Inexpensive and efficacious interventions that avert childhood deaths in sub-Saharan Africa have failed to reach effective coverage, especially among the poorest rural sectors. One particular example is insecticide-treated bed nets (ITNs). In this study, we present repeat observations of ITN coverage among rural Kenyan homesteads exposed at different times to a range of delivery models, and assess changes in coverage across socioeconomic groups.We undertook a study of annual changes in ITN coverage among a cohort of 3,700 children aged 0–4 y in four districts of Kenya  annually between 2004 and 2006. Cross-sectional surveys of ITN coverage were undertaken coincidentally with the incremental availability of commercial sector nets (2004), the introduction of heavily subsidized nets through clinics (2005), and the introduction of free mass distributed ITNs (2006). The changing prevalence of ITN coverage was examined with special reference to the degree of equity in each delivery approach. ITN coverage was only 7.1% in 2004 when the predominant source of nets was the commercial retail sector. By the end of 2005, following the expansion of heavily subsidized clinic distribution system, ITN coverage rose to 23.5%. In 2006 a large-scale mass distribution of ITNs was mounted providing nets free of charge to children, resulting in a dramatic increase in ITN coverage to 67.3%. With each subsequent survey socioeconomic inequity in net coverage sequentially decreased: 2004 ; 2005 , and 2006 with near-perfect equality . The free mass distribution method achieved highest coverage among the poorest children, the highly subsidised clinic nets programme was marginally in favour of the least poor, and the commercial social marketing favoured the least poor.Rapid scaling up of ITN coverage among Africa's poorest rural children can be achieved through mass distribution campaigns. These efforts must form an important adjunct to regular, routine access to ITNs through clinics, and each complimentary approach should aim to make this intervention free to clients to ensure equitable access among those least able to afford even the cost of a heavily subsidized net. Noor and colleagues found low levels of use of insecticide-treated mosquito nets when nets were mainly available through the commercial sector. Levels increased when subsidized nets were introduced and rose further when they were made available free. Malaria is one of the world's most important killer diseases. There are over a million deaths from malaria every year, most of those who die are children in Africa. Frequent attacks of the disease have severe consequences for the health of many millions more. The parasite that causes malaria is spread by bites from certain species of mosquito. They mostly bite during the hours of darkness, so sleeping under a mosquito net provides some protection. In some countries where malaria is a problem, bed nets are already used by many people. A very much higher level of protection is obtained, however, by sleeping under a mosquito net that has been impregnated with insecticide. The insecticides used are of extremely low toxicity for humans. As insecticide-treated nets (ITNs) are a relatively new idea, people do need to be persuaded to buy and use them. ITNs must also be re-impregnated regularly, although long-lasting ones that remain effective for 3–5 y (or 21 washes) are now widely distributed. The nets are inexpensive by Western standards but the people who are most at risk of malaria have very little income. Governments and health agencies are keen to increase the use of nets, particularly for children and pregnant women. The main approach used has been that of “social marketing.” In other words, advertising campaigns promote the use of nets, and their local manufacture is encouraged. The nets are then sold on the open market, sometimes with government subsidies. This approach has been very controversial. Many people have argued that ways must be found to make nets available free to all who need them, but others believe that this is not necessary and that high rates of ITN use can be brought about by social marketing alone.It has been known for more than ten years that ITNs are very effective in reducing cases of malaria, but there is still a long way to go before every child at risk sleeps under an ITN. In Kenya, a country where malaria is very common, a program to increase net use began in 2002, using the social marketing approach. In 2004 most of the nets available in Kenya were those on sale commercially. In October 2004 health clinics started to distribute more heavily subsidized ITNs for children and pregnant women and, in 2006, a mass distribution program began of free nets for children. The researchers, based at the Kenya Medical Research Institute (KEMRI), wanted to find whether the number of children sleeping under ITNs changed as a result of these changes in policy. They also wanted to see how the rate of net use varied between families of different socioeconomic levels, as the poorest children are known to be most likely to die from malaria.This is a large study involving 3,700 children in four districts of Kenya. The researchers conducted surveys and then calculated the rates of net use in 2004, 2005, and 2006. In the first survey, when nets were available to most people only through the commercial sector, only 7% of children were sleeping under ITNs, with a very big difference between the poorest families (3%) and the least poor (16%). By the end of 2005, the year in which subsidized nets became increasingly available in clinics, the overall rate of use rose to 24%. By the end of 2006, following the free distribution campaign, it was 66%. The 2006 figure was almost exactly the same for the poorest and least poor families.The rate of net use in the districts in the survey is much higher than expected, even though one-third of children were still not protected by ITNs. The sharp increases—particularly among the poorest children—after heavily subsidized nets were introduced and then after the free mass distribution suggests that this is a very good use of the limited amount of funds available for health care in Kenya and other countries where malaria is common. If fewer Kenyan children have malaria there will be cost savings to the health services. While some might claim that it is obvious that nets will be more widely used if they are free, there has been heated debate as to whether this is really true. Evidence has been needed and this research now provides strong support for free distribution. The study has also identified other factors which will be important in the continuing efforts to increase ITN use.http://dx.doi.org/10.1371/journal.pmed.0040255.Please access these Web sites via the online version of this summary at malaria and on insecticide-treated nets (in English and Spanish)The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish). MedlinePlus brings together authoritative information from the US National Library of Medicine, National Institutes of Health, and other government agencies and health-related organizationsThe MedlinePlus encyclopedia contains a page on malaria  and from the Roll Back Malaria Partnership on the use of insecticide-treated netsInformation is available from the World Health Organization on Medical Research Institute see the organization's Web siteFor information about the KenyaThe BBC Web site has a “country profile” about Malaria Atlas Project Web site, which is funded by the Wellcome Trust, UK and is a joint project between the Malaria Public Health & Epidemiology Group, Centre for Geographic Medicine, Kenya and the Spatial Ecology & Epidemiology Group, University of Oxford, UKMalaria data and related publications can be found on the Kenya Ministry of Health, Division of Malaria Control Web site has useful information on malaria epidemiology and policies for KenyaThe  The gulf between levels of childhood mortality in sub-Saharan Africa and access to simple, cost-effective interventions known to significantly reduce mortality is immoral . For oveDuring the early days of RBM, the technical advice to countries provided by World Health Organization (WHO) was to create an “enabling environment” that allowed malaria-endemic countries to embrace multiple approaches to providing ITNs . These aThe best-practice debate has been driven by personal opinion ,7 or datPrior to the launch of Kenya's National Malaria Strategy in April 2001 , access In January 2002 the UK Department for International Development (DFID) awarded PSI-Kenya US$33 million over 5 y to socially market partially subsidised ITN within the existing retail sector. The programme, named PSI Coverage Plus, was the only major operational ITN