sequences-method adapted from Koda et al. 2000(0.02 MB XLS)Click here for additional data file.Table S3First Round and Extension Primers for G6PD, HbS, A-61C and C-101G(0.02 MB XLS)Click here for additional data file.Figure S1Bpm1 restriction digest of plasmid colonies(0.22 MB TIF)Click here for additional data file.


Estimating the burden of malaria in Sudan is important for evidence-based planning of malaria control. Estimates of malaria burden in terms of DALYs (Disability Adjusted Life Years) were not developed locally. This study synthesized information from different sources to calculate malaria incidence, mortality and DALYs lost in Sudan in 2002.A search for local studies and reports providing epidemiological data on malaria in Sudan was conducted. Preliminary estimates of incidence rate, case fatality rate and mortality rate were developed from the data found. The preliminary estimates were processed in the disease modelling computer software, DisMod II, to produce internally consistent mortality and incidence rates, which were used to calculate DALYs lost due to malaria.Malaria incidence in Sudan was estimated to be about 9 million episodes in 2002 and the number of deaths due to malaria was about 44,000. 2,877,000 DALYs were lost in Sudan in 2002 due to malaria mortality, episodes, anaemia and neurological sequelae. Children under five years of age had the highest burden. Males had the highest incidence and mortality, but females lost more DALYs.Formal health system data underestimated malaria burden. The burden estimates can be useful in informing decision making, although uncertainty around them needs to be quantified. Epidemiological research is needed to fill data gaps and update the estimates. Estimating the burden of malaria is highly needed for evidence based planning of malaria control. In Sudan, malaria has been the subject of a large amount of epidemiological, entomological and biomedical research. Malaria surveillance, as part of the general reporting of health events from health facilities or specific surveillance for epidemic preparedness, provided a wide range of information. This resulted in multiple and diverse sources of information about malaria burden in Sudan, each source serving the purpose for which it was established. The problems with these sources are non-representativeness, variability of the sensitivity and specificity of the diagnostic criteria used and variability of the indices measured. Some may suffer from underreporting. These sources, therefore, could not directly provide a single valid national indicator of malaria burden. Official figures of incidence and mortality reflected cases reported only from the formal health system. Malaria health burden results from both disability and mortality, so that multiple statistics are needed to describe it. DALYs (Disability Adjusted Life Years) are composite indicators that summarize disability and mortality information in a single number . The DALNo local efforts have previously been made to measure the burden of malaria in terms of DALYs in Sudan, using locally-derived epidemiological data. As part of a project to measure the burden of diseases in Sudan, this study synthesized information about the epidemiology of malaria from different sources to calculate national incidence rate, mortality rate and DALYs lost due to malaria.The study measured the burden of malaria in 2002 for all Sudan. The burden was attributed to mortality and disability from malaria episodes, anaemia and neurological sequelae, as defined in the GBD 2002 , and calRelevant published and unpublished studies were found by electronic search in local databases and Pubmed, using the keywords: malaria, Sudan, Africa, case fatality, age distribution, sex distribution, anaemia, cerebral malaria, and neurological sequelae. Postgraduate thesis archives were hand-searched for thesis not indexed in local databases. Research institutes, governmental and non-governmental organizations were contacted and relevant reports obtained. Reference lists were examined for more research papers. Studies and reports were relevant if they provided at least one of the following: incidence, prevalence, mortality rate, case fatality ratio, age or sex distribution of cases or deaths, remission proportion, average duration and disability proportions. There was no restriction regarding the time of study. Studies identified were reviewed systematically and those selected for use in the calculations were the most representative and with the best methodological quality where applicable.The total population by age and sex for Sudan in 2002 was interpolated from the 1998 and 2003 population projections of the Sudan Central Bureau of Statistics . Age- anFigure Figure Age-sex specific preliminary incidence, mortality and case fatality rates were processed in DisMod II. This is a disease modelling software that uses input estimates to calculate an internally consistent set of epidemiological estimates . The outAdjusted odds ratios of cerebral malaria  were useThe age-specific proportions