Preparatory to a community trial investigating how best to deliver rectal artesunate as pre-referral treatment for severe malaria; local understanding, perceptions of signs/symptoms of severe malaria and treatment-seeking patterns for and barriers to seeking biomedical treatment were investigated.19 key informant interviews, 12 in-depth interviews and 14 focus group discussions targeting care-givers, opinion leaders, and formal and informal health care providers were conducted. Monthly fever episodes and danger signs or symptoms associated with severe malaria among under-fives were recorded. Respondents recognized convulsions, altered consciousness and coma, and were aware of their risks if not treated. But, these symptoms were perceived to be caused by supernatural forces, and traditional healers were identified as primary care providers. With some delay, mothers eventually visited a health facility when convulsions were part of the illness, despite pressures against this. Although vomiting and failure to eat/suck/drink were associated with malaria, they were not considered as indicators of danger signs unless combined with another more severe symptom. Study communities were familiar with rectal application of medicines.Communities' recognition and awareness of major symptoms of severe malaria could encourage action, but perceptions of their causes and poor discrimination of other danger signs – vomiting and failure to feed – might impede early treatment. An effective health education targeting parents/guardians, decision-makers/advisors, and formal and informal care providers might be a prerequisite for successful introduction of rectal artemisinins as an emergency treatment. Role of traditional healers in delivering such medication to the community should be explored. P. falciparum parasites from maturing to the more pathogenic sequestered stages, organ failure, and high risk of death (15–20%) for hospital admissions Most malaria deaths occur in children in rural areas of sub-Saharan Africa Reviews of research conducted to understand people's perception and treatment of fever - the primary symptom of malaria and other childhood illnesses non per os children with rectal artesunate as emergency treatment for children with such symptoms at the community level in rural Tanzania. The intervention is ongoing and its results will be presented separately. This paper reports, therefore, the findings of the formative study.As the rectal artemisinins are now recommended as emergency pre-referral treatment for children who cannot take drugs by mouth, in settings where parenteral treatment is not immediately accessible The study was conducted in Mtwara Rural district in Mtwara region south of Tanzania, which is mainly inhabited by the Makonde ethnic group. The district has a total population of 212,019 and approximately half of the population  does not have access to health facilities in close proximity. Malaria transmission in the district is perennial and falciparum malaria is the leading cause of outpatient attendances (53%) as well as in-patient (39%) attendances, mostly under-five children. Health care services include a regional hospital, 4 health centres and 30 dispensaries. Dispensaries treat uncomplicated malaria among other illnesses and refer severe cases to health centre or hospital. Trained village health workers (VHW) and Traditional Birth Attendances (TBA) deliver community based health services.A census of 67 out of 112 villages that do not have access to a dispensary or health facility in close proximity in Mtwara Rural district was conducted in 2004. Four rural communities/villages were purposively selected taking into consideration the geographic diversity of the district and accessibility to a health facility. Administratively, each village is divided in to 3–6 hamlets depending on the size of the village. The total population of the selected communities was 6915 of which 15.9% (n = 1099) were children under the age of 5 years (data from census).Qualitative approaches consisting of key informant interviews (KII), focus group discussions (FGD) and in-dept interviews (IDI) were used. Interviews and discussions were held by Kiswahili speaking research assistants and a social scientist.Using semi-structured and open-ended individual interviews, fifteen KII were involved in the study. The targeted respondents included health care providers , elders, opinion leaders/community leaders and parents of under-five children. The question guides for the KII directed them to record local diseases that could lead to death especially in children, local understanding of the signs and symptoms of malaria with special emphasis on severe illness suggestive of severe malaria, the underlying symptoms or illness-specific causes, patterns of symptom-specific actions and treatment seeking pathways by parent/guardians, factors causing delay in seeking care from health facilities and the decision making hierarchy at household level for seeking care outside the home. Local experience with use of rectal medicines was also investigated.Through KII and participants of FGD, traditional healers who attend severely ill children in their communities were identified. In each of the four study villages one traditional healer was interviewed as the local specialist in treating children with symptoms associated  with severe malaria. Traditional healer interviews were semi-structured and focused on exploring healers' descriptions of local illness and healing concepts, especially the associated Kimakonde terminologies, and treatment practices mentioned by community key informants and focus group participantsUsing guided themes, 14 FGD of 6–8 participants per group were conducted with homogenous groups deliberately chosen to represent those most directly involved in providing care and making treatment decisions for severely ill young children. Mothers, who currently had a child under the age of five years at the time of the study (young mothers' group), women over 45 years, who, in their roles as mothers, grandmothers, mothers-in-law, aunties, etc, exerted influence over mothers of under-fives (old mothers' group), fathers who had a child under the age of 5 years at the time of the survey and (fathers' group) and health workers of Kitere and Mahuranga Health Centres  serving the study communities were targeted. The content of FGD guide was similar to that provided to the key informants. Each FGD session took an average of 45 minutes.For the in-depth interviews (IDI), 12 mothers of under-five children whose children were admitted to the Regional Hospital with a diagnosis of severe malaria  were interviewed to obtain clinical history and treatment-seeking behavior for the illness. The interviews focused on (i) mother's recognition of signs and symptoms with which the child presented during the actual illness, (ii) mother's description of sequences of symptom presentations and of actions taken in seeking care during the child's present episode. Each IDI lasted on average 40 minutes. This group of mothers is referred to as “hospital mothers” in the paper.In addition to the qualitative survey, monthly surveillance of febrile episodes with or without danger signs or symptoms/signs suggestive of severe malaria  among under-five children in the study communities were recorded for 4 months using quantitative methods. All households with children under the age of five in each community were mapped during the census and a surveyor was recruited for each hamlet and trained to conduct a monthly fever and danger-sign survey using a structured questionnaire.The interviews were held in Kswahili, tape recorded and transcribed. The unit of analysis for the KII and IDI was the individual and it was the whole group for the FGD. The data were analyzed manually by applying codes within the structure of the thematic guide. The data were then structured into categories and put into a grid before identifying patterns, consensus, variation, contradiction and associations between variables The study was approved by the Medical Research Co-coordinating Committee of the National Institute for Medical Research (NIMR), Tanzania. Permission was obtained from the regional, district and village authorities. Verbal consent was obtained from the participants.The structure of study findings first considers symptom recognition, disease concepts and aetiology of two main perceived leading causes of death in children, malaria and convulsions. The second section discusses patterns of treatment for severely ill children with an emphasis on symptoms associated with malaria, convulsions and other bio-medically recognized symptoms of severe malaria. In the first section, information from the focus group discussions and from community key informant interviews forms the bulk of the data considered supplemented by and triangulated with data from traditional healer interviews, health care provider FGDs) and the quantitative survey data. In the section that follows, data from the interviews with hospital mothers are introduced and considered in conjunction with and in relation to data collected from all other study sources.The characteristics of the participants in the KII, FGD groups and IDIs are presented in homa, commonly translated as ‘fever,’ and homa kali ‘fierce fever’ were terms used interchangeably by the respondents with the word malaria. Although the term homa sometimes has other implications in the wider taxonomy of disease, homa and homa kali always carried a primary association with mwili joto or ‘hot body’ . Both terms, malaria and homa, were reported to be current and carry the same meaning in the vernacular Kimakonde as well as local Kiswahili discourses. Another term used to describe malaria was homa ya maleria.The local terminologies for common illnesses are summarized in kigongo and chidumba were mentioned as Kimakonde equivalents of malaria/homa in several FGD and by a small number KII, although both traditional healers and health centre staff who mentioned kigongo and chidumba categorized them as separate illnesses affecting both children and adults: kigongo severe febrile illness and chidumba as flu/cold.The terms Likonde (a Kimakonde term) was commonly used to describe convulsion illnesses affecting children although the respondents were also familiar with the term degedege, a Kiswahili term for convulsion illnesses in children. Other local terms for convulsion illnesses mentioned included liuni  and Kukwijula .homa with fever, vomiting, shivering, confusion/delirium and headache being the most commonly mentioned symptoms  or hot head (kichwa joto) with many informants also mentioning chills and/or shivering (kutetemeka). A few stated that relatively mild raised temperatures were normally interpreted as a sign of malaria, with very high fevers attributed to other illnesses, particularly to those associated with evil spirits or witchcraft. Through the monthly community surveys of underfive morbidity, a total of 1054 fever episodes were reported among the 1099 under-five children during the period of March–June 2005, the peak of malaria season, indicating that febrile illness in children under 5 years is very common in the area.There was substantial agreement between all categories of participants on the main symptoms associated with malaria/symptoms . High fekutapika) especially when frequent was listed as a symptom of malaria/homa, but consistently not of degedege/likonde:The quantitative survey revealed that 27% (n = 281) and 22% (n = 232) of the reported fever episodes among under-fives in the study communities were accompanied by vomiting and failure to drink/suck/eat, respectively. Vomiting (“(a) child is not supposed to vomit frequently, it can be either two or three times, but (if it is) more than that the parent realizes that the child has been attacked ” “(b) for the symptoms of malaria, I have to start by looking at what happened to my child. It started with crying, later on vomiting, headache, then colicky abdomen. I rushed to the hospital, the doctor said that the child had malaria and that due to vomiting intravenous fluids were needed, but even before one unit was started — (the child died)” Kunyong'onyeka/kulegea) was mentioned by both categories of respondents as a symptom associated with malaria/homa and/or degedege/likonde makang'a, a condition suffered by adults. Most respondents described makang'a as growths on the genitalia  although traditional healers mentioned that such growths might also be detected inside the nose or ear. Whether it was their location or seriousness was not probed.A child suffering from loss of appetite, paleness, inability to eat/drink/suck coupled with fever usually raises suspicion of Degedege/likonde equivalent to convulsions was reported to be the second most frequent illness leading to death in the study communities (degedege/likonde among the reported under-fives' fever episodes. All categories of respondents reported that this condition affects young children between the age of weaning or crawling and five to six years of age. Care-givers gave a detailed description of degedege/likonde episode:munities . The com(a) “the child has jerky movements, then (…) falls down, or if s/he doesn't fall down s/he can look with a staring gaze or looks as if s/he has been attacked by an evil spirit and thereafter the eyes roll side to side while the black spot in the centre looks pale” .