distribution initiative between 2002 and 2004 and aimed to target urban and rural retail outlets with Supanet ITNs across all malaria-endemic districts in Kenya. A two-tier pricing system of 350 Kenya Shillings (KES)  in urban settings versus KES100 (US$1.3) in rural settings was implemented. The programme's aims were to increase community awareness of the value of ITNs thus creating a “net culture”; force existing retail prices down; and increase clients' willingness to pay for nets through a sustainable, unsubsidised commercial market [In June 2004, DFID approved an additional US$19 million to PSI to establish a parallel distribution system of heavily subsidised ITNs to children and pregnant women through Maternal and Child Health (MCH) clinics, recognizing that these vulnerable groups might not be able to access socially marketed commercial sector nets. The programme began in October 2004, and during the first 6 mo Supanet ITNs were bundled with separate Powertab net treatment tablets (for every 6 mo) and distributed to MCH attendees. In May 2005 an additional US$37 million was committed by DFID to PSI to procure and distribute Supanet-branded long-lasting insecticidal nets (LLINs), Olyset and Permanet. These public sector nets were heavily subsidized pretreated nets  and branded with the MoH logo [In February 2002 the MoH responded to the first call of the Global Fund to Fight AIDS, TB and Malaria (GFATM) for funding applications to secure five million nets and net treatments to provide free of charge to children under the age of 5 y and pregnant women. This application was unsuccessful. During round four of the GFATM awards in April 2004, Kenya's application was successful and US$17 million was approved to procure and distribute 3.4 million LLINs (Olyset and Permanet brands) free of charge to children under the age of 5 y. This represented, at the time, the largest successful award for free distribution of LLINs in Africa. The implementation of the free mass distribution of LLINs was arranged in two phases during 2006. During the first phase, 21 of Kenya's 70 districts were selected for distribution of LLINs from 8 to 12 July 2006 and integrated with the national measles catch-up vaccination campaign. Health facilities and centralised non-health facility posts were identified by the Kenya Expanded Programme on Immunisation and used as delivery points of both measles vaccine and LLINs to each child under the age of 5 y. A second mass distribution of LLINs, not integrated with any other intervention, took place from 25 to 27 September 2006 in 24 additional districts using previous mass vaccine campaign delivery centres as distribution points.The study was carried out in four districts purposively sampled in collaboration with the MoH to provide detailed longitudinal milestone data on changing access to interventions proposed within the Kenya National Malaria Strategy between 2001 and 2006 ,22. The http://www.esri.com/) and each polygon attributed to population totals derived from the last national census in 1999 [Within each district, rural enumeration area (EA) boundaries were digitized with ARCGIS 9.0 ; source of drinking water; type of sanitation facility; homestead size; and persons per sleeping room see .http://www.stata.com) and ARCGIS 9.0 (ESRI). All information specific to the EA, homestead, and mother or guardian were linked to the relevant child through the use of a primary homestead identifier consistent across all data sets. To account for unequal probabilities of selection of EAs, all results were weighted (weight = 1/probability of selection) and precision of proportions  were adjusted for clustering with EA as the primary sampling unit. A χ2 test was performed to compare net use proportions across subgroups within and between survey years. For comparisons of socioeconomic groups within a survey year, the Pearson χ2 statistic, accounting for clustering, was used. This statistic is turned into an F-statistic using the second-order Rao and Scott correction and p-values interpreted the same way as the Pearson χ2 statistic for data without clustering [Data entry and storage were undertaken using Microsoft Access, and analysis was undertaken using STATA version 9.2  derived from the first principal component were used to construct the wealth index . WeightsEthical approval was provided by the National Ethical Review Board IRB .A total of 2,761 homesteads were selected across the 72 rural communities located in the four districts in 2004. Three homesteads refused participation in 2004 (0.01%); 11,050 observations of children under the age of 5 y were made across the three survey years in 2004/5, 2005/6, and 2006/7. Over the three years, 155 (1.4%) usually resident children were visiting elsewhere at the time of the annual surveys, 66 (0.6%) children's parents or guardians refused to be interviewed and 133 (1.2%) children died before the census rounds in 2005/6 and 2006/7.In 2004/5 the weighted proportion of children usually using a net, adjusted for clustering, was 13.9%, similar to the proportions of children using a net the night before the survey . The smap < 0.001). Children living in the poorest homesteads had proportionately the largest (six-fold) increase in ITN use over the 12 mo interval, rising from 2.9% in 2004/5 to 17.5% in 2005/6 (p < 0.001); however, children living in the least poor homesteads were still twice as likely to have slept under an ITN the previous night as those in the poorest homesteads . The poverty concentration index declined from 0.131 in 2005/6 to 0.000 in 2006/7  and the PSI-MCH clinics (41%) . Within n 2006/7 . The conn 2006/7  further n 2006/7 .By the end of September 2006, the three principal net distribution strategies  were all operating in parallel, providing an opportunity to examine socioeconomic targeting of each of the delivery mechanisms . In 2006We have shown that a concerted, multi-pronged approach to ITN delivery in rural areas of Kenya over 3 y resulted in over 60% of children sleeping under a net treated with insecticide, surpassing the original RBM target set in Abuja in 2000 [In recent years, there has been a consensus among national ministries of health, development partners, and other stakeholders that access to health interventions should be made pro-poor . AlthougUnlike single cross-sectional surveys of ITN coverage, the value of the present study is its longitudinal observations among the same homesteads exposed at different times to different systems of delivery. Such data help inform debates on whether ITN delivery should be free or subsidized and whether they should be provided through routine clinics, mass campaigns, or the private sector. Our data clearly show that the most effective means to rapidly scale up ITN coverage, particularly among the poor, is through targeted free distributions, preferably coincidental with other campaigns such as mass vaccination. However, it does not necessarily follow that this should represent the only mechanism by which treated nets are distributed to rural communities.The PSI programme of heavily subsidized net delivery through over 3,000 clinics since October 2004 has reported over 5 million sales of nets . AlthougTo effectively integrate routine versus mass campaign ITN distribution requires careful planning. It is important that when planning integrated free mass campaigns, each component of the package is not jeopardized by the other, for example delaying immunization to meet the needs of ITN distribution. In Kenya, funding for the donor-supported PSI programme ends in 2007. Funds are available from the GFATM and the World Bank Booster Programme during 2007 and 2008 to continue LLIN distribution through mass campaigns. Replacing or “permanently” treating existing non-LLINs, covering future vulnerable pregnant women and their infants and expanding to pockets of the country where coverage has remained low all require long-term sustainable financing planned against projected spatially defined needs. These combined approaches require long-term evaluations, modelled on the approach presented, to monitor the synergies between delivery strategies to make sure they retain the equity required to reach those most in need. Whether funding for combined models of delivery comes from national budgets, direct budgetary support from donors, or through mechanisms such as the GFATM, Presidents Malaria Initiative, or the World Bank is beyond the scope of this paper. What we can say is that if funding is not secured for clinic supply and catch-up mass campaigns for LLIN delivery beyond 2008 the impressive, rapid progress toward the RBM target of 80% coverage by 2010 in Kenya will be lost.Table S1(45 KB DOC)Click here for additional data file.