of severe anaemia amongst severe malaria cases were calculated using adjusted odds ratios of severe anaemia in the same way as neurological sequelae. Studies reported the ratio of severe anaemia to mild and moderate anaemia -22, and According to GBD methods , DisMod • YLL = number of deaths X standard life expectancy at the average age of death• YLD = number of incident cases/episodes X average duration X disability weights• DALY = YLL + YLDStandard life expectancies of the GBD 1990  were useTable In both males and females the highest incidence was in children 5 – 14 years of age. It declined afterwards and increased again in males in the age group 45 – 59. The incidence was higher in males than in females in all age groups. These age and sex patterns reflect the original patterns in the studies used in estimation of the preliminary incidence rates.The mortality rate of malaria was the same in males and females Table . This waThe total number of deaths in 2002 was estimated to be about 44,000, far exceeding the numbers reported from the formal health system  in the same year . This waTotal DALYs and DALYs/1000 by age and sex are shown in Table The comparability of the results to results of similar studies in other countries was limited by the different methods, case definitions and disability weights usedDisMod II ensured that the estimates used in the final burden calculations were internally consistent. However, large discrepancies were previously found when an empirical data set was compared to DisMod II results and were attributed to data inaccuracies and past disease trends . This emThe validity of the results of this study depends on the validity of the data used in their synthesis. Although the best sources available were used, inadequate reports sometimes limited proper study appraisal. In some instances, no single study proved to be the best or most representative, and the results of all the available studies were summarized in one estimate.The data sources used ranged from health facility based studies to nationally representative population based studies. The former were not representative of the population concerned and some population based studies, NGO surveillance data and national surveys did not cover all the country. Some figures were borrowed from studies in other African countries. However, non representative and non local studies were resorted to only where no local estimates could be found or were too unreliable to use.Sudan states were classified by endemicity level although administrative boundaries between states did not coincide with boundaries between the different endemicity areas, according to a climate-based distribution model .Most of the studies used were conducted before or after 2002. This could have affected the validity of the results because their use was based on the assumption of a stable epidemiological and demographic status, which cannot be assumed about malaria in Sudan.Information about neurological sequelae due to malaria was found only for children less than nine years of age. The findings were generalized from these groups to all age groups due the lack of studies on the other age groups. Findings from northern Sudan were generalized to southern Sudan for the same reason.Disability weights reflect societal preferences for health states. In spite of the extensive analysis in the GBD to ensure that the disability weights developed were highly reliable , they arThe search conducted in this study revealed the lack of representative and acceptably valid mortality data as well as representative data on case fatality, remission and complications of malaria in Sudan.The study revealed underreporting of malaria episodes and deaths to the formal health system, with the consequent underestimation of malaria burden. Children less than five years of age had the highest mortality rate and DALYs, emphasizing the known effect of malaria on this population group. Females lost more DALYs than males in all age groups, which altered the picture displayed by the incidence rates alone. The epidemiological estimates and DALYs calculations in this study form a basis for comparing interventions that affect mortality and morbidity differently, by comparing the amount of burden averted by them. The DALYs would mark the position of malaria among the rest of the diseases, if compared to DALYs due to other diseases. Uncertainty around the estimates should be considered when using them for decision making and further work should quantify this uncertainty to facilitate utilisation of the results. The data gaps revealed in this study call for more epidemiological research to fill them. This will allow regular updating of the burden estimates by replicating the calculations using updated input data.The author(s) declare that they have no competing interests.SIA designed the methodology, supervised and participated in the data collection and performed the calculations. EM and KMA revised and endorsed the methodology and the results. All authors read and approved the final manuscript.