(b) “the child starts with high fever (mwili joto), seizure (kukwijula), followed by fixed gaze” likonde dume) and female (likonde jike). The male type, named by many as liuni, was described as more severe and likely to result in serious sequelae such as mental retardation or death. The passage of yellow stools was frequently mentioned as a symptom of convulsions, most often (but not exclusively) associated with the more severe ‘male’ convulsions.Two types of convulsions with fever were locally recognized, male (degedege/likonde from a chronic seizure disorder named as chihundahunda/kifafa (epilepsy), which was understood to affect adults as well as older children; however, the distinction between the two presentations was not always made; a number of informants stated that improperly treated degedege/likonde could progress to chihundahunda/kifafa and traditional healers implicated the same evil spirits as responsible for degedege/likonde in the later development of chihundahunda/kifafa.Some informants consistently differentiated kuchanganyikiwa), irritability and delirium (kuweweseka). Kushtuka  was a frequently recognized symptom in convulsing children although sometimes mentioned in connection with malaria, as observed by an older mother:Changes in mood or mental status were widely reported among the symptoms of malaria/homa, including mental confusion  the child starts to rise in body temperature, then she may babble deliriously  and startle easily, have jerky movements (kushtuka)” .Kigongo (severe febrile illness). The presentation of a child in coma was reported as an infrequent condition:One traditional healer , who mentioned loss of consciousness, listed it among the symptoms of “That problem (loss of consciousness) does not  happen (in this village), but if it did happen we would pour water over the patient then send (him/her) to the traditional healer” This is supported by the quantitative survey which revealed that only 1% (14/1054) of the reported fever episodes was reported to have been accompanied by altered consciousness including coma. Respondents sometimes mentioned links between loss of consciousness and convulsions:“Coma (kuzimia) - occurs in children with convulsions and locally is known as kujuka. It is like dying but the one who is dead never comes up again but those in coma regain consciousness later on” .homa was the most frequently mentioned illness that could lead to death in children, symptoms suggestive of severe malaria were generally not categorized as malaria by study respondents. Coma (kuzimia/kujuka) was mentioned by only one respondent as a possible symptom of severe malaria; when other respondents were prompted to discuss coma, they asserted that coma was not associated with malaria. Moreover, the onset and progression of malaria/homa was generally seen as gradual. Any rapid death in the communities was viewed with suspicion, and not necessarily associated with malaria:Although malaria/“When the child is taken by god after (only) two or three days of illness, they (family members) say ‘no, this can't be malaria, probably it is sorcery, it couldn't be malaria affecting (the child) in such a (short) period of illness’ .The onset of degedege/likonde was variously described as gradual, with initial signs including high fever and excessive crying, or as very rapid, with the first symptom being a seizure:“A child will be playing, eating as usual, and all of a sudden develops jerky movement (kushtuka), the whites of the eyes will then protrude. In hospital this condition is called degedege” .degedege/likonde was caused - or believed to be caused - by supernatural agencies; often evil spirits (subiani or chipatupatu). A traditional healer stated:Thirty-six percent of the FGDs and 47% of the Key Informants reported that “convulsions are caused by a demon known as ‘kufali’ ” This condition was also associated with sorcery/witchcraft or god's curse,’ or ‘god's plan’ as noted by a village health worker:“Degedege itself – the experts  say it is caused by evil spirit “shetani”. Sometimes they tell you the child is bewitched” degedege/likonde, 57% of the FGDs  and 40% of the key informants related it to malaria. A few respondents were specific in describing degedege/likonde as advanced or late-stage malaria, as in the following statement made by a key informant in Msakala:While supernatural causation was prominent in the discourse about “Convulsion (degedege) is caused by mosquitoes; in other words, we can say it is matured (form of) malaria ” .‘Malaria’ and illnesses caused by ‘evil spirits’ were often explicitly described as mutually exclusive, though it was clear that differentiating between the symptoms of the two could be difficult and might require specialist knowledge:“There are some people  who can tell you whether the disease is malaria or it is (caused by) an evil spirit” .duka) are abundant in the villages and many of them stock and sell antipyretics/analgesics. No evidence of retail outlets selling antimalarials or other medicines was available in any of the study villages. There were traditional healers with different specialties: (i) healers ‘of books’ are scriptural healers whose healing practices centre around the use of books and writing and are main providers of charms to prevent degedege/likonde; (ii) healers ‘of evil spirits’ are mediums and diviners who contact and exorcise or appease the malevolent spirits responsible for causing a patient's ill health; (iii) herbalists use herbal remedies and are the main traditional providers of treatment for convulsing children in the study villages; (iv) witchcraft: secretive agents of intentional harm, is greatly feared.There were no health facilities, private dispensaries, drug shops or pharmacies situated within any of the study villages, although distance to the nearest Primary Health Care facility varies from 3–10 km. Small general goods shops  to the patient” Lethargy was said to increase the care-giver's level of concern and might therefore contribute to a decision to seek care outside the home; however, respondents did not appear to associate this symptom more with one type of treatment system than another.kutapika) and/or failure to eat/drink/suck as symptoms of malaria/homa that did not necessitate outside treatment on their first occurrence but which raised the level of care-giver's concern; however, little information was conveyed about how “vomiting” or “failure to eat/suck” were managed. A few respondents mentioned trying to tempt a sick child to eat by offering different types of food, or forcing liquids on the patient, but in general vomiting or failure to eat/suck seemed to be often ‘bundled’ with other likely more alarming symptoms, than as events of particular importance in themselves.Along with high fever, several respondents spoke of vomiting (Seven of the 12 mothers with currently admitted children mentioned vomiting on the first day of their child's current illness  and six o 3) mentioned makang'a as the reason she had sought treatment from a traditional healer before taking her child to the regional hospital. It was the child's paternal grandparents who had raised the possibility of makang'a and on day two of the illness they decided that the mother should be examined and treated by the traditional healer. The child's failure to improve following this treatment was the reason given for taking the child to the regional hospital two days later.Treatment of makang'a  often led to consultation with traditional healers to examine and treat them. Treatment of the child was affected by excising the growths found on the parent. A traditional healer stated that without such treatment the entire family was vulnerable to the curse and the child was likely to die. One of the hospital mothers  to the traditional healer” .Kigongo acknowledged using brewed herbal tea as well as spraying a herbal preparation over the patient's nose. None of the mothers of children currently admitted to hospital listed coma/loss of consciousness among the events occurring at any point in their children's illness.A traditional healer  who mentioned loss of consciousness as a symptom of degedege/likonde, participants in all FGDs and 47% of the Key Informants pointed to the traditional healer as the first resort for convulsing children. Participants in the health care providers' focus groups confirmed the practice but dissociated themselves from it. As one health worker quoted:For “With degedege many rush to traditional healer. They don't believe that it can be treated at hospital” Forty-percent of the Key Informants and 42% of the focus groups (young and old mothers) mentioned a number of home treatments derived from traditional healing practices, which is initiated at home on an emergency basis when there was a significant barrier (e.g. night time). Smoking the convulsing child using elephant dung was a commonly mentioned practice:“You get elephant dung, you heat it in a small piece of pot and the smoke goes through to the child, and, god willing, the seizures can be alleviated” nkuhu). Dousing the convulsing child with cold water, urinating on or immersing the child in the sea water were usually described as emergency measures rather than complete treatments. Only one of the twelve hospital mothers mentioned dousing or immersing her convulsing child in water.The presenting symptoms and corresponding treatment pathways for 12 children with severe malaria admitted at Mtwara hospital are summarized in degedege/likonde) children even for those who associated convulsions with malaria:Most respondents identified first resort to the traditional healer as the prevailing norm for treatment of convulsing …..in children convulsions are mostly treated by traditional healer” Rituals involving smoking, fanning, smearing, therapeutic marks on skin or skin incisions, and preparation of herbal potions were all associated with traditional healers' treatment of convulsing children. Key informants and FGD participants provided detailed descriptions of such treatment practices, as captured in the quotes below:“ takes a nyungo mat made like a big bowl and uses it as a fan, laying down the child and fanning him/her on the head until the body cools down, then (s/he) takes certain leaves, crushes them and uses them to bathe the child, then leaves the child to sleep” .