Anaemia is difficult to recognize clinically, even by health workers.Ethnographic studies from East Africa suggest that cerebral malaria and anaemia are not classified in local knowledge as malaria complications, but as illnesses in their own right. Cerebral malaria 'Ethnographic longitudinal cohort field study for 14 months, with monthly home-visits in families of 63 newborn babies, identified by community census, followed throughout April – November 2003 and during follow-up in April-May 2004. Interviews with care-takers (mostly mothers) and observational studies of infants and social environment were combined with three haemoglobin (Hb) screenings, supplemented with reports from mothers after health facility use.General danger signs, reported by mothers, e.g. infant unable to breast-feed or sit, too weak to be carried on back – besides of more alarming signs such as sleeping all time, loosing consciousness or convulsing – were well associated with actual or evolving moderate to severe anaemia (Hb ≤ 5–8 g/dl). By integrating the local descriptions of danger symptoms and signs, and comparing with actual or evolving low Hb, an algorithm to detect anaemia was developed, with significant sensitivity and specificity. For most danger signs, mothers twice as often took young children to traditional healers for herbal treatment, rather than having their children admitted to hospital. As expected, pallor was more rarely recognized by mothers, or primary reason for treatment seeking.Mothers do recognize and respond to symptoms and danger signs related to development of anaemia, the most frequent complication of malaria in young children in malaria endemic areas. Mothers' observations and actions should be reconsidered and integrated in management of childhood illness programmes. In areas with intense malaria transmission, such as the lowlands of north-eastern (NE) Tanzania, anaemia is the most common manifestation of severe malaria , peakingThe World Health Organization (WHO) strategy for anaemia detection, under the Integrated Management of Childhood Illness (IMCI) , includedegedege' [While fever is recognized as the common manifestation of, even equalled to, malaria, and usually instigates prompt health seeking by caretakers, anaemia is a 'silent burden'  hiding begedege' -14, in segedege' . Ethnogregedege' -17.To investigate if pallor, weak body and possibly other ''danger signs" were known to caretakers of new-born babies, and which actions were taken against them, and at what stage, a longitudinal study was undertaken in NE Tanzania, with an ethnographic approach.An ethnographic approach was used, aiming at identifying practical knowledge of mothers, i.e. their 'clinical' skills for symptom or sign awareness, and the application of that knowledge at the time of illness . The stuFollowing a community census in early 2003, covering 2,500 households in Muheza town, Tanga Region, NE Tanzania, in which infants born within the five preceding months of the onset of the high malaria transmission season (in May) were eligible. 111 live-born infants were identified, and followed for a full annual cycle of malaria risk, acknowledging that vulnerability to malaria starts from about the age of four months, when maternal protective antibodies decline. Thus, infants' may include small children up to the age of 18 months. Because of other ongoing research projects involving infants, and migration of mothers to the countryside after delivery or marriage, only 63 infants were eligible to, and accepted inclusion by caretakers, while two had died, reportedly from pneumonia.Kiswahili, by the principal investigator and a local, respected female interpreter, during one-monthly home-visits of one hour duration, combined with observations of infant, care-takers and social environment. In case of illness, in-between visits took place.For each infant, caretakers, mostly mothers, were interviewed in-depth, in the local joint language, ® , supplemented by passive detection of Hb at health facility in case of clinic visits, as reported by mothers.On three occasions, by the end of the long rains, (high transmission period), the short rains, and at the onset of the next long rains, in August, November, and April, respectively, levels of Hb were estimated, by finger-prick blood and HaemoCueInfection and nutritional factors intertwine in anaemia development, but were deliberately not examined in this study, in order to avoid the caretakers being "induced" or the infants "over-researched".Ethical clearance was granted by the Scientific Committee of the Tanzania National Institute of Medical Research, NIMR, and the Tanzanian Commission for Science and Technology, COSTECH. Mothers volunteered after written consent, and were ensured of the confidentiality of the information they offered, and guaranteed anonymity. The principal investigator (PI) being a trained anthropologist, not a medical doctor, implied that medical investigations and interventions were only undertaken at health facilities and after referral, in response to care-takers' preferences. However, the project offered free access and admission to the local Maternal and Child Clinic (MCH) at/to the local district hospital. Following the procedure of the latter, infants with Hb ≥ 7 g/dl were told to mothers to have 'enough blood', while children with Hb < 7 g/dl, or with difficulties in breathing or prostration during interview were offered referral to hospital.Kiswahili terms most frequently used by mothers.Mothers' general knowledge of illness symptoms and signs in their babies reflected experience from having another child, previous clinic attendance, school attendance and traditional beliefs. Table Homa signifies febrile illness of any kind, joto kali high fever, and homa kali includes strong febrile illness sometimes with vomiting, diarrhoea and/or convulsions. Mchango is illness with convulsions, accompanied or starting with kustuka, meaning startled, frightened or shocked. Mchango is feared, because if untreated it may develop into kifafa, grand mal seizures of epilepsy, but this can be avoided by herbal prevention and treatment given during symptoms and at each following new moon, as tea, baths and fumigations of mother and child. Kulegea, weakened body, means becoming soft, as an overripe fruit, and is often accompanied by vomiting and diarrhoea. When breast-milk is vomited or not taken the milk is considered bewitched by a person with evil eyes: a form of witchcraft called zongo, which is also the name of the illness. The healer treats this by cleansing the mother together with her baby, who is given purgatives. Zongo is much feared, and speaking about it was a matter of trust,, and it took time for the investigator to realize and adapt to this. However, the regular visits and long-term investigation over more than a year made it clear that zongo was frequent, and that zongo is an important danger sign recognizing prostration or lethargy, cf. Table Kulegea was also sometimes thought to be caused by too strong  anti-malarials, as a side-effect of quinine, amodiaquine or sulfadoxine-pyrimethamine.Kiswahili word for anaemia upungufu wa damu, meaning lack of blood, was mainly used by hospitals as a diagnosis, while mothers were hesitant to suggest themselves that their child lacked blood, even if most had visited health facilities. However, some learning experiences of attending health facilities were reflected in mothers, who knew how to look for paleness of the body, kupauka, and considered kupauka as a condition arising from homa kali, strong fever  had to be reflected in pro- as well as retrospect; occasionally Hb was known from unplanned visits to health facilities. The median time between symptom appearance, reported by mothers, and actual screening for Hb was five weeks. A total of 21 infants developed moderate-severe anaemia (Hb < 8 g/dl) within six weeks from mothers' recognition of illness. Mothers' knowledge on 'malaria', fever and 'anaemia' and possibly related symptoms and signs were only asked for during an actual illness episode, and general interviews on malaria were deliberately not undertaken, to avoid induction of answers and knowledge bias, as indicated above. Interestingly, the kulegea (weakened or soft body), mchango (convulsions), zongo  and combinations hereof or other symptoms, including pallor. The main manifestations and actions taken by mothers appear from Table About two thirds of the 63 children had at one point suffered from what mothers considered severe manifestations: Mothers' recognition of symptoms lead to urgent action in a vast majority of cases, but 52 % went for traditional treatment plus minusallopathic ('western') health care. For manifestation mothers considered severe, almost three of every four mothers used traditional healing. Among 30 taking herbal/traditional treatment, 70% did so in conjunction with allopathic treatment with anti-malarials, at clinic or hospital.Paleness was recognized on 21 occasions by mothers, in 14 instances within six weeks of actual Hb estimation, which proved that 10 children in fact had low Hb; this manifestation was often accompanied by other manifestations, as indicated in Tables Furthermore, there was an increasing correlation between low Hb and general danger signs/symptoms, from being soft/weak and unable to sit on back, over breastfeeding failure to prostration and convulsions: from 58 to 82 % of infants had low Hb with presence of these manifestations, recognized early by their mothers.kulegea, zongo, and mchango, were pooled and are given in Table Maternal reports of general danger signs, besides of paleness, were significantly associated with present or developing moderate-severe anaemia. Those signs indicated in Table Thus, in more than 2/3 of infants with breast-feeding failure, prostration/lethargy and/or convulsion, anaemia was imminent, and should be addressed when managing infants and young children, whether febrile or not. So far, the problem is indeed addressed by the mothers, but not as anaemia.The present study confirms that symptoms and signs that may be related to malaria, including to anaemia are frequent among infants in Muheza town. Muheza district is an area of intense malaria transmission , where tKiswahili, particularly prostration/lethargy, unable to sit or to be carried on mothers back, or sleeping all time. These signs, designated kulegea or zongo, or convulsing, called mchango, appeared useful entry points for mothers' recognition, and action, and – in future – for health education and potential inclusion in the local Integrated Management of Childhood Illness (IMCI) algorithms, together with the less sensitive and specific pallor or paleness; kupauka. It should be noted that most mothers did not know or use the vocabulary used by local health staff for anaemia, namely upungufu wa damu.It is encouraging that in spite of more frequent use of traditional versus allopathic health systems, mothers were able to identify danger symptoms and signs early, and took action against them within 24 hours, as described in details elsewhere . As expected, paleness was less useful to indicate present or emerging low Hb, while more general danger signs, with well-known significance in the local language, degdege that are taken primarily to traditional healers, as others have indicated [The present study was not undertaken to try correcting what may be considered mothers' misconceptions and superstition regarding causes of infant illnesses, but it underscored that it is not only cerebral malaria-related symptoms such as ndicated -14. Whenndicated  that parKiswahili, it was found that the following signs: weakness, and more specific and sensitively, prostration/lethargy, inability to sit or be carried on mothers' back, or convulsions, correlated well with actual or developing low Hb. Furthermore, it was found that in cases where mothers feared that their children suffered from the much feared zongo or mchango, they mainly consulted traditional healers, who treated both mother and child. Mothers' concepts and observations deserve being incorporated in the local algorithms for integrated management of infant and young child illness, and traditional healers whom are often approached before or along with the established allopathic health system should be considered to be integrated as well, to help reducing the persistent high infant mortality in the area.This longitudinal, prospective ethnographic study found that mothers to infants in Muheza town, a high-transmission area, with anaemia being the main manifestation of malaria, and where the infant mortality remains high in spite of decades of research and interventions, are able to recognize danger signs related to malaria, including anaemia, early on, and to take treatment seeking action, often within 24 hours. By using the local joint language, FR planned and carried out the study and wrote 1.st draft of manuscriptICB outlined the 2.nd version of the manuscript, and together withHS helped designing the study and analysing the data, and supervised it.