Understanding the pathogenesis of malaria in pregnancy and its consequences for both the mother and the baby is fundamental for improving malaria control in pregnant women.The study aimed to investigate the role of ABO blood groups on pregnancy outcomes in an area of unstable malaria transmission in eastern Sudan.A total of 293 women delivering in New Half teaching hospital, eastern Sudan during the period October 2006–March 2007 have been analyzed. ABO blood groups were determined and placental histopathology examinations for malaria were performed. Birth and placental weight were recorded and maternal haemoglobin was measured.P = 0.9), parity  and placental malaria infections. In all parity blood group O was associated with a higher risk of past  placental malaria infection. This was also true when primiparae were considered separately .114 (39.7%), 61 (22.1%) and 118 (38.2%) women were primiparae, secundiparae and multiparae, respectively. The ABO blood group distribution was 82(A), 59 (B), 24 (AB) and 128 (O). Placental histopathology showed acute placental malaria infections in 6 (2%), chronic infections in 6 (2%), 82 (28.0%) of the placentae showed past infection and 199 (68.0%) showed no infection. There was no association between the age (OR = 1.02, 95% CI = 0.45–2.2; Among women with all placental infections/past placental infection, the mean haemoglobin was higher in women with the blood group O, but the mean birth weight, foeto-placental weight ratio was not different between these groups and the non-O group.These results indicate that women of eastern Sudan are at risk for placental malaria infection irrespective to their age or parity. Those women with blood group O were at higher risk of past placental malaria infection. It has been estimated that 90% of the global malaria burden occurs in sub-Saharan Africa, where 40% women are exposed to malaria infections during pregnancy . MalariaPlasmodium falciparum preferentially trigger rosette formation depending on the red blood cell blood group [P. falciparum infections during pregnancy [Host genetic factors modulate the risk and severity of infection via specific mediators, which can be different in various epidemiological settings. Cell adhesion plays a fundamental role in placental malaria pathophysiology . Cell suod group . Howeverregnancy .Recently, ABO blood groups have been reported to be one of the host risk factors for placental malaria infections and they are associated with other materno-foetal outcomes . The curThe study was conducted between October 2006 and March 2007, at the labour ward of New Halfa Teaching Hospital, eastern Sudan. After informed consent, women with a singleton baby were approached to participate in the study. Those with antepartum haemorrhage, hypertensive disorder of pregnancy (diastolic blood pressure > 90 mm Hg) and diabetes mellitus were excluded. A structured questionnaire was administered to collect information about socio-demographic characteristics, mode of delivery and birth outcome. Obstetrical examinations were performed and the findings recorded . The babies were weighted immediately following birth on a Salter scale to the nearest 10 g; scales were checked for accuracy on a weekly basis.P. falciparum parasites per 200 white blood cells was counted and double-checked blindly by an expert microscopist. Maternal haemoglobin concentrations were estimated by HemoCue haemoglobinometer . Maternal blood groups were investigated by the agglutination method.Maternal, placental and cord blood films were prepared, the slides were Giemsa-stained and the number of asexual et al [Full thickness placental blocks of around 2–3 cm were taken from the placenta, kept in neutral buffer formalin for histopathology examinations. Placental malaria infections were characterized based on the classification of Bulmer et al : uninfect-test, ANOVA  or by the Mann-Whitney test . X2 and Fisher's exact tests as appropriate. Multiple linear regressions were used to analyse placental malaria infections as a dependent variable and age ≤ 19 years, parity  and maternal blood group (O versus non O) as possible influencing factors. The others factors included in the analyses were: maternal blood group (O versus non O), parity, placental infection, maternal haemoglobin, birth weight and foeto-placental ration. P ≤ 0.05 was regarded as significant.Data were entered in computer using SPSS for windows and double-checked before analysis. Means and percentages were compared either by Students' The study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum.During the study period, there were 578 deliveries. Of these, 24 were twins and four were triplets, 550 singleton deliveries. The data of placental histopathology, maternal haemoglobin, placental and birth weight were complete in 293 women; these data were included in the final analyses. Out of these 293 women, there were 114 (39.7%), 61 (22.1%) and 118 (38.2%) primiparae, secundiparae and multiparae, respectively. The mean (SD) age was 25.6 (6.4) years and 57(19.4%) of the women were teenagers (age ≤ 19 years). Of the 293 women, 82 (28.0%), 59 (20.1%), 24 (8.2%) and 128 (43.7%) had blood group A, B, AB and O, respectively. The mean (SD) haemoglobin was 11. 