“For the child who eats, the medicine is mixed with porridge; for the child who is unable to eat they do therapeutic marks on the skin  or smear the child with medicines on the mother's breast” degedege/likonde, an act followed by visits to clinics or hospitals once the child was ‘over the crisis’:Half of the FGDs and 20% Key Informants positioned the traditional healer as a first-stage treatment for “if (the child) gets better you take her/him to the hospital” Of the 12 children admitted at hospital, 58% were reported to have had convulsions. Although many mothers had used traditional medicine either as home remedies (n = 5) or at traditional healers (n = 1), they also used biomedical treatment, hence the eventual resort to hospital, and admission of the child. The amount of time spent at the traditional healers was decided by the healer and was not seen as ‘negotiable’ by the care-givers and could vary from few hours to days:“They don't stay  for a long time. It may be for one hour or more and then they recover. And our traditional healer here in this place treats every type of disease” KII .“At the traditional healer (you stay) until he declares that he has failed to treat. There is therefore no specific amount of time” Three of the twelve hospital mothers, those who had taken more than three days to seek care since the onset of symptoms, reported consultation with traditional healers with only one for convulsions. Several respondents described from personal experience or stated as a general truth that traditional healers retained patients in their care for lengthy periods even when treatment appeared to be failing. A teacher in one of the communities noted that“There is no traditional healer who can permit his/her patient to seek alternative treatment even if s/he finds the condition of the patient is not improving” A father remarked that:“(…) after one week the (child's) condition was not good so we asked permission to leave but the old lady  refused (…) eventually we had to give an amount of money which she required  and then we left” homa, for which no preventive measures were described, numerous informants mentioned methods of protecting children from degedege/likonde before, during or after an episode of the condition:In contrast to malaria/“When the child is born there are some protective measures which should be done. Local herbs are applied to pierced skin on the sides of the child and this will protect the child against likonde. If the parents did not perform this, the child will be affected by likonde” “When the child falls sick s/he is given a protective charm to wear and grows with it until it falls off” Participants in five FGDs from 3 communities believed that injections, given to a convulsing child, could be fatal. An older mother noted that“If you inject a convulsing child you increase the poisoning of that child. Convulsion has poison and injection too, so you will be poisoning the child suddenly, you will kill him/her. "Despite beliefs about the dangers of injection use in convulsing child, some care-givers sought care from health facilities, sometimes directly contravening the advice of elders:“Let me talk. My child was suffering from convulsions and as usual my mother came. She told me that if I took the child to hospital I would come back without the child, but I told her that what she was talking about was out-of-date. And I took my child  and on the second day he was all right” .The three mothers who went to the regional hospital on the first day of their child's illness  reportedThe majority of the key informants (73%) and participants of the FGDs (86%)stated that decisions to seek care for severely ill children were made by fathers. If the father of a child was not around, the mother or grandparent made the decision. Mothers and fathers both reported seeking advice and hands-on assistance from family elders and neighbours, especially when symptoms developed at night. The in-depth interviews with the hospital mothers confirm this pattern of decision-making: 5 cases by fathers, 2 cases by mothers, 2 cases by both parents and the remaining 3 by relatives. The decision making process was not further probed to establish whether the pattern seen for hospital admissions and described in interviews was due to financial control, distance or the severity of the illness.Spatial and temporal considerations dominated discussions on factors inhibiting access to formal health facilities. Transport from the village to the nearest health facility was largely by bicycle, and many respondents complained of inadequate access:“Today my child can become ill, I'll go to my neighbor to ask for the bicycle and he will tell me that his bicycle is out of order [not working]. I'll proceed to another one and the answer will be the same, and that is when you decide to go on foot” In the rainy season roads were reported to be difficult or impassable in Kawawa, Chemchem, and Kihamba:“…during rains, you can't ride a bicycle, the road becomes muddy and walking takes a long time. So during rains, transport is really a problem .Perceived severity was sometimes a key indicator for resort to the nearby traditional healer while less urgent cases (no clear immediate danger) taken to the health facility.“People go to the local traditional healer when the patient is seriously ill because the hospital is at a distance. He might want to take the patient to the dispensary but it is very difficult (because of distance), so (he feels) it is better to rush the patient to the nearby traditional healer who treats even evil spirits — he might be of help” .Uncertainty about the likely course of illness was also cited as a factor that could cause delay in seeking treatment. Thus respondents often spoke of the decision to seek care for ‘common’ symptoms such as high fever in terms of a care-giver's personal ability to “tolerate” the child's illness during a watch-and-wait period:“Some believe others not” “some can tolerate (waiting) while others can not” “I can't wait while the child is sick — I can't! My child is sick and I want him to get medicines!” Night was a significant barrier to move out of the home, with respondents mentioning fear of wild animals and sense of unspecified dangers “after sunset” as factors that would effectively prohibit most people from venturing out until morning. Neighbors were the stand-ins for medical providers during night time hours, being called on for advice, reassurance, and practical help during long night vigils with sick children. One of the 12 hospital mothers mentioned fear of moving in the night alone as the main reason she had not taken her convulsing child to the hospital straight away, instead she called in the neighbors who assisted with emergency ‘first aid’ by smoking elephant dung and immersing the child in a bucket of water.Cost of care was frequently raised to explain delays in accessing both traditional and biomedical treatment. In the case of treatment at the traditional healer's place, cost could cause a double delay: first as a barrier to accessing care from the traditional healer as the parents have to secure money to pay the service and, if treatment was initiated before the family had managed to ‘find’ the money to pay for it, as a barrier to leaving the traditional healer's place in order to seek hospital care:“At the traditional healer, payment is needed and if there is nothing in the house the mother will delay while trying to raise some money. If they happen to go to the traditional healer (without money) and be accepted, two things may happen. Mother and child will remain at the traditional healer's place while father is looking for money, or mother will have to leave some of her clothes (as surety) and come back to collect them when she gets some money” Out of the 12 hospital mothers, 33% reported “lack of money” as the main reason for their delay in getting the child to hospital. Although consultations and treatment were free for under-fives as part of government policy, money was needed for transport, and also sometimes more directly for the treatment itself. Several FGD and key informant respondents reported unofficial costs associated with health facilities, often arising from a poor supply situation that resulted in patients having to purchase the needed medicines or equipment from private suppliers:“You can reach there (dispensary) and find that one syringe costs five hundred shillings (0.4 US$), and if you don't have that money it means you can't get treatment” Another potentially significant delay factor was seen in relation to the care-giver's lack of decision-making power or independent access to the required resources (mainly money), with many respondents mentioning the mother's need to wait for the father's permission before embarking on treatment:“(The) mother will not take a sick child to the hospital unless the father returns home. (…) Since the father may have many wives it is quite possible that the sick child is not from the household where the father slept that night. The child will not be taken to hospital until the father is consulted, “the child is sick, should we buy him an aspirin or take him to the hospital?” .makang'a treatment first.Two of the 12 hospital mothers stated the absence of the father to give permission or provide them with money was the main cause of delay in taking their sick children to hospital. It was also mentioned that conflicting treatment preferences between care-givers and decision-makers were a cause of delay: one hospital mother reported that although she had wanted to take her child to the hospital sooner, elderly members of the extended family had advised her to go to the traditional healer for Despite the many barriers and constraints people faced in seeking care from health facilities, 40% of the Key Informants and 36% of the FGDs stated that when a child was seriously ill such obstacles simply had to be overcome:“You can't stay idle waiting for rain to stop; you know how serious the child's fever is. (…) some have bicycles; others, like us, do not. Therefore it is possible that even ‘though it is midnight you carry the grandchild and off you go (on foot)” chidudu, soap for constipation and diazepam for convulsions . All these respondents also thought that rectal medicine would be acceptable in their communities. The remaining respondents were either not familiar with the practice (4 Key Informants) or were not probed for response (2 FGD and 1 Key Informant).When respondents were asked if they had heard or had direct experience of rectal application of medicines, 86% of the focus groups and 67% of the Key Informants stated that they were familiar with some type of rectal medicine, either traditional or biomedical. These included rectal use of herbs for abdominal distention and homa symptoms corresponded to the biomedical definition of uncomplicated malaria, and was considered treatable at health facilities. Although the description of degedege/likonde was consistent with the biomedical description of convulsions, a prominent feature of severe malaria, it was perceived as a separate disease associated with evil spirits and witchcraft, a perception noted in other parts of Tanzania kuzimia) was rarely mentioned spontaneously and was referred to in the context of traditional healing. Lethargy was seen as a sign of severe illness and might provoke action outside the home. However vomiting and failure to eat/suck/drink raised concern but were not seen as indicators of severe illness; when combined with fever they were managed within the home. Vomiting was rarely mentioned in mother's accounts of symptoms; it was considered a mundane symptom, overshadowed by more disquieting symptoms that followed. The early occurrence of vomiting and failure to eat, while increasing anxiety, did so only in the presence of other more dominating symptoms and thus pointed away from the possible development of more serious complications associated with degedege/likonde instead of alerting care-givers to the increasing potential of severe disease.This study provides new information on the pattern of danger signs in children in a hyperendemic area of malaria in Tanzania, and documents local perception and common treatment-seeking behavior related to each of these danger signs, or their combination. The communities' description of malaria/Our findings reveal that the study communities