On the island of Bioko , insecticide-treated nets (ITNs) have been the main tool used to control malaria over the last 13 years. In 2004, started an indoor residual spraying (IRS) campaign to control malaria. The purpose of this study is to asses the impact of the two control strategies on the island of Bioko , with regards to Plasmodium infection and anaemia in the children under five years of age.Two transversal studies, the first one prior to the start of the IRS campaign and the second one year later. Sampling was carried out by stratified clusters. Malaria infection was measured by means of thick and thin film, and the packed cell volume (PCV) percentage. Data related to ITN use and information regarding IRS were collected. The Pearson's chi-square and logistic regression statistical tests were used to calculate odds ratios (OR)In the first survey, 168 children were sampled and 433 children in the second one. The prevalence of infection was 40% in 2004, and significantly lower at 21.7% in 2005. PCV was 41% and 39%, respectively. 58% of the children surveyed in 2004 and 44.3% in 2005 had slept under an ITN. 78% of the dwellings studied in 2005 had been sprayed. In the 2005 survey, sleeping without a mosquito net meant a risk of infection 3 times greater than sleeping protected with a net hanged correctly and with no holes (p < 0.05).IRS and ITNs have proven to be effective control strategies on the island of Bioko. The choice of one or other strategy is, above all, a question of operational feasibility and availability of local resources. The fight against malaria in Sub-Saharan Africa has to be transectoral and tackled from the clinical, social and vectorial points of view, involving the local health structure  and international support programmes -3.The vector control campaigns are based on environmental sanitation and suitable environmental management, the implementation of educational programmes and the use of insecticides, either to impregnate fabrics  or to spray (indoors and outdoors). The majority of these directives are currently the basic pillars of the fight against vectors, except for the use of pesticides, which has to be highly regulated and their handling subject to strict control, according to the recommendations of the WHO .Despite the good results from controlling the transmission during the 50 s and 60 s with widespread spraying activities with insecticides from the organochlorine family (such as DDT) in various regions of the world ,6,7, (iiInsecticide-treated nets (ITNs) appeared to be an alternative to indoor residual spraying (IRS) and proved to be a good tool to reduce the morbidity-mortality among the most vulnerable groups (children and pregnant women) -15. The Despite what has already been stated, control campaigns based on IRS are being implemented in many countries of Sub-Saharan Africa. These are obtaining excellent results, in particular in those countries where the transmission of malaria is not constant or seasonal ,21.On the island of Bioko , ITNs have been the main tool used to control malaria over the last 13 years (1991–2004). At the start of 2004, a private initiative, funded by oil companies, was set up under the aegis of Equatorial Guinea's Ministry of Health, to implement a huge campaign to control malaria on the island. The campaign was based on IRS with pyrethroids.The aim of this study was to assess the impact of the two control strategies currently used on the island of Bioko with regards to Plasmodium infection and anaemia in children under five years of age.Equatorial Guinea, a Central African country located in the Golf of Guinea, consists of an island region and a continental region. Bioko (N 3° 43' and E 8° 43') is the main island, measuring 2017 km2 and with approximately 60,000 inhabitants. It is 30 km from the coast of Cameroon and its landscape is very mountainous, due to the volcanic origin of the island, with two main peaks: Mount Basilé (3012 m) in the north and Mount Biao (2009 m) in the south  and the second, one year later (February 2005). Sampling was carried out by stratified cluster survey: multi-stage in 2004 and two-stage in 2005. During the first survey, one child per household was chosen at random (non self-weighted sample), while all the children from the selected households in the second survey were sampled (self-weighted sampling by cluster).A blood sample was taken from the selected children to determine the malaria infection by means of the microscopical examination of a stained thick and thin film. Each sample was studied by two qualified laboratory technicians and a third technician was called in when there was a discrepancy in the result. Temperature and packed cell volume (PCV) percentage were also measured, with anaemia defined as a PCV percentage below 30% . A curatA database was created in Access 2000 to computerize the data collected on the form. STATA 8.2 statistical software was used to analyse the data. The non self-weighted design of the sample was taken into account and the data was weighted in accordance with the selection probabilities. The cluster design effect was also taken into account when calculating the 95%confidence intervals. The comparisons between groups were carried out using the Pearson's chi-square test. Logistic regression analysis was used to calculate the Odds Ratios (OR) of the infection's potential predictors and to compare the results between the two surveys. A p-value <0.05 was considered to be statistically significant.During the study conducted in February 2004, the number of selected households was 168: 143 in the urban area and 25 in the rural area, and 168 children under 5 were surveyed. In February 2005, the number of households selected for the study came to 279: 150 in the urban area and 129 in the rural area, and 433 children were surveyed.Plasmodium falciparum, 95% in the first study and 86% in the second.Out of the 162 blood samples taken in 2004 (six refused to have their blood taken), 66 were positive for malaria, meaning a prevalence of 40% [95% IC: 26–53%]. In 2005, the prevalence was significantly lower at 21.7% [95% IC: 13.1–30.3%], based on the 432 samples collected (114 positive). When the crude parasite rate (CPR) for the two years is compared, there are significant differences depending on location, age and use of nets. However, there were no significant differences in the percentage of children with anaemia  of the children surveyed in 2004 and 44.3% (95% IC: 31–57.7%) of those surveyed in 2005, had slept under a mosquito bed net the previous night. There were significant differences in the use of the mosquito net between the older and younger children, but no differences were found according to location in either of the two surveys  of the mosquito nets were not hung. With respect to hanging, 13 were incorrectly hung or were too small compared to the size of the bed (5%). 25% of the mosquito nets checked had holes.On the other hand, 78% of the dwellings studied in 2005 had been fumigated with insecticide the previous year and 31% (52) of those had been fumigated more than once. In order to determine the impact of using mosquito nets on the CPR among the children surveyed in 2005, three groups were established: children not protected by a mosquito net (268), children protected by a net without holes and correctly hung (107) and children protected by nets that were not hung correctly and/or had holes (57). There was significant difference of the CPR of these three groups. When the CPR of the surveyed children was compared to the IRS, it was noted that there were no significant differences in the CPR of the children from fumigated homes with respect to the CPR of the children from non-fumigated homes, 20% and 29%, respectively  during the dry season and exophilic behaviour (resting outdoors) during the rainy season [A factor to be taken into account with respect to the success of the control campaigns based on IRS would be the resting habit of the vector species ,31. On ty season . As the On the other hand, a clear association between sleeping or not under a mosquito bed net and Plasmodium infection was noted in both surveys. This data coincides with the results obtained in all the experimental studies conducted with ITNs ,14,15,18The difference found in the risk of infection between the different age groups could be associated to differences in the immunological status and the use of ITNs. It is known that, during the first months of life, the risk of infection is lower because there is still a degree of immunity from the mother. The risk of infection first increases with age and then decreases when the individual himself reaches a degree of immunity due to the numerous contacts with the parasite . With reWith respect to the prevalence of anaemia, a significant reduction was not noted as the result of using ITNs or IRS, contrary to what was seen in other studies ,15. The IRS and ITNs on the island of Bioko have proven to be effective control strategies. In some trials carried out with ITNs, where high population coverage was reached, a similar community protection effect was noted to that obtained using IRS ,15,37. TThe sustainability of any control strategy is conditioned by the availability of skilled national human resources and by the degree of economic investment, whether from private or state resources. The presence of economic interests in an area of high endemicity (e.g. the oil industry on the island of Bioko) fosters the implementation of control programmes with a high investment level, mainly with private resources . The disGP was involved in the design of the second survey, participated in the data collection and drafted the manuscript. MD was involved in the design of the surveys, performed the statistical analysis and interpretation, and drafted the manuscript. EC was involved in the design of the first survey, participated in the data collection and helped to draft the manuscript. LM was involved in the design of both surveys, participated in the collection of the data and helped to draft the manuscript. ML, CM, JO and AN participated in the collection of the data and carried out the laboratory diagnosis. JR participated in the design, the interpretation of statistical analysis and drafted the manuscript. AB participated in the design of the surveys and has given approval of the version to be published. JC participated in the design, the interpretation of statistical analysis, and coordinated the draft of the manuscript. All authors read and approved the final manuscript.