4 (2.1) gm/dl. The mean (SD) birthweight was 3,063.4 (545.4) gm, while the mean foeto/placental ratio was 5.6 (1.2). Maternal and placental blood films were positive in four cases and the blood films for malaria were positive in one maternal, placental and cord setting.P = 0.9), parity  and placental infections .Six (2%), 6 (2%) and 82 (28.0%) of the placentae showed acute, chronic, past infection on histopathology examination respectively, while 199 (68.0%) of them showed no infection. There were no associations between the age . This was also true when primiparae were considered separately  of 51 primiparae women with group O versus 10 (15.9%) of 63 with non-group O, P = 0.05,  of 128 women with blood group O versus 37 (22.4%) of 165 with non-O group, % of 128 The mean birth weight and foeto-placental ratio were not significantly different between those with and without placental malaria infections or those women with blood group O and non-O group, Table .P = 0.02), this difference was statistically significant even when primiparae were considered separately ,  with blood group O were at higher risk for past placental malaria infections. While significantly higher haemoglobin was observed in women with past placental malaria and blood group O, the other parameters  were not different between the two groups. Surprisingly, in this study the birthweight was not associated with histopathological placental malaria infections and the explanation for this remains unclear. These women had very low prevalence of peripheral, placental and cord microscopically detected parasitaemia, hence the role of submicroscopic parasitaemia on low birthweight cannot be excluded. Previously, a large burden of submicroscopically-detected parasitaemia was observed to be associated with morbidity and mortality among pregnant and non-pregnant individuals in the same geographical area ,16.P. falciparum infection. It was established that parasitized erythrocytes form rosettes more readily with red blood cells (RBCs) of A, B, or AB groups than with blood group O and this parasite-triggered RBC rosette formation is associated with the severity of clinical disease and with the development of cerebral malaria [P. falciparum leading to changes in immune response [Recently, Loscertales and Brabin  have obs malaria ,18. Preg malaria  – and a  malaria . Furtherresponse  or facilresponse .P. falciparum malaria and non-O blood group had significantly lower haemoglobin levels compared with patients with group O blood [In the current study, women with past placental infection had a higher mean haemoglobin when they were of O blood group. This correlates with the previous findings where non-pregnant patients with severe  O blood . The hig O blood  have alsThese results indicate that women of eastern Sudan are at risk for placental malaria infection irrespective to their age or parity. Those with blood group O were at higher risk of past-chronic placental malaria infection. Among women with past-chronic placental infection, the mean haemoglobin was higher in women with the O blood group, but the mean birthweight and foeto-placental weight ratio were not significantly different between these groups.IA and SB carried out the study and participated in the statistical analysis and procedures, ZMZ participated in the statistical analysis, MP coordinated and participated in the design of the study, statistical analysis and the drafting of the manuscript. AM and MS participated in the lab work. All the authors read and approved the final version.


Malaria and hypertension are major causes of maternal mortality in tropical countries, especially during first pregnancies, but evidence for a relationship between these syndromes is contradictory.In a cross-sectional survey of Tanzanian parturients, the rate of hypertension was similar in placental malaria (PM)-positive (11/85 = 13%) and PM-negative (73/602 = 12%) individuals. However, we found that PM was associated with hypertension in first-time mothers aged 18–20 y but not other mothers. Hypertension was also associated with histologic features of chronic malaria, which is common in first-time mothers. Levels of soluble vascular endothelial growth factor receptor 1 (sVEGFR1), a preeclampsia biomarker, were elevated in first-time mothers with either PM, hypertension, or both, but levels were not elevated in other mothers with these conditions. In first-time mothers with PM, the inflammatory mediator vascular endothelial growth factor (VEGF) was localized to maternal macrophages in the placenta, while sVEGFR1, its soluble inhibitor, was localized to the fetal trophoblast.The data suggest that maternal–fetal conflict involving the VEGF pathway occurs during PM, and that sVEGFR1 may be involved in the relationship between chronic PM and hypertension in first-time mothers. Because placental inflammation causes poor fetal outcomes, we hypothesize that fetal mechanisms that promote sVEGFR1 expression may be under selective pressure during first pregnancies in malaria-endemic areas. Children of mothers with placental malaria were more likely to exhibit parasitemia within their first year. First-born children were at a lower risk, regardless of whether their mothers had placental malaria. Plasmodium falciparum. One of the most serious consequences of infection is that this parasite can multiply in the placenta of a pregnant woman. This “placental malaria” is very harmful to the mother and to the fetus; it leads to low birth weight and is estimated to be responsible for the deaths every year of about 200,000 babies within their first year of life. A woman who is pregnant for the first time is most likely to suffer from placental malaria, and to have her placenta become highly infected and extremely inflamed. If she later becomes pregnant again, she will be protected to some extent by antibodies she has developed against the parasite.Malaria is one of the world's most serious health problems. It causes about 1 million deaths every year, and most of these deaths are in children. Several different parasites can cause