were familiar with rectal application of medicine, mainly traditional medicine but also biomedical, an observation reported by Kaona and Tuba Care-givers indicated that home treatments were often initiated as first aid before urgently seeking treatment from a traditional healer, or as ‘a holding measure’ undertaken if ‘expert treatment’ could not be accessed immediately (e.g. during the night). When the moment of crisis (the convulsion) was past and/or access barriers were removed, care-givers were seen as likely to seek follow-up care either at traditional healers or at a biomedical health facility or both. The fact that respondents who associated convulsions with malaria yet would seek care from traditional healers raises concern. Similar discrepancies between knowledge and treatment practice in convulsions have been reported from other parts of Tanzania The quantitative data in this study confirm findings elsewhere that febrile illness is common in children The WHO and UNICEF Integrated Management of Childhood Illness (IMCI) Guidelines developed in the mid-1990's to assist clinical discrimination between children needing urgent hospital management, and others needing symptomatic therapy recommend hospital referral for all children with danger signs including: convulsions, inability to drink, vomiting everything, unconscious or lethargy While there is an extensive, coherent body of evidence collected for the purpose of developing, validating and refining IMCI facility guidelines The findings of the study have implications for the introduction of community-based interventions that improve management of severe disease, including the introduction of rectal artemisinins as pre-referral treatment at community level. Local communities' ability to recognize convulsions, altered consciousness and coma, their awareness about the risks of such conditions if not treated, and the fact that mothers are opting to go to a health facility even when children are convulsing might be facilitating factors for the introduction of artesunate suppositories. In addition, the practice of rectal administration of medicines by the study communities could be an added advantage. Biomedical options that are more convenient than traditional options for emergency care are easier to introduce- particularly where time, distance (dangers of movement at night), and money are involved. Emergency treatment with an artesunate-based suppository at the community level could enable the severely ill child to have treatment while the parents seek transport and funds for definitive care. However, poor discrimination of vomiting, lethargy and failure to feed as early signs of severe disease might call for health education with special emphasis on danger signs. Targeting such education to care-givers of young children, decision-makers especially men, advisors, formal and informal care providers might be a prerequisite for successful introduction of rectal artesunate as an emergency treatment. Traditional healers are part of the community and are easily accessible temporally and spatially and therefore might pose a major barrier to rapid use of the drug in a sick child and adhering to referral advice. Mechanisms involving traditional healers in delivering artemisinin-based suppositories, a medicine that holds great promise for Africa, should be explored.With the aim of facilitating the successful introduction of rectal artesunate through a well-grounded understanding of symptom recognition and treatment practices for “severe malaria,” the study collected qualitative data from several categories of people in different settings. The collection of data relied heavily on informants' reports of normative behavior in response to questions posed in a context far removed from the immediate stress and uncertainty of managing a child's actual illness which could lead to overstating perceived norms, underestimating, or downplaying the extent to which people normally deviate from them. There is a likelihood that the researchers themselves were perceived as representing the health service and had vested interests in the MoH/biomedical health system, which would tend to skew responses towards expression of greater acceptance of biomedical disease concepts and treatment practices.Only “hospital mothers” whose children were out of immediate danger and pronounced as recovering - those for whom the move to hospital proved most rewarding – were interviewed. This might have contributed to a reporting bias with the likely tendency being to over-report recourse to biomedical therapies while under-reporting treatment delays or recourse to ‘traditional’ or other treatments. We also interviewed mothers who sought care at the highest level of the health system. This excludes those who did not make to the hospital for one reason or the other (a selection bias).All interviews and focus group discussions were conducted in Kiswahili, the official language of Tanzania, while the vernacular language of the study area is Kimakonde. However, fluency in spoken Kiswahili is nearly universal among study participants who expressed themselves without apparent difficulty in all topic areas explored.


Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria.In falciparum malaria sequestration of erythrocytes containing mature forms of 11  parasites per body, and was over six times higher in severe malaria  than in patients hospitalised without signs of severity . Parasite burden was highest in patients who died . The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony—but in severe malaria is unrelated to stage of parasite development.We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai–Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 × 10Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes. Measuring sequestered parasites using plasma PfHRP2 concentrations may provide a more accurate estimate of total parasite mass and hence severity in falciparum malaria. Plasmodium falciparum ) was four times higher if mature-stage parastites predominated (n = 79); 1.6 × 1012 (2.5 × 1011 to 5.4 × 1012) parasites compared with 4.0 × 1011 (6.7 × 109 to 2.1 × 1012) parasites if mature-stage parasites did not predominate (p = 0.002 by Mann-Whitney U test) .The number of sequestered parasites was also assessed in relation to the parasite stage in the peripheral blood smear. We have reported previously that in patients with severe malaria a predominance of mature stages (trophozoites and schizonts) in the peripheral blood  . Overall11 (2.3 × 1011 to 3.5 × 1011) versus 1.7 × 1012 (1.3 × 1012 to 2.3 × 1012) parasites/body (p < 0.001). Within the group of patients with severe malaria, those who died (n = 28) had over twice the geometric mean parasite load compared with patients who survived (n = 139): 3.4 × 1012 (95% CI 1.9 × 1012 to 6.3 × 1012) versus 1.5 × 1012 (95% CI 1.2 × 1012 to 2.0 × 1012) parasites/body  the median (interquartile range) number of sequestered parasites was ten times lower than in patients with severe malaria (n = 167): 9.0 × 1010 (6.7 × 109 to 4.1 × 1011) compared to 9.1 × 1011 (6.1 × 1010 to 3.2 × 1012) parasites/body . Within the patients with severe disease, the median (interquartile range) calculated number of sequestered parasites was twice as high in the fatal cases than in the patients who recovered: 1.4 × 1012 (4.3 × 1011 to 1.4 × 1013) versus 7.6 × 1011 (1.2 × 1010 to 2.7 × 1012), but this difference was of borderline statistical significance .The estimated total parasite biomass increased in proportion to severity of the disease.The geometric mean (95% CI) parasite load was six times lower in patients hospitalised without evidence of severe malaria than in patients with severe malaria: 2.8 × 10n = 73) had a significantly higher calculated total body parasite load than patients with a lower lactate concentration (n = 94): geometric mean (95% CI) 2.8 × 1012 (2.0 × 1012 to 4.1 × 1012) versus 1.3 × 1012 (0.9 × 1012 to 1.8 × 1012) parasites/body . The calculated sequestered parasite biomass was also significantly higher in patients with high plasma lactate concentrations  than in those with an admission lactate concentration below 5 mmol/l  . There was no association between the circulating parasitaemia  and plasma lactate concentration.= 0.012) . There wn = 87), defined as those having a total plasma bilirubin above 42.8 μmol/l [n = 80). Finally, a slight but significant negative correlation existed between admission Hct and the calculated log10 total parasite biomass , but there was also a weak, though significant, correlation between Hct and the log10 total circulating number of parasites . The group of severe anaemic patients as a whole did not have a significantly higher number of either circulating or total parasites in their blood  for which parasites are circulating  at an overall parasite multiplication rate of one . That is,If the time of admission to hospital and stage of parasite development (in hours) are unrelated, then the overall ratio of circulating parasites But the parasite numbers are usually expanding, particularly in uncomplicated malaria before treatment. In the present model we have assumed a multiplication rate of eight per cycle for preceding cycles. If the parasite age distribution is normally distributed with a standard deviation of 4 h, as estimated previously , then thcir) that is predictably released into peripheral blood plasma in acute falciparum malaria. The estimated total parasite biomass was clearly associated with disease severity and outcome in univariate analyses. In a logistic regression analysis with outcome as dependent variable, the parasite load estimates did not predict disease severity better than conventional measures:GCS, plasma lactate, and serum creatinine outweighed the contribution of parasite load to the regression model, suggesting a close causal relationship between these variables. The estimated total parasite biomass was also associated with other well-established markers of severity. In contrast with this, peripheral blood parasitaemia and the derived total number of circulating parasites were not associated with disease outcome, nor with other important measures of severity such as admission plasma lactate concentrations. It should be noted that some of the criteria defining severe disease do include the level of the peripheral blood parasitaemia, which is variably related to total biomass, although in the model peripheral parasitaemia was not used in the calculations of parasite biomass. Pre-treatment with antimalarial drugs affecting PfHRP2 production cannot explain the differences in the estimated total parasite biomass between patients with severe versus uncomplicated malaria: documented pre-treatment was relatively rare , and this did not differ between the groups.The positive association between sequestered parasite biomass and disease severity fits with the widely accepted view that the sequestration of erythrocytes containing the mature forms of the parasite in the microvasculature of vital organs is the central pathological process in falciparum malaria. The greater the number of sequestered parasites, the more severe is the disease. The cytoadherence of parasitised erythrocytes to the endothelial lining of the microcirculation is very efficient. Mature forms of the parasite are rarely seen in the peripheral blood smear used to assess the patient, whereas they are central to pathology. Overrepresentation of late stages in the peripheral blood smear is a prognostic factor for fatal outcome, because it represents a larger sequestered parasite biomass ,11. Marc−15 and 11.6 × 10−15 g. The antigenicity of PfHRP2 is not constant among parasite isolates because of variation in the expression of antigenic motifs. The calculations also assume a parasite multiplication factor of eight, which was derived from observations in early studies with experimental infection of humans with P. falciparum as a treatment for syphilis [d).Scatterplot showing the correlation between the sequestered parasite biomass  and the