Malaria microscopy, while the gold standard for malaria diagnosis, has limitations. Efficacy estimates in drug and vaccine malaria trials are very sensitive to small errors in microscopy endpoints. This fact led to the establishment of a Malaria Diagnostics Centre of Excellence in Kisumu, Kenya. The primary objective was to ensure valid clinical trial and diagnostic test evaluations. Key secondary objectives were technology transfer to host countries, establishment of partnerships, and training of clinical microscopists.A twelve-day "long" and a four-day "short" training course consisting of supervised laboratory practicals, lectures, group discussions, demonstrations, and take home assignments were developed. Well characterized slides were developed and training materials iteratively improved. Objective pre- and post-course evaluations consisted of 30 slides  with a density range of 50–660 parasites/μl, a written examination (65 questions), a photographic image examination (30 images of artifacts and species specific characteristics), and a parasite counting examination.To date, 209 microscopists have participated from 11 countries. Seventy-seven experienced microscopists participated in the "long" courses, including 47 research microscopists. Sensitivity improved by a mean of 14% (CI 9–19%) from 77% baseline (CI 73–81 %), while specificity improved by a mean of 17% (CI 11–23%) from 76% (CI 70–82%) baseline. Twenty-three microscopists who had been selected for a four-day refresher course showed continued improvement with a mean final sensitivity of 95% (CI 91–98%) and specificity of 97% (CI 95–100%). Only 9% of those taking the pre-test in the "long" course achieved a 90% sensitivity and 95% specificity, which increased to 61% of those completing the "short" course. All measures of performance improved substantially across each of the five organization types and in each course offered.The data clearly illustrated that false positive and negative malaria smears are a serious problem, even with research microscopists. Training dramatically improved performance. Quality microscopy can be provided by the Centre of Excellence concept. This concept can be extended to other diagnostics of public health importance, and comprehensive disease control strategies. Quality of laboratory medicine has always been a barrier to effective health care in the developing world. The issues and potential solutions have been recently illustrated by Petti, Bates and colleagues ,2. LaborMalaria microscopy remains the reference or "gold" standard for malaria diagnosis in clinical trials (drug and vaccine), new diagnostic methodology evaluation, and for clinical care for much of the world today. It is known that microscopy is an imperfect gold standard -5. The aNew diagnostic methods are now available, including malaria rapid diagnostic tests (RDTs) and PCR ,5,6; howSensitivity and specificity of the new diagnostic techniques have been evaluated based on microscopy. Variability in the accuracy of microscopy certainly accounts for some of the wide spectrum of reported sensitivity and specificity of these new devices -7. BecauErrors with microscopy and the need for uniform training, quality assurance, and standardized reporting methods have been noted in the literature ,9. A recWhere microscopy is available in the clinical setting, physicians often treat patients despite negative blood film reports, because of lack of confidence in the report . False pThis manuscript details problems, outlines pitfalls, and provides solutions for successful, validated malaria microscopy. These solutions will help provide valid results in the clinical trial setting when microscopy is used as the primary endpoint. The solutions presented are certainly not unique to microscopy. Lessons learned, solutions proposed, and the Centre of Excellence concept can be widely adapted to improve clinical trials and health care worldwide, especially in the developing world.The Malaria Diagnostics Centre of Excellence in Kisumu, Kenya was established in March 2004. The primary mission for the Centre of Excellence is to ensure valid microscopy when it is used to determine the primary endpoint in clinical trials or to assess new diagnostic methods. This mission is being accomplished through standardized and quality training, consistent methods documented in standard operating procedures (SOPs), a critically evaluated rereading paradigm (for blood film reading), quality control and assurance, and objective assessment and documentation of each microscopist's performance, in both the formal testing setting and in everyday job performance.Additional objectives are to train microscopists working in the developing world in the clinical setting, to transfer technology to host countries, to become internationally recognized, and to establish partnerships with organizations with similar missions. Transfer of technology to host countries is occurring through mentoring and motivating host country leadership, training trainers for other locations, and transferring training materials to other centers. The intention is to become internationally recognized through training leaders, offering/advertising capability, and by conducting, presenting, and publishing operational research results in areas of need.Seven expert microscopists from four continents  were identified beginning in 2001. Standardized training was developed through their experience, review of relevant training materials, and iterative improvement based on constructive feedback and training results over two years. Useful books and plates are available from WHO for purchase or download ,15.Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, true negatives, and serially diluted P. falciparum slides (for counting). Training slide sets have been generated for each organization to have on site for on-going training. Negative slides are prepared from non-exposed people newly arriving to endemic areas to avoid the issue of the low-parasite density healthy cases common in malaria endemic areas. Fully validated species identification and counting examinations are under development.Well-characterized slides were developed for formal slide reading examinations. Slide examinations comprised well characterized slides with low density parasitaemia of A written examination was developed and now consists of 65 multiple choice questions and a question bank to assess microscopists' knowledge of key concepts and facts. A digital picture test evaluation was also developed to teach and evaluate microscopists' ability to correctly identify characteristics of artifact and of various growth stages of each malaria species. PowerPoint presentations were developed to train didactic and visual concepts necessary for accurate microscopy. The test questions, pictures and presentations have been iteratively improved over time.Over the last four years, SOPs have been developed and iteratively improved at the Kombewa research site near Kisumu. These include SOP's for sample collection and reception, slide preparation, buffer preparation, stain preparation and staining of slides, slide reading, rereading paradigm, slide storage, and quality control. These SOPs are compared with the SOPs of the participants, and provided to course participants to take home.The venue for training is the Walter Reed Project Guesthouse lecture hall, located in Kisumu, Kenya. Laboratories at the nearby sites are used for laboratory practicals. Courses are conducted by experienced facilitators and senior microscopists. Currently, the Centre has three full-time staff , a part-time physician director, part-time laboratory officer, and additional three to six expert microscopists available for teaching. A twelve-day "long" course was designed for first time training, and a four-day "short" course is available as a refresher for those who performed well in the long course.Training targets malaria microscopists  from research and clinical laboratories. Entry requirements include at least a diploma or certificate in medical laboratory technology and one year of experience as a malaria microscopist. Attendees are currently required to be proficient in written and spoken English.Essential equipment included 23 microscopes, four computers, and two computer projectors. Fifteen high quality power point presentations with lecture notes, a slide bank of well-characterized malaria smears, a picture bank, WHO Bench Aids for the Diagnosis of Malaria , WHO BasTeaching methods consist of supervised laboratory practicals, open laboratory time, lectures, group discussions/presentations, demonstrations, and take home assignments. Objective assessment include a pre- and post-training written test, positive-negative slide test, species identification slide test, counting slide test, artifact and species identification picture test, and slide preparation practical. The agenda for the "long" course is presented in Table P. falciparum, one was P. ovale, and one was P. malariae. The density range of the positives was 50–660 