malaria; the most serious is   Another problem that is common in tropical countries and also causes many deaths during pregnancy is preeclampsia—high blood pressure (hypertension) and protein loss in the urine. This is also a condition that is most common in first-time mothers. The causes of preeclampsia are not clear, but many factors are probably involved. Among the theories that have been proposed are that inflammation in the placenta might play a part, and that there may be a “conflict” between the needs of the mother and those of the fetus.The researchers wanted to see whether placental malaria might be a factor in the development of preeclampsia. This association has been suggested before, but there has been no clear evidence.Working with pregnant women in Tanzania, they found that, overall, women with placental malaria were no more likely than other women to develop hypertension. However, for those women who were aged 18–20 and pregnant for the first time, having placental malaria was associated with hypertension. The researchers also measured levels of a substance called sVEGFR1 , which is known to increase before and during preeclampsia and is thus considered to be a biomarker for the condition. sVEGFR1 levels were high in first-time mothers with either placental malaria or hypertension, or both, but levels were not raised in other mothers with these conditions. A related substance, VEGF, which is known to be involved with the process that causes inflammation, was high in first-time mothers with placental malaria, but not in those who had preeclampsia alone.The researchers believe that their findings support the view that, in younger first-time mothers only, placental malaria can cause preeclampsia and that this results from a conflict between the mother and her fetus. Action to reduce the chance of such women getting malaria would have the additional benefit of lowering their chance of developing preeclampsia. The findings have also led the researchers to propose possible mechanisms as to how placental malaria leads to preeclampsia. They have made suggestions regarding the further research that is now needed.http://dx.doi.org/10.1371/journal.pmed.0030446.Please access these Web sites via the online version of this summary at malariaWikipedia, an online encyclopedia that anyone can edit, has pages on The World Health Organization (WHO)UNICEF (the United Nations children's organization)MedlinePlus brings together authoritative information from the US National Library of Medicine, National Institutes of Health, and other government agencies and health-related organizations.The Wellcome Trust Plasmodium falciparum preferentially affects first-time mothers: rates of up to 70% occur in regions of sub-Saharan Africa, where low birth weight due to PM is estimated to cause 200,000 infant deaths each year. Among the dozens of malaria parasite species that infect mammals, only P. falciparum is known to sequester in the placenta during naturally occurring infections [Placental malaria (PM) caused by fections . During fections . First-tHypertensive disorders of pregnancy occur primarily in humans, and are estimated to cause 10%–15% of maternal deaths . PreeclaMalaria is well known to decrease blood pressure in non-pregnant individuals, but the relationship between PM and blood pressure is not clear . During Serum levels of placentally derived soluble vascular endothelial growth factor receptor 1 (sVEGFR1) are elevated prior to  and duriPlacental samples and clinical information were provided by Tanzanian women aged 18–45 y delivering at the Muheza Designated District Hospital, Muheza, Tanga region. Sample donors were among those recruited to participate in a birth cohort study known locally as the Mother-Offspring Malaria Study. Women provided signed informed consent before joining the study, and those with chronic debilitating disease and their children were excluded. Clinical information was collected by project nurses and assistant medical officers on standardized forms. Study procedures involving human participants were approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Board of Seattle Biomedical Research Institute and the National Medical Research Coordinating Committee in Tanzania.Hypertension was defined as systolic or diastolic blood pressure greater than or equal to 140 mm Hg or 90 mm Hg, respectively. Blood pressure was abstracted from hospital delivery cards as the maximum measurement recorded during the delivery hospitalization. Blood pressure measurements were not taken during active labor. Urine protein assessment was not routinely performed in the hospital. Immediately prior to delivery, peripheral blood was collected in citrate phosphate dextrose, and plasma was separated and frozen at −80 °C. The placenta was collected at delivery, and a full thickness biopsy from the middle third of the placental disc was frozen in liquid nitrogen and stored at −80 °C. PM was detected by microscopy of Giemsa-stained thick and thin smears of placental blood extracted from placental tissue by mechanical grinding. Placental parasite density was quantified as percent IE.Cryosections (5 μm) of placental tissue were Giemsa stained and assessed by examining greater than 90 60X fields per section. Malarial pigment is a brown heme-derived product that refracts under polarizing light and persists in acellular fibrinoid in the intervillous space. Pigment deposition in fibrinoid was quantified by determining the proportion of fields with pigment present. Inflammation was qualitatively scored by the presence of inflammatory cells in the intervillous space.Soluble VEGFR1 levels in peripheral