percentage of late stages (trophozoites and schizonts) in an admission peripheral blood slide in 337 patients with falciparum malaria. The sequestered and total parasite biomass were derived from plasma PfHRP2 concentrations as discussed in the text .Click here for additional data file.Figure S2sr = 0.44, p < 0.001).Scatterplot showing the correlation between the PfHRP2-derived sequestered parasite biomass on admission and admission plasma lactate concentrations in 337 patients with falciparum malaria .Click here for additional data file.Plasmodium;P. falciparum causes the most severe disease. After a person is bitten by an infected mosquito, the parasites enter the person's red blood cells (erythrocytes), and then these infected red blood cells get stuck in small blood vessels, blocking the blood flow and causing many of the problems associated with malaria. The parasites multiply inside the red cells, which eventually split and release more parasites.Malaria is caused by parasites of the genus It is possible under the microscope to see the number of parasites in the circulating red blood cells but hard to measure how many are inside red blood cells stuck in the small blood vessels at any one time. Knowing the number of parasites blocking up the blood vessels might give a better idea of how severe the disease a patient has. An indirect measure of the number of unseen parasites in the body is a protein, PfHRP2, which the parasites produce and which is released into the blood when the red blood cells split.P. falciparum malaria and looked mathematically to see whether the PfHRP2 measurements could be linked to the total number of parasites each person had in their body. They found that people with severe malaria had high total numbers of parasites hidden in their red blood cells, as measured by the PfHRP2 levels.They measured the amount of PfHRP2 in the blood of 337 adult patients with Further work will need to be done to reproduce these findings in other groups of people with malaria. However, estimating the total number of parasites in this way may make it possible to predict more accurately the likely severity of a person's disease.The World Health Organization has a set of Web pages on malaria:http://www.who.int/topics/malaria/en/http://www.nlm.nih.gov/medlineplus/tutorials/malaria/htm/index.htmMedlinePlus has an interactive tutorial on malaria:


Rogerson discusses a new study that found that children with the haptoglobin (Hp) 2-2 genotype had a greater fall in hemoglobin over the malaria season than children with other Hp genotypes. In malaria-endemic countries of Africa, anaemia is very common in pregnant women and in children under five. Although anaemia is multifactorial—causative factors include iron deficiency and other nutritional deficiencies, helminth infection, and HIV—malaria is clearly an extremely important factor. Over half of malaria-related deaths are attributed to severe malaria anaemia  concentration less than 50 g/l) . SeveralThe pathogenesis of malaria anaemia remains incompletely understood. Dyserythropoiesis , intravascular haemolysis of infected red cells, and destruction of both parasitized and uninfected erythrocytes by splenic macrophages are all important ,4. InterThe glycoprotein haptoglobin (Hp) is the body's main tool for removing circulating, toxic free Hb during intravascular haemolysis. Hp binds rapidly and with high affinity to free Hb, with the resultant complex being taken up by CD163, which is expressed on monocytes/macrophage lineage cells. Binding of Hp/Hb complexes to CD163 leads to cytokine secretion by macrophages [The pathogenesis of malaria anaemia remains incompletely understood.2Hp gene arose by duplication of a region of the gene encoding the α chain of 1Hp, leading to three phenotypes—1-1, 2-1, and 2-2. Hp1-1 leads to smaller, more abundant dimers with a higher affinity for Hb, and to Hp/Hb complexes with a lower affinity for CD163. Studies have associated the Hp2-2 phenotype with increased morbidity from cardiovascular disease, diabetes, HIV infection, and other conditions—perhaps because concentrations of Hp2-2 are lower, Hp2-2 is less able than Hp1-1 to penetrate extravascular spaces, and Hp2-2 is less able to prevent oxidative damage or immune activation than the other phenotypes [In humans, unlike other animals, Hp exists in two forms. The enotypes .Hp genotype and severe malaria or associations between Hp genotype and mild disease [Hp genotype; although, in one study, there was a suggestion of an association with severe malaria, especially severe anaemia.Given the importance of intravascular haemolysis in malaria infection, studies of Hp in malaria are important, but results to date have been inconclusive. Earlier studies based on phenotyping suggested Hp1-1 was associated with severe or symptomatic infection ,9. For e disease ,12. ThesPLoS Medicine, Atkinson and colleagues have taken a different approach [Hp genotypes, children who were 2/2Hp had a greater fall in Hb (by 4 g/l) over the malaria season than did other children. Hp genotype significantly predicted Hb levels at the end of the malaria season in multivariate analysis, together with iron status and several other variables. A separate analysis examining factors influencing the magnitude of the fall in Hb gave similar results. Additionally, children with 2/2Hp genotype were more likely to be parasitaemic. The authors suggest that the effect of Hp genotype on the fall in Hb  may be more significant than that of sickle cell trait (HbAS) or glucose-6-phosphate dehydrogenase deficiency. On the other hand, sickle cell trait and glucose-6-phosphate dehydrogenase deficiency have been more compellingly associated with protection from severe malaria than have Hp genotypes [In a new study in approach . Workingenotypes .Hp genotype influence Hb fall? The authors offer several interesting suggestions. Because destruction of uninfected erythrocytes may be especially important in development of malaria anaemia, this mechanism is one plausible route. Oxidative damage to uninfected cells might be more marked in Hp2-2 individuals since Hp2-2 proteins bind less efficiently to Hb, and since levels of Hp2-2 are lower and more easily depleted, increasing premature destruction of erythrocytes. Another reason Hp genotype may influence Hb levels might be because complexes of Hp2-2 with Hb, but not other Hp/Hb complexes, enter monocytes (which express less CD163 than macrophages), stimulating cytokine release by these circulating cells. Thus, different Hp types inducing different cytokine responses may affect the duration of inflammatory, marrow-suppressive cytokine production following malaria. Persistent marrow suppression, as judged by inappropriately low reticulocyte responses after resolution of infection, is a feature of malaria [2/2Hp genotype has been associated with increased iron accumulation in haemochromatosis—in one study but not in others —suggestIs a 4 g/l difference in Hb fall clinically relevant? Probably. Although the effects of this fall on the proportion of children suffering moderate or severe anaemia are not given in the study, the population distribution of Hb levels is wide. A fall in mean Hb may translate into increased numbers of individuals with symptomatic disease, although this effect is not demonstrated in this study. From a public health perspective, interventions that result in a rise in Hb of similar magnitude—such as intermittent presumptive treatment in pregnancy —are endo


Insecticide-treated bed nets (ITNs) are known to be highly effective in reducing malaria morbidity and mortality. The effectiveness of ITNs is largely influenced by behavioural factors and not much is known regarding such factors under programme conditions.This descriptive study was nested into a large ITN effectiveness study in rural Burkina Faso. During two cross-sectional surveys in the dry and rainy season of 2003, random samples of young children from nine representative villages (n = 180 per survey) were investigated for compliance with ITN protection and related behaviour. Data were collected through direct observations and through interviews with mothers.ITNs were perceived as very important for protection against mosquitoes and malaria particularly during the rainy season, but there were problems with their use during the dry season. Young children usually slept with their mother under the ITN and self-reported compliance was 66% and 98% during dry and rainy season, respectively . Important reasons for low compliance during the dry season were high temperatures inside houses and problems related to changing sleeping places during the night.Under programme conditions, compliance with ITN protection in young children is sufficient during the rainy season, but is rather low during the hot and dry season. Greater emphasis needs to be placed on information/education efforts to make people aware of the fact that the risk of contracting malaria may persist throughout the year. Nei Chin (the Canon of Medicine) [Malaria has accompanied humankind for as long as 5,000 years, the first written evidence of the disease dating as far back as 2,700 BC in the Chinese medical classic edicine) . Protectedicine) .In recent years, these two techniques have been combined into the powerful tool of insecticide-impregnated bed nets and curtains (ITNs). ITNs have consistently been shown to be very effective and sustainable in reducing malaria morbidity and all-cause mortality in children of different malaria endemic areas -7. They The impact of ITN interventions under real life conditions is known to be influenced by a number of important socio-economic, cultural and ecological determinants -16. MostCentre de Recherche en Santé de Nouna (CRSN), Burkina Faso between May and August 2003 [The study took place in the research area of the ust 2003 . Nouna, ust 2003 . The areAnopheles breeding sites, favouring the Anopheles gambiae complex and causing entomological inoculation rates (EIR) of 100 to 1,000 infected bites per person per year [The rainy season usually lasts about five months from June to October with 600 mm to 800 mm of total rainfall. During this period the whole area becomes flooded, with the development of suitable per year . The dryper year .th birthday. Children were individually randomized to ITN protection from birth until their 5th birthday (group A) versus from month six until their 5th birthday (group B). A prototype long-lasting ITN (PermaNet®) was used which was shown to be not really long-lasting [This descriptive study was nested into a large ITN effectiveness trial which included all children of the rural CRSN study area born between 2000 and 2003 . A total-lasting . As a co-lasting . Morbidi-lasting ,7. FieldThis study comprised two cross-sectional surveys in nine representative villages of the overall 41 villages included in the ITN trial. Surveys took place in May 2003 (hot dry season) and July/August 2003 (rainy season). Results are based on answers to a comprehensive questionnaire combined with unannounced visits during sleeping hours.After stratification for the three health-centre defined sub-areas, the nine study villages were selected from the 41 trial villages (excluding the six sentinel villages) to represent the study population in its socio-cultural, demographic and geographical diversity. In each of the nine survey villages and independently for the dry and rainy season survey respectively, 20 ITN trial children were randomly selected for this study from the list of the trial participants (n = 180 per survey).The surveys in the nine villages always started by direct observations during an unannounced visit of a trained field worker to the respective study household, who was accompanied by the responsible village informant. These visits took place in the late evening (around 9.00 PM) or early morning (around 5.00 AM), when young children are expected to sleep. During visits, the utilisation of ITNs was verified and noted down on a standardized form and additionally documented with a digital