parasites/μl, with a mean of 220 and median of 142. Of the positive slides, one was 50 parasites/μl, five were between 100–200 parasites/μl, and five were between 201–660 parasites/μl.A written test comprising 65 questions was given as a pre- and post-test. The following areas were covered: Good Clinical Laboratory Practice/standard operating procedures (32% of test questions), malaria parasite morphology/stages (31%), slide preparation and staining (14%), microscopy results interpretation (11%), microscope care/knowledge (5%), general malaria knowledge (3%), sensitivity/specificity calculation (2%), and density determination (2%). A practical slide reading examination of 30 slides was given before and after the course. Nineteen slides were true negative, 11 were positive, and of the positive slides, nine were Staphylococcus aureus and fungi) at varying densities (1/100 high power fields -2 per high power field). Negatives were validated as negative by sampling criteria and by giving each of the 209 participants the opportunity to challenge the results with the Centre experts.Negative slides were collected in Jakarta and Bangkok from healthy individuals in malaria free areas, who had not traveled to malaria endemic areas or taken malaria prophylaxis in the past two years or had malaria in the last 10 years. In Kenya, slides were collected from newly arrived previously non-exposed individuals within five days of arrival to Kenya. This was to ensure that the donors were truly negative. Thirty-two percent of all true negative smears were spiked with artifact . MR4 positives were determined to be positive and species identified by two expert readers in Indonesia with confirmation by three independent expert readers at the Centre. These were supplemented by slides collected in Kenya containing P. falciparum, P. malariae, and P. ovale. These were independently confirmed positive by one expert from Thailand and three experts at the Centre. Positive smears were further validated by giving each of the 209 participants the opportunity to challenge the results with the Centre experts (asking staff to identify the parasites for them).Sixty-six percent of positive smears were from the Malaria Research and Reference Reagent Resource Center (MR4) collection effort in Cambodia and contained The slides used in practical slide reading examinations were identical, but renumbered in the post-examination. Participants were given five minutes per slide for reading. One minute was provided between slides. They were instructed to read 200 high power fields before calling the smear negative. The presence or absence of parasites and the species was recorded.P. falciparum, four P. malariae, five P. ovale, two Plasmodium vivax, and three mixed species with various stages present.Ability to properly identify species and artifact was assessed in a photographic image examination. The images included 11 without and 19 with malaria parasites. Of those without parasites, various artifacts were present. Images with parasites were distributed as follows: five A slide preparation practical emphasized correct sample collection methods for making malaria blood smears, smear preparation, preparation of stains, and individual slide staining. Participants were then expected to make smears using their own SOPs and the Walter Reed Project SOPs. Five smears from each SOP were then assessed on a standardized form, and the participant assigned a passing or failing grade.Written examination questions were scored as one point each. Practical slide reading examinations were reported as sensitivity, specificity, and species error. Sensitivity was calculated as Sensitivity = . Specificity was calculated as Specificity = . Data were entered into, verified in, and calculations performed with Microsoft Office Excel 2003. Further statistical analyses were performed and tables constructed with SPSS 14.0 for Windows. Pre- and post-test means were compared with paired sample T-tests. Means were compared with ANOVA, and proportions with Fisher's Exact Test (2-sided). P values were not corrected for multiple comparisons.To date, 209 microscopists have participated from the following countries: Kenya (178), Uganda (10), Tanzania (5), Rwanda (1), Burundi (1), Malawi (6), Cameroon (3), Mali (2), Nigeria (2), and Thailand (1). One hundred and fifty eight participants (76%) have been primarily research and 51 (24%) have been primarily clinical. Leadership/expertise has participated from Kenya (4), Uganda (1), Nigeria (2), Mali (1), Cameroon (1), Zanzibar (2), Thailand (1), Peru (1), USA (1), and Indonesia (1).Data from the courses between July 2005 and January 2006 are reported here. These data include 77 participants in the long course (including 10 who trained twice) and 23 participants in the short course (including five who trained twice). Of these, 69% were conducting primarily research. Participants were from Kenya (81), Tanzania (5), Malawi (4), Rwanda (1), Burundi (1), Cameroon (2), Nigeria (2), Mali (2), and Uganda (2). On a questionnaire, participants reported a mean (median) of years of laboratory education 3 (3), years employed as a laboratory technician or technologist 9 (6), and years employed as a malaria microscopist 5 (3). Twelve percent reported that they were laboratory supervisors, 30% reported that they had a malaria proficiency program in their laboratory, and 84% reported that they used SOPs in their laboratory.Pre- and post-training scores and percent improvement by organizational group and course type from the most recent participants are presented in Additional File Staphylococcus aureus with fungi at varying densities). On the pre-test, 26% of those spiked with artifact were read as false positives, while 18% of those not spiked were. On the post-test, 8% of those spiked with artifact were read as false positives, while 6% of those not spiked were. The slides with the highest density artifact had higher rates of false positives. Spiked slides did not appear to affect the species reported, except for a slightly higher rate reported as P. vivax in the post-test. The false positive smears reported as P. vivax and P. ovale increased overall in comparing the pre-test with the post-test, while the reporting of P. falciparum decreased.Thirty-two percent of all true negative smears were spiked with artifact  densities of those with false negatives on the pre-test were 174 (128) parasites/μl compared with 216 (142) parasites/μl for the true positives. The densities were 133 (120) and 216 (142) parasites/μl for the false negatives and true positives on the post-test, respectively.Short course participants were selected from those who previously scored well in a long course. Comparison between the long and short course scores in Additional File The percentage of participants achieving sensitivity and specificity cut points on final examination by organization and course type is presented in Additional File CoE microscopy training appeared to substantially contribute to obtaining a level of 90% sensitivity and specificity on the pre-test, post-test, or both. Those that passed neither had a mean (sd) of 0.14 (0.50) courses attended previously, while those that passed just the post-test had a mean of 0.5 (1.0) courses attended previously, and those that passed both had attended 1.1 (1.6) course previously (p = 0.004). Use of SOPs, having a malaria proficiency program in the laboratory, and number of Good Clinical Practice trials conducted appear to correlate with ability to pass at a 90% sensitivity and specificity level . Years of training, years of experience, being a laboratory supervisor, and Good Clinical Practice courses did not appear to affect ability to score well.These data clearly illustrate that malaria microscopy is problematic. The ultimate goal should be its replacement by affordable, more robust diagnostic techniques, where valid microscopy is not achievable. The Centre will help ensure that microscopy is providing a valid "gold standard" comparator as these new tools continue to become available and need evaluation for regulatory approval and operational assessment. As long as microscopy is used to determine the primary endpoint in malaria prevention and treatment trials, great lengths must be taken to ensure that good products are not inadvertently discarded. A 1% false positive rate with serially collected smears can under estimate protective efficacy by 10–30% .Many of the participants describe the training as a life changing experience. They report they did not know they were not accurately diagnosing malaria. After attaining good test scores, they say they can now work with confidence. They report it is not just malaria diagnosis that will be improved, but all laboratory procedures they are involved in. Certification is strongly desired.The results are encouraging, but it remains to be seen if improved results in the classroom setting translate to the field, and if this concept is sustainable. Certification criteria are currently being developed along with a plan to certify