plasma were determined in duplicate by ELISA kit DVR100A . Levels were corrected for dilution volume in anticoagulant, and over-diluted samples were detected by low potassium concentration as measured by EasyLyte Plus  and excluded.GGGGAAGAAATCCTCCAGAA(fwd), AGCCTTTTTGTTGCAGTGCT(rev), (63-bp product); VEGF : CCCACTGAGGAGTCCAACAT(fwd), TGCATTCACATTTGTTGTGC(rev), (106-bp product); and cytokeratin 7: GGCTGAGATCGACAACATCA(fwd), CTTGGCACGAGCATCCTT(rev) (103-bp product). Real-time PCR was performed in duplicate using SYBR green master mix and an ABI Prism 7000  with an annealing temperature of 60 °C. Threshold cycles (CT) were normalized to CT of KRT7, and t-tests performed on normalized CT values. Data are presented as fold-difference from control gene, calculated by log2.Total RNA was extracted from frozen cryosections using RNeasy minikits . RNA quality was assessed by Agilent 2100 Bioanalyzer , resulting in 28/18s ratios of 1.1 to 1.5. cDNA was synthesized using Superscript III enzyme  and anchored oligodT20 primers. Exon-spanning primers were designed for sVEGFR1:Cryosections (5 μm) were fixed in paraformaldehyde, blocked in chicken and goat sera , and variously probed with mouse anti-VEGFR1 extracellular domain (R&D Systems) at 1:400, which detects both the membrane-bound and soluble isoforms of VEGFR1, mouse anti-VEGF clone VG1  at 1:200 and rabbit anti-placental lactogen  at 1:1,000 dilution. Secondary antibodies included Alexafluor 488 chicken anti-mouse antibody  and TRITC goat anti rabbit antibody . DAPI (Sigma) was used to define nuclear DNA. VEGF was also visualized using DAB Envision + kit (Dako), and sections were counterstained with Giemsa.t-test was used for the analysis of continuous variables. Mann-Whitney test was used to examine parity and placental pigment deposition. Peripheral plasma sVEGFR1 concentration and parasite density (n + 1) were log-transformed prior to analysis. Odds ratios were calculated using logistic-regression analysis. Regression coefficients were calculated using simple regression analysis. Interactions between variables were determined using factorial ANOVA. Spearman rank correlation was used to examine the correlation between sVEGFR1 quantitative PCR and ELISA measurements.Analyses were performed using Statview . Student's We examined the relationship between PM and hypertension in northeastern Tanzania. The analysis protocol for the study was to examine the relationship of demographic factors including parity and age to hypertension in women stratified by PM status. In first-time mothers, who are at risk for both PM and preeclampsia, we examined the histological features of PM that were associated with hypertension. We examined the expression of sVEGFR1 in women stratified by parity, PM status, and hypertension.Placental samples and clinical information were provided by women delivering at the Muheza Designated District Hospital, in an area of intense malaria transmission . Analysep = 0.005). The interaction term (parity × PM) had a similar effect that was also significant (p = 0.030). When data were stratified by parity and median age of first-time mothers, PM-positive women were at significantly increased odds of hypertension compared with PM-negative women for first-time mothers aged 18–20 y , but not older first-time mothers , or mothers of later pregnancy .When women of all ages were analyzed in aggregate, blood pressure did not differ between PM-positive and PM-negative women , consistn = 32, p = 0.002). PM-positive first-time mothers with hypertension had significantly increased amounts of pigment compared with PM-positive first-time mothers without hypertension. Hypertension was associated with low placental parasite density, particularly in first-time mothers. Placental parasite density was negatively associated with diastolic blood pressure . Thus, hypertension in PM-positive women was associated with features of chronic disease, such as heavier pigment deposition and lower parasite density.We examined placental pigment deposition and placental parasite density in PM-positive women to determine whether chronic forms of PM were associated with hypertension . As prevp < 0.001, n = 49), and were significantly elevated in first-time mothers with PM, hypertension, or both. sVEGFR1 transcript levels were specifically elevated in tissue that had maternal inflammatory cells in the intervillous space  of peripheral blood smear diagnosis for detecting PM. Further, we do not know whether the normotensive women with elevated sVEGFR1 levels would have gone on to develop hypertension if they had not delivered.We observed VEGFR1 localization in the trophoblast but not in the maternal inflammatory cells. Trophoblast VEGFR1 expression has been reported ,27; howeP. falciparum malaria during human history may have contributed to the elevated sVEGFR1 levels observed in healthy first versus second pregnancies [The role of sVEGFR1 in healthy pregnancy is not known; however, VEGF administration in one study caused pregnancy loss in mice . VEGF ingnancies , and thegnancies –39 or Sognancies ,41 ancesIn conclusion, hypertension occurs in young first-time mothers with chronic malaria, and their elevated sVEGFR1 levels suggest they are suffering from preeclampsia. Clinical studies should address the relationship between peripheral parasitemia, sVEGFR1 levels, and blood pressure longitudinally over the course of gestation, and should examine the effects of VEGFR1 gene polymorphisms on these outcomes. Effective prevention of PM could lower the incidence of hypertension and should be examined in interventional trials.Table S1(28 KB DOC)Click here for additional data file.