camera. At the same time, the families were informed about the timing of the research team's visit on the following day, when the forms and the digital camera were then handed over to the research team upon their arrival.A pre-tested structured questionnaire was then administered by the research team, consisting of a trained fieldworker and the investigator (CF), to the mothers/caretakers of the study children. In addition to demographic and socio-economic parameters, the questions addressed mainly aspects of behaviour regarding malaria prevention and treatment with particular emphasis on specific ITN use.Did the study child sleep under the ITN last night?' as well as by the direct observations during the respective nights.ITN compliance was evaluated by the question 'Data entry was done by the investigator (CF) directly after the survey visits. The data were analysed with EpiInfo 3.0 (October 2003) with simple proportions used to describe the parameters investigated.The study protocol was approved by the Ethical Committee of the Heidelberg University Medical School and the local Ethical Committee in Nouna, Burkina Faso. Informed community consent was sought through explanations and discussions during village meetings before the study was implemented. Oral informed consent from interviewees was a prerequisite for the interviews and the direct observations.Table The study ITNs were found in all of the 360 households visited during the two surveys. Beside the one ITN given per household for protection of the cohort child against mosquitoes, 85/180 (47%) and 75/180 (42%) of respondents reported the existence of at least one additional, but untreated bed net in their household during the dry and the rainy season time points respectively. Respondents indicated that other physical barriers commonly employed to prevent mosquitoes from biting were covering clothes and/or light blankets, which were mentioned by virtually all interviewees. Moreover, 158/180 (88%) and 171/180 (95%) reported the use of insecticide coils or insecticide spray in their households during the dry and the rainy season time points respectively. Traditional methods such as using specific plants as mosquito repellents were mentioned by some 10% of caretakers, both during dry and rainy season time points. 4/180 (2%) and 23/180 (13%) of respondents mentioned evacuation of stagnant water for mosquito control during the dry and the rainy season time points respectively.The use of modern and traditional malaria prevention medications was reported by ten percent of respondents. Chloroquine and antipyretics were the modern drugs used at irregular intervals for prevention, while specific plants for drinking and bathing were mentioned as traditional prevention methods.The perceived advantages and disadvantages of ITN and bed net use in general were to a large degree dependent on the season. Table While 105/180 (58%) of respondents complained about the disadvantage of heat and the corresponding lack of convenience during the dry season, this proportion was only 15/180 (8%) during the rainy season. Practicability was influenced by the tendency to sleep outside the house during the dry season, the problems in fixing and keeping the bed net outside and the difficulty to remount the net in the early morning hours when many people withdraw inside the house due to falling temperatures. As a consequence, mothers and young children often slept either unprotected during the first or the second half of the night depending on where the bed net was mounted initially, the majority of them preferring to leave the net mounted in the house and to retire underneath when moving indoors early in the morning. Other stated reasons for not using the ITN during the dry season were the lack of mosquito nuisance in 13/180 (7%) and travelling in 5/180 (3%) of respondents.Table Of the dry season respondents who clearly reported that the study child was not under ITN protection during the previous night, approximately half stated that the ITN had not been used for less then one week while one quarter gave periods up to one month and the other quarter for more than one month. The three rainy season respondents, who had reported that the child was not protected with the ITN during the previous night, all declared that this was an exception and that their children were usually sleeping under the ITN.The reasons given for only part-time protection of children during the previous night of the dry season were mainly prolonged household activities during which the child is commonly carried on the back of the mother. Additionally, due to high temperatures inside the house where the ITN is situated, mothers and their children tend to sleep outside during the first night hours but few mount their net there. In those cases where the ITN was successfully installed outside the house, children were unprotected in the cold morning hours when the mothers moved back with them inside the house where nets were generally not remounted.Direct observation revealed that people were very creative in fixing the ITNs both inside and outside the house. The great majority of the villagers usually managed to mount their nets correctly, only a few reported to sometimes use the ITN for a limited time period as a sort of "cover cloth" for the child when they do not have sufficient time to install it immediately. Of those who reported full compliance with ITN use, 67/118 (57%) and 117/177 (66%) said to tack it correctly under the mat or mattress during dry and rainy season surveys respectively. The proportion of ITN found to be tacked correctly under the mat/mattress during direct observations was 6/62 (10%) and 48/142 (34%) during the dry and rainy season surveys respectively.During the dry season, 104/180 (58%) of the study children slept primarily on mats set outside the house. Only a few households reported moving the actual beds outdoors. Children, however, were reported to use both mats and beds, as they were often moved between indoors and outdoors in the course of the night. This pattern was also confirmed by the direct observations. During the rainy season instead, 135/180 (75%) of children consistently slept on beds inside the house.None of the study children slept alone under the ITN, instead they shared it in most occasions with their mother and eventually other siblings and only exceptionally with their father Table . When chDuring the dry season, 99/118 (84%) of children were reportedly put under the ITN after dinner  and 106/118 (90%) did stay there until sunrise . During the rainy season, the corresponding figures were 150/177 (85%) and 161/177 (91%).This study has explored factors related to the acceptance and use of ITN in a rural area of Burkina Faso and identified behavioural aspects that could influence the implementation of ITN in malaria control programmes. Unlike previous studies exploring compliance with ITN use ,21,27-29The most encouraging finding emerging from the study relates to the fact that two years after the beginning of the trial, the ITNs which had been distributed were without exception still present in the 360 households visited on the occasion of this study. In addition, there was no evidence that an ITN had been taken away from the child to whom it had been assigned to be solely used by another household member. In virtually all instances, respondents reported that the ITN was jointly used by the study child, his/her siblings, and their mother. Such findings appear most favourable when considering that under similar circumstances, 28% of bed nets distributed free of charge in Burundi were found to be resold to neighbouring Tanzania .In addition to the ITNs distributed as part of the trial, non-treated conventional bed nets were also found to be used by about 45% of study respondents. This figure confirms the net prevalence assessed at baseline prior to implementation of the ITN trial  and indiThe results from this study suggest that bed nets are used throughout the year, although important differences in compliance exist between the rainy and the dry season. Seasonal changes in temperature influence sleeping patterns and thus, as a consequence, people's bed net use. ITN compliance was found to be very high in the rainy season but to drop substantially in the hot dry season. Similar seasonal variations in the compliance with ITN use had already been observed across different endemic regions, indicating that bed net use may vary between 20 and 76 per cent during the hot dry season to over 90 per cent during the rainy season ,21,27-29Asking respondents to motivate their decision to sleep or not to sleep under the net allowed a better understanding of possible obstacles to the continuous compliance with ITN use. Since children sleep with their mothers, changes in adult sleeping preferences and patterns across seasons inevitably influence whether the child is protected throughout the night. This study indicates a certain easiness to comply with ITN use during the rainy season when people sleep indoors. It also confirms previous findings citing heat is an obstacle to sleep indoor and under a net ,38-40 anThe totality of respondents in the study reported protection against mosquito nuisance as the primary reason for sleeping under a net. In line with findings from prior research, such wish to protect oneself from mosquito nuisance is also likely to explain why in combination with ITN use, people reported using a variety of other means of protection against mosquitoes, ranging from covering themselves with thick clothes and blankets ,43 to usAn interesting feature emerging from the study is the high percentage of respondents reporting that ITNs offer protection against malaria. In addition, a large percentage of respondents reported having spent less money on insecticides and malaria drugs following ITN use. This high awareness that ITN use reduces malaria transmission is most likely due to the fact that the distribution of the ITNs took place in an area which, thanks to a series of malaria studies including the ITN effectiveness trial, has been sensitized over a number of years on the importance of using ITNs to protect children from malaria. This finding represents an encouraging element for the development of communication and sensitization campaigns as it shows that when repeatedly and correctly informed, people do appreciate novel information on the advantages of ITN use even when malaria transmission may not necessarily be conceptualized in relation to mosquito bites ,47,48.Such high awareness that ITNs offer protection against malaria could potentially be used to enhance compliance with ITN use also during the hot dry season. Compliance with ITN use is in fact likely to drop in the hot dry season as net use becomes rather inconvenient due to climatic changes and as the incentive to protect oneself from mosquito nuisance is reduced given the lower mosquito density ,29,49. AThe effectiveness of ITN interventions depends to a large degree on human behaviour as a factor in health, influenced by socio-cultural traditions as well as economic and environmental determinants. The failure of previous malaria control programmes has been attributed to the lack of adequate consideration given to these aspects which must be taken into account and should be routinely incorporated as an essential feature into control programmes.In conclusion, compliance with ITN protection in young children under programme conditions was shown to be sufficient during the rainy season but rather low during the hot and dry season in a malaria holoendemic area of Burkina Faso. Greater emphasis needs to be placed on information/education efforts to make people aware of the fact that the risk of contracting malaria often persists throughout the year.C Frey, C Traoré, B Kouyaté and O Müller designed the study. C Frey and C Traoré conducted and supervised the field work. C Frey, M De Allegri and O Müller analysed the data. All authors contributed to the writing of the paper. O Müller was the principal investigator.