microscopists at a basic level from a limited number of institutions in Kenya in the next twelve months. The plan is to also offer an advanced level and expert level of certification in the near future. Certificates will be valid for two years. For a broader impact, the plan is to train trainers from other Centres to enable development of other Centres of Excellence.Government sector laboratory technicians are having more difficulty achieving acceptable scores in the long training course , false negatives (sensitivity), species error (wrong species or missed mixed infections), and counting error ,9,18-22.Prestudy assessment of microscopists using a very limited number of slides may help, but will not completely solve the problem. For example, using blinded assessments of a well-validated 15 slide test set, qualified microscopists were identified for a recent malaria diagnostic pivotal trial. Subsequently, internal QC mechanisms determined that one senior microscopist still made errors in excess of 10%, requiring re-interpretation of hundreds of slides and a substantial delay in completion of the analysis .False negative smears are expected at low parasite densities (e.g. < 100 parasites/μl) by chance . InsuffiSpecies errors are common with low density infections, especially when only ring forms are seen ,21. MissSubstantial variability in counting results is a common problem. Counting errors may impact clinical trials if parasite density is an endpoint . O'Meara and colleagues have explored the factors affecting variability and assessed a grid method for improving accuracy .Several factors will lead to improved endpoints and confidence in results in both the clinical trial and clinical setting. Suggestions are as follows for clinical trial setting: proficiency training and testing leading to certification of microscopists at varying levels of expertise, a rereading paradigm specifically designed for the situation, SOPs, quality control and assurance, laboratory standards, and reporting guidelines.RDT: Rapid Diagnostic Test, QA/QC: Quality Assurance/Quality Control, ACTs: Artemisinin-based combination therapy, MR4: Malaria Research and Reference Reagent Resource Center, NIH: National Institutes of Health, SOP: Standard Operating Procedure, WHO: World Health Organization, CoE: Centre of Excellence, GCLP: Good Clinical Laboratory PracticesCO served as the Principle Investigator for funding and execution of project; led evaluation of a problematic clinical trial, conceived the Malaria Diagnostics Centre of Excellence concept, assisted with CoE design, teaching, data collection, analysis, interpretation of data and wrote this manuscript. AN was the lead visiting expert microscopist, and assisted with CoE design, teaching, data collection, analysis, interpretation of data. PO is lead microscopist/laboratory technician at the CoE who assisted with CoE design, teaching, data collection, analysis, interpretation of data. CA is lead administrator of CoE, assisted with CoE design, teaching, data collection, analysis, interpretation of data. KA was a key facilitator of the CoE during its inception, led CoE design, assisted with teaching, data collection, analysis, interpretation of data. WPO was a visiting scientist to the CoE, assisted with teaching, conception and design of data capture, revised manuscript critically for substantial intellectual content. SR was responsible for day-to-day management of CoE, assisted with CoE design, teaching, and data collection. KM was laboratory supervisor, Western Kenya; assisted with day-to-day management of CoE; teaching, and data collection. JC analyzed data; and revised manuscript critically for substantial intellectual content; EC assisted with CoE design, teaching, and interpretation of the data. CL was a lead visiting expert microscopist, and assisted with CoE design, teaching, data collection, analysis, and interpretation of data. JO was a CoE microscopist, who assisted with CoE design, teaching, and data collection. PM was a visiting microscopist who assisted with CoE design, teaching, data collection, analysis, interpretation of data. JSO and MLO were the Kenyan expert microscopists who interpreted all smears, proctored all laboratories, and were critical to developing all training materials. BO is Director of the CoE who led CoE design, teaching, data collection, analysis, interpretation of data. All authors read and approved the manuscript.Pre-test scores and percentage improvement following training for each examinationClick here for filePercentage of participants achieving sensitivity and specificity cut points on initial examination Percentage of participants achieving sensitivity and specificity cut points on final examination Improvement of participants achieving sensitivity and specificity cut points on initial examinationClick here for file


In 2003, artesunate-amodiaquine (AS+AQ) was introduced as the new first-line treatment for uncomplicated malaria in Burundi. After confirmed diagnosis, treatment was delivered at subsidized prices in public health centres. Nine months after its implementation a study was carried out to assess whether children below five years of age with uncomplicated malaria were actually receiving AS+AQ.A community-based study was conducted in Makamba province. Randomly selected households containing one or more children under five with reported fever onset within fourteen days before the study date were eligible. Case-management information was collected based on caregiver recall. A case definition of symptomatic malaria from observations of children presenting a confirmed malaria episode on the day of the survey was developed. Based on this definition, those children who had probable malaria among those with fever onset in the 14 days prior to the study were identified retrospectively. Treatment coverage with AS+AQ was then estimated among these probable malaria cases.Out of 195 children with fever on the day of the study, 92 were confirmed as true malaria cases and 103 tested negative. The combination of 'loss of appetite', 'sweating', 'shivering' and 'intermittent fever' yielded the highest possible positive predictive value, and was chosen as the case definition of malaria. Out of 526 children who had had fever 14 days prior to the survey, 165 (31.4%) were defined as probable malaria cases using this definition. Among them, 20 (14.1%) had been treated with AS+AQ, 10 with quinine (5%), 68 (41%) received non-malaria treatments, and 67 got traditional treatment or nothing (39.9%). Most people sought treatment from public health centres (23/99) followed by private clinics . The median price paid for AS+AQ was 0.5 US$.AS+AQ was the most common treatment for patients with probable malaria at public health centres, but coverage was low due to low health centre utilisation and apparently inappropriate prescribing. In addition, AS+AQ was given to patients at a price ten times higher than the subsidized price. The availability and proper use of ACTs should be monitored and maximized after their introduction in order to have a significant impact on the burden of malaria. Artemisinin-based combination therapies (ACT) are recommended by WHO throughout sub-Saharan Africa as replacement for failing first-line antimalarials. Their advantages and potential to reduce malaria morbidity and mortality have been well described ,2. SubstPlasmodium falciparum epidemic with 630 to 1,190 deaths per day [In Burundi, malaria is the major health problem in terms of both morbidity and mortality . During  per day . Efficac per day . As a co®) or a thick smear, and outpatient consultation fees, was theoretically available in public health centres at a heavily subsidized price (0.05 US$ for children below 5 years and 0.1 US$ for adults). In Burundi in 2004, the gross national income per capita was estimated at 90 US$ [In Makamba province, situated in the South of the country and one of the most affected by malaria, numerous agencies, including Médecins Sans Frontières (MSF), were involved in the financing, provision and monitoring of this new treatment. A full course of treatment, including both a confirmed diagnosis by means of a rapid test . Disappointing coverage may result from (i) low access to health care in the population affected and (ii) inadequate diagnosis, prescription and use of the new treatment in health centres. The Roll Back Malaria Initiative has set a goal of achieving 60% coverage of childhood fevers with effective, prompt antimalarial treatment. Nine months after its introduction in Burundi, a study was carried out to assess the coverage of AS+AQ among uncomplicated malaria cases in children less than five years of age.Plasmodium falciparum is the most common species observed. The Burundian Ministry of Health and local authorities approved the study.The study was carried out in Makamba province in the south of Burundi bordering Tanzania and lake Tanganyika  . The proClassically, studies of antimalarial treatment coverage have used cross-sectional designs in which patients or caregivers of children are asked to report on past febrile episodes and on their management ,12. One Accordingly, two simultaneous community-based studies were carried out:(i) a cross sectional malaria case management study. Here, households of children under five years with reported fever onset in the 14 days prior to the study day were sampled, and their caregivers were asked to recall the children's symptoms and case management.