During blood feeding, the mosquito injects saliva into the vertebrate host. This saliva contains bioactive components which may play a role in pathogen transmission and in host-vector relationships by inducing an immune response in the vertebrate host. The evaluation of human immune responses to arthropod bites might also represent a research direction for assessing individual exposure to the bite of a malaria vector.Anopheles gambiae bites in young children living in a malaria endemic area. Immunoblots were performed with An. gambiae saliva to detect anti-saliva Ab bands and the evolution of immunogenic bands at the peak of, and following, the transmission period.The present study examined the antibody (Ab) IgG response during the season of exposure to Anopheles Ab was directed against a limited number of salivary proteins . Specific IgG responses to mosquito salivary proteins were variable among exposed individuals; nevertheless, two major bands (175 and 72 kDa) were observed in all immune-responder children. Analysis of the intensity of immunogenic bands revealed that IgG levels against the 175 kDa band were significantly higher during the peak period compared to the end period malaria transmission.The results showed that anti-Anopheles bites. The use of immunoblots coupled with evaluation of band intensity could be an adequate tool for distinguishing immunogenic salivary proteins as candidate markers of bite exposure. Furthermore, this study may open the way to design new epidemiological tools for evaluating the risk of malaria exposure.This preliminary work supports the potential of using anti-saliva immune responses as a measure of exposure to  Anopheles vector. Among the 60 Anopheles species which transmit malaria worldwide, Anopheles gambiae is the most competent and the primary vector of Plasmodium falciparum in sub-Saharan Africa [P. falciparum can be measured by several diagnostic tests, such as thick smears or rapid diagnostic tests  [Morbidity and mortality of malaria are closely linked to exposure of the human host to the n Africa . Human iipstick) . The evaipstick) .An. gambiae [Anopheles species and little is known about the human host response to Anopheles saliva. The Ab response to Anopheles stephensi saliva has been detected in allergic individuals [An. stephensi saliva, which is recognized by sera from individuals exposed to malaria [An. gambiae was found in children living in a malaria endemic area and was correlated with the intensity of exposure to An. gambiae bites [Anopheles bite. Thus, the objective of the present work was to describe immunogenic proteins of An. gambiae saliva so as to elucidate whether detection of immunogenic components depends upon the malaria transmission season.During the bite, the female mosquito injects saliva containing bioactive molecules such as vasodilatators, anti-clotting and anti-hemostatic proteins, which permit a successful blood meal . During  gambiae -8. In pa gambiae ,10. Inde gambiae . Interes gambiae -14, and  gambiae . Surprisividuals . Owashi  malaria . A recenae bites . That stP. falciparum transmission from September to November [The present study was carried out in Niakhar, a rural area of Senegal situated 115 km east of Dakar. This site is a dry savannah with a rainy season from July to October. The region is typical of the Sahel and sub-Sahel regions of Africa, where the occurrence of malaria is unstable, with a season of November . Sera weNovember . Sera fr®II apparatus .All water used was of 18 MΩ quality and was produced using an EASYpureAn. gambiae s.s. were bred in an insectarium in Dakar (IRD-UR77). Saliva from 10- to 20-day-old uninfected An. gambiae females was collected as previously described [Adult female escribed ,21. SaliOne-dimensional electrophoresis (1-DE) was carried out using Criterion XT- Bis-Tris 4 to 12% acrylamide gels . Saliva from 48 mosquitoes was loaded on each gel and run with XT-MES buffer according to the manufacturer's instructions. Gels were silver-stained as described by Poinsignon et al. .Gels were transferred onto a PVDF membrane  for 2 h, 400 mA. Free binding sites on the membrane were blocked by incubation in 5% dry milk dissolved in TBS for 45 min at room temperature. After washing three times with TBS Tween 0.1% (TBST), membranes were incubated overnight at 4°C with serum samples diluted 1/100 in blocking buffer. The membranes were then incubated with alkaline-phosphatase mouse monoclonal anti-human IgG antibody at a dilution of 1/5000  for 2 h at room temperature after three washes in TBST. The membranes were revealed with chemoluminescent substrate  and exposed to CL-Xposure film (Pierce) for 5 s. Prestained standards  were used to determine the relative molecular weights of the electrophoresed components. The peak and post-season serum samples of each subject were consistently examined in the same immunoblot experiment, and a positive control serum (pool of responder children) was used for each immunoblot. The films were scanned by an "Image scanner II"  and the intensities of immunogenic bands were analysed by the Scion Image Program (Scion Corporation). A numeric value was calculated for each band by analysing the mean intensity of staining. Results represented a ratio sample band/standard band and were expressed as integrated OD (iOD).® (Graphpad Software Inc). After verifying that values in each group did not assume a Gaussian distribution, the non-parametric Wilcoxon matched pair was used to compare the intensity of immunogenic bands between September and December. Differences were considered significant at P < 0.05.Data were analysed with a Graph Pad PrismAn. gambiae, which showed numerous bands ranking from 10 kDa to > 100 kDa  and December . Immunoblots revealed substantial individual variations in intensity and number of detected immunogenic proteins. The IgG Abs reacted with a few salivary proteins with molecular masses of 175, 115, 72 and 30 kDa  was measured for each paired serum from studied children. The comparison of intensities of the IgG Ab bands between peak- and post-malaria season sera Figure  showed aAn. gambiae salivary proteins. Interestingly, two major bands (72 and 175 kDa) were detected in all positive individuals. The results also showed a significant decrease in the intensity of the 175 kDa immunogenic band between the peak and the end of the malaria season.The present study reported that children living in a malaria endemic area developed an IgG response against a limited number of Anopheles salivary glands; one is an anti-inflammatory factor described in An. stephensi salivary extracts [An. gambiae saliva [This immunogenic band did not appear to be strongly expressed in saliva, as it did not match abundant proteins clearly visible on silver-stained gel. Along this line, sparse reports mentioned high molecular masses protein in extracts , and theextracts . A putatextracts . But thee saliva ,8. DiscrAnopheles saliva. Previous studies in the field of allergy to mosquito bites have reported considerable variations of Abs response associated with mosquito species and population densities [versus 60% in the low exposed village. However, a spatial heterogeneity in malaria transmission is also observed elsewhere depending on the repartition of individuals at the village level [Anopheles mosquito that may be insufficient to elicit anti-saliva Abs [Anopheles bites [Anopheles exposure are needed to determine whether one immunogenic band could reflect a "pattern" of specific exposure. Several epidemiological parameters, such as genetic background or the possibility of co-infections could also involved in the variations in immunogenicity observed between children [Surprisingly, only half of the children developed IgG Abs against ensities ,25,16. Hge level . Some ofliva Abs . In addies bites . Finallyes bites ,28 who hes bites . The reschildren -31.Anopheles salivary antigens was transient, as previously observed in the same area [Anopheles exposure, especially in areas with low intensity of exposure. Interestingly, the seasonal influence was significant for the 175 kDa band suggesting that this band could represent a good candidate for a marker of intensity of exposure to Anopheles bites. The results also showed a positive association with the intensity of immunogenicity for the 175 kDa band and the malaria morbidity in children over the same period. Indeed, the children developing malaria morbidity showed the higher intensity for this band in September which decreased during the malaria season. A previous study reported that the children presenting high IgG Abs in September are those will suffer from malaria attack within the next three months [Anopheles saliva in children living in malaria endemic area. Few studies have described the anti-saliva Abs response to mosquito bite in children [Anopheles bites have common immunogenic bands suggesting that major common epitopes could be found in exposed populations. Theses results represent the first step toward the identification of immunogenic protein as a marker of exposure. Studies in other areas of malaria transmission and within a larger population were necessary to reinforce this hypothesis. But the methodology presented here, associating immunoblots and measurements of band intensity, appears very convenient for initial screening of molecules with an immunological and epidemiological interest. It provides qualitative and semi-quantitative data on the immunogenic components detected. Furthermore, such an approach could be applied to other vector-borne diseases which represent public health priorities in the developing world.The results presented here show a seasonal decrease in the intensity of immunogenic bands suggesting that the Ab response to ame area . As a maame area  and cleaame area . This tre months . Howeverchildren  whereas Anopheles vectors [The last ten years have witnessed a renewed interest in host/vector studies for controlling vector-borne diseases. Major efforts have been made in sequencing the genomes of most "killing" vectors  and the  vectors ,23,34. TSC carried out immunoblot analysis, participated in the design of the study and drafted the manuscript.FR conceived and coordinated the study and helped to draft the manuscript.SD carried out protein preparation and immunoblots.TN carried out mosquito saliva collection.FXS participated in gel electrophoresis.DB provided the scientific environment in Dakar and revised the manuscript.FS participated in the conception and coordination of the study and helped to draft the manuscript.All authors approved the final draft of the manuscript.


Because of the nature of the analysis used in this study , no conc


A high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA) but no intracellular parasites, support the idea of pitting.in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC) co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide). Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive.An Co-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% ± 1.31% macrophages with engulfed trophozoites) as opposed to erythrophagocytosis (5.33% ± 0.95%) (P < 0.01). Following the interaction, 26.9% ± 8.1% of the RBCs were spherocytes as determined by flow cytometric reduction in eosin-5-maleimide binding which detects RBC membrane band 3. The median of patient RBCs with pitted parasites  was more than 3 times  that of RESA+, PI+ RBCs  (P < 0.01). RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation.It is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes. In the malaria holoendemic area of Lake Victoria basin, the estimated inoculation rate averages one infective bite daily and, as a consequence, prevalence rates of asexual stage malaria parasites in children is almost 100% . In an aPlasmodium falciparum malaria [Animal experiments conducted in late 60s suggested that the spleen can remove intraerythrocytic parasites while leaving the host erythrocyte intact, a process referred to as 'pitting' ,5. This  malaria ,6 has re malaria ,6. More  malaria . Whether malaria  that areP. falciparum in the RBC leads to membrane insertion of parasite antigens such as RESA and P. falciparum erythrocyte membrane protein-1 (Pf EMP-1), and also induces profound modifications to the erythrocyte integral membrane proteins such as Band 3, which then becomes the sites for deposition of immunoglobulins or complement [The growth of mplement . All themplement -12. A complement . The triin vitro model system of studying the process of pitting in an environment that is devoid of host factors is also described. This model may provide a method of identifying RBC membrane changes that trigger macrophages to engage iRBCs and, thereby, initiate the pitting process. The in vivo relevance of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital.This paper reports on high prevalence of spherocytes in children that are constantly exposed to malaria and provides evidence that this may be caused by pitting of malaria parasites. An P. falciparum parasitaemia and a haemoglobin level of ≤ 5 g/dL, and two controls, a symptomatic control (SC) and asymptomatic control (AC), each matched by age and gender to a case. SC were children with asexual P. falciparum parasitaemia and haemoglobin greater than 5 g/dL. AC were healthy children recruited from the same village as the cases. From each group, 25 thin blood films were examined by microscopy for abnormal RBC morphology. The haematologist slide's examiner had no prior information on the clinical status of the children. Blood specimens were also obtained from another group of 25 patients who were recruited in a case control study at Kisumu Ditrict Hospital. In this study, cases as defined above were matched with symptomatic controls of similar age and gender (± 2 months). In both studies, the protocols were approved by the Ethical Review Committee of the Kenya Medical Research Institute (KEMRI).Giemsa-stained thin blood smears were made from patients enrolled in a case control study whose details have been described earlier ,12. BrieP. falciparum, NF54, was cultured in normal O+ RBC from healthy donors and maintained in vitro using continuous culture conditions [g and the pellet re-suspended in 6 mL 5% D-sorbitol. After 10 min incubation at room temperature, the cells were washed twice in malaria culture medium, diluted to 5% haematocrit and cultured as described above. This treatment was repeated every 48 hours until all the parasites were at ring stage as confirmed by microscopy. At 8% trophozoite parasitaemia, the iRBC were harvested by Percoll gradient purification method. Briefly, Percoll gradients were prepared by adding 10 mL of 90% Percoll to the bottom of a 45 mL Oakridge tube. Subsequent 5 mL layers of the 80%, 70% and 40% gradients were added consecutively. Parasites were suspended in 5 mL of RPMI 1640 and layered on top of the 40% gradient. This preparation was centrifuged in a SS34 fixed angle rotor  at 10,000 rpm for 20 minutes, at 20°C. Trophozoite infected erythrocytes were removed from the second interphase and washed twice in RPMI 1640 containing 0.5% bovine serum albumin.A laboratory strain of nditions . Parasitnditions . Briefly5 cells/mL in a humidified incubator at 37°C, 5% CO2.THP-1 cells were obtained from American Type Culture Collection  and were maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum, 0.05 mM 2-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 1.0 mM sodium pyruvate, 2.5 g/L D (+) glucose and 2 g/L sodium bicarbonate at cell densities of 3 × 106/mL in 6 mL medium containing TNF-α at a final concentration 4 ng/mL. This treatment did not affect cell viability as determined by trypan blue exclusion. Activated monocytes were washed once in RPMI and co-incubated with 1 × 106 trophozoite iRBCs in a water bath for 1 hour at 37°C. The cells were washed twice by centrifugation and the resulting pellet resuspended in 80 μL of RPMI. Giemsa-stained thin smears were made using 10 μL of the cell suspension. Thereafter, the smears were examined by microscopy to identify: 1) pitted malaria parasites, 2) spherocytes and 3) the number of RBCs with and without the parasites.Three triplicate THP-1 cultures were grown overnight (17–19 hours) in sterile 15 mL polypropylene tubes at a concentration of 3 × 10The EMA dye binds quantitatively to the RBC integral membrane protein Band 3 and can be used to reveal RBC membrane loss . EMA pre6) were washed in PBS containing 1% BSA. The cells were then incubated with 1 mL 0.05% glutaldehyde for 30 minutes at room temperature. The glutaldehyde fixed cells were washed and permeabilized with 500 μL 1% Saponin in PBS for 5 minutes at room temperature. After two washes the cells were either resuspended in PBS containing 1 μg/mL RESA Mab, clone 28/2  and or in PBS containing 50 μg/mL propidium iodide and incubated at room temperature for 30 minutes. The cells were then washed twice with PBS and a FITC conjugated goat anti-mouse IgG-Fab fragment  added and cells incubated for 30 minutes at room temperature. RBCs were gated on the basis of their logarithmic forward and side scatter characteristics. 10,000 events were acquired and the proportions of RBCs that were RESA positive detected in the FL1 channel and those that were PI positive in the FL3 channel.For flow cytometry, patient RBCs  added and the slides incubated as above. The slides were washed twice with PBS and examined using a BX40 fluorescent microscope , equipped with a Magnafire digital camera.Welch corrected t test was used to compare the number of monocytes with engulfed parasites against those with engulfed iRBCs from a set of six experiments. Results are given as mean and standard error of the mean (SEM). For detection of proportion of RBCs that were both RESA+ and PI- and those that were RESA+ and PI+, the % RBCs population identified by flow cytometry was multiplied by total RBC count in each sample as determined by Coulter. The Mann-Whitney test was then used to compare proportion of RBCs that were both RESA+ and PI- and those that were RESA+ and PI+. Dye binding results are expressed as median channel fluorescence (MCF) and SEM. The Kruskal-Wallis test was used to compare the dye binding results for the un-interacted iRBCs, interacted normal RBC and interacted iRBC.As shown in Table The success of pitting experiment was dependent on the ability to: 1) identify pitted malaria parasites from intact iRBCs co-incubated with the monocytes, and 2) detect increase in the number of uninfected RBCs as a result of this interaction. This required having a stage of malaria parasite that does not rupture during experimental manipulation and having a near 100% purified iRBCs before the interaction. The preferred method of preparing different stages of malaria parasites is by Percoll gradient. It was technically impossible to separate non-infected RBCs from ring stage iRBCs. But, while it was possible to obtain pure preparation of schizonts iRBCs by Percoll, during the 1 hour co-incubation with monocytes, many were found to have ruptured and it was, therefore, difficult to tell whether the engulfed schizonts were extracted from iRBCs or were from already ruptured parasites. Pure preparation of trophozoites iRBCs was easy to obtain by Percoll separation and they remained intact during the period of interaction with monocytes as confirmed by light microscopy. Therefore, all the experiments were conducted using trophozoites iRBCs.For some reason, pitting was optimal when trophozoite iRBCs and monocytes were used at a ratio of 1:3. After 1 hour of interaction at this ratio, 14.0% ± 1.31% of the monocytes (mean of six experiments) were noted to contain malaria parasites in their cytoplasm as opposed to only 5.33% ± 0.95% found to have engulfed whole iRBCs  lower than that of un-incubated iRBC Figure  and 2B. EMA staining did not reveal presence of spherocytes in the RBCs of the 25 patients attending Kisumu District Hospital (results not shown).Patient RBCs were probed for "footprint" of previous parasitization using anti-RESA Mab while patent infection was detected by propidium iodide Figures  and 4. UA retrospective examination of blood films made from children with malaria revealed high prevalence of spherocytes regardless of their clinical status or parasitaemia Table . It is nModel human monocytes THP-1) were used to demonstrate removal of intra-erythrocytic malaria parasites (Figure  were usein vitro study, Kumaratilake et al interacted human peripheral leukocytes with cultured P. falciparum and showed that extraction of intraerythrocytic malarial parasites was preferred to ingestion of infected erythrocytes [in vitro set up where macrophages were interacted with the much bigger trophozoite stages. It is conceivable that unlike in the trophozoite stages, pitting of the small ring stages would not involve removal of sizeable RBC membrane. Another explanation is that EMA is not sensitive enough to detect the small numbers of spherocytic cells that emanate from pitting. Fluorescent microscopy did however identify RESA+, P. falciparum negative RBCs that were spherocytic (Figure In an hrocytes . Howeverc Figure . EMA wouP. falciparum induces profound modifications to the erythrocyte membrane. It causes oxidative damage to proteins such as Band 3 while intraerythrocytic parasite growth causes insertion into the membrane of antigens such as RESA and Pf EMP 1 [In vivo, these alterations, particularly the haemichrome-mediated oxidation of Band 3, are thought to mark the iRBCs for immunoglobulin and complement deposition and subsequent erythrophagocytosis [in vitro model, erythrophagocytosis also occurred (Figure The growth of Pf EMP 1 ,9,20,22.d Figure  indicatiP. falciparum negative RBCs that are longer than that of parasitized RBCs [Patient RBCs were probed for "footprint" of previous parasitization using anti-RESA Mab. The proportion of previously parasitized RBC was consistently higher than that of RBCs with patent infection as determined by flow-ctytometry Figure  and immuzed RBCs .in vitro data from this study indicate that pitting of malaria parasites can produce spherocytes. Taken together with other studies that indicate that pitting of malaria parasites is common [In conclusion, s common ,19, and s common , it is ps common . It couls common . The neoin vitro model described here may prove useful in dissecting out those RBC membrane changes that trigger macrophages to engage iRBCs and thereby initiate the pitting or erythrophagocytosis process.Finally, the JNW designed the research, analysed the data and wrote the paper. SLS examined and interpreted the blood smears and helped write the paper. SBA performed the research. CWG helped in study design and writing of the paper.