(ii) a study to develop a local, symptom-based retrospective case definition of malaria. Children under five years reported as febrile on the day of the study were sampled. Among these, a gold standard diagnosis of true uncomplicated malaria was established, and caregivers were asked to describe the children's symptoms. Combining those symptoms most predictive of true malaria, the best possible symptom-based definition of malaria was identified, which was then applied to children in the case management sample so as to restrict the analysis to 'probable malaria cases'.In practice, the two studies used partially overlapping samples, since many children with fever in the previous 14 days were also febrile on the day of the study.Households containing one or more children under five years of age, with reported fever onset one to fourteen days before the study date were eligible. Data were collected on all such children present in the household, so as to classify the level of access to health care in different areas within Makamba province (data not shown in this paper). Lot Quality Assurance Sampling rules were used to select an equal-sized and thus non-self-weighting sample of 24 households within each of the 18 health centre catchment areas ('lots') (SampleLQ v1.10) . Within After providing written informed consent, caregivers were interviewed in Kirundi using a standardized, pre-piloted, back-translated questionnaire. Information was collected on household socio-demographics, signs and symptoms in the febrile children, and case management during febrile episodes.Each observation was then aggregated into an overall province-wide sample, applying population weights so as to account for differing lot population sizes. The sample would thus consist of at least 432 children with a history of fever (18 lots × 24 households × at least one child per household). Even if only 1/3 of these had subsequently fitted the symptom-based malaria definition, this sample is still sufficient to estimate coverage with a precision no worse than ± 10%.While sampling households as part of the case-management study, any children with reported fever on the study date were identified. After obtaining written informed consent, caregivers were asked whether the children exhibited any of a pre-determined list of signs and symptoms. The same pre-determined list of signs and symptoms was used as for the case management study. In addition, clinical assistants examined the children for signs of malaria  and other febrile pathologies , and thick blood slides were taken. An expert technician read all blood slides, and 5% were blindly re-read for quality control. For ethical reasons, sick children were treated on site or given a voucher for free treatment at the nearest health centre.Febrile children with positive slides with parasite count ≥2,000/μL and no signs of a severe illness not due to malaria,  and children with positive slides with parasite count < 2,000/μL and no signs of another non-malaria febrile illness, were considered true malaria cases; all others were considered true negatives. Against this gold standard classification, various combinations of caregiver-reported symptoms (i.e. more than one symptom occurring together in the same child) were tested by calculating their positive (PPV) and negative (NPV) predictive values. In order to establish the most specific symptom-based definition of malaria PPV was favoured over NPV as the main function of this definition was to exclude as many false positive malaria cases as possible. The chosen combination of symptoms was then applied to the case management sample so as to retrospectively identify children whose reported fever was probably due to malaria.Based on a Kenyan study , it was Data were entered on EpiData version 3.0  and checked for inconsistencies. Analysis was done using Stata 8.0 . The treatment coverage with AS+AQ among probable malaria cases and the case management of probable malaria cases were expressed as percentages weighted by population size. Probable malaria cases were taken as the denominator of the coverage estimate. A strict definition of coverage was used: AS+AQ prescription in a public health centre and by the second day after onset of fever (approximately 48 hours). A more lenient definition was also applied, namely having received AS+AQ under any circumstances.The study was carried out with the permission of the Ministry of Health, Bujumbura Burundi. A written informed consent was obtained from all who participated in the study.Data collection took place between July 30 and August 20, 2004. A total of 526 children were included in the case management sample and 195 in the symptom-based malaria definition sample  and 103 as true negatives. Among all symptoms reported by the caregivers, loss of appetite, sweating, somnolence, shivering and intermittent fever had a PPV higher than 50% . The most frequented treatment places were public health centres , followed by private clinics . All the other possibilities for case management – i.e. treatment already available at home ; treatment bought at markets, private pharmacies or hospitals ; traditional treatment  – were mentioned less than 10% in the interviewed households. However, 37  did not receive any treatment.AS+AQ was prescribed in public or private health centres, and was not available in markets or private pharmacies. Among the 23 children who received treatment in a public health centre, 5  got AS+AQ and 5 quinine  as first-line treatment, while 13  did not receive any antimalarial treatment.Altogether, 64.2%  of children received some drug treatment. The median price people had to pay for AS+AQ treatment was 0.5 US$ IQR: 0.3–1.3 US$), but varied from 0.4 US$ (IQR: 0.2–0.6 US$) in public health centres to 1.3 US$ (IQR: 0.5–1.5 US$) in private health centres. The median price for quinine was 0.6 US$ (IQR: 0.2–1.0 US$). For treatment other than antimalarials  households paid between 0.3 US$ and 5.0 US$ . Ideally, such cross-sectional studies should take place more than once a year.It should also be noted that the study was carried out in only one of the 16 provinces in Burundi, and thus conclusions cannot be drawn for the entire country.With the development of ACT and the change of national malaria protocols in many African countries, a big step has been taken towards effective control of this deadly disease. However, this study demonstrates that this effort may not be sufficient, and illustrates the importance of monitoring coverage and developing better methodologies for doing so. Better access to care is essential to improve treatment coverage; diagnostic and prescribing practices in health centres need to be supervised, and malaria treatment guidelines need to be enforced, including free availability of ACT. Where these conditions are not met, the deployment of new and highly efficacious regimens will probably have a negligible impact on malaria control.The author(s) declare that they have no competing interests.SG participated in the design of the study, conducted the field part and the analysis and wrote the manuscript. SC participated in the study implementation, its analysis, data interpretation, and manuscript revision. KE participated in the study implementation and manuscript revision. AL carried out all microscopic examination of blood samples. CB interpreted the study findings and revised the manuscript. JPG had been involved in the study design and revised the manuscript critically. FC conceived the study, had been involved in drafting the manuscript and revised it critically. All authors read and approved the final manuscript.


As efforts are currently underway to roll-out insecticide-treated bednets (ITNs) to populations within malarious areas in Africa, there is an unprecedented need for data to measure the effectiveness of such programmes in terms of population coverage. This paper examines methodological issues to using household surveys to measure core Roll Back Malaria coverage indicators of ITN possession and use.ITN coverage estimates within Anseba and Gash Barka Provinces from the 2002 Eritrean Demographic and Health Survey, implemented just prior to a large-scale ITN distribution programme, are compared to estimates from the same area from a sub-national Bednet Survey implemented 18 months later in 2003 after the roll-out of the ITN programme.Measures of bednet possession were dramatically higher in 2003 compared to 2002. In 2003, 82.2%  77.4–87.0) of households in Anseba and Gash Barka possessed at least one ITN. RBM coverage indicators for ITN use were also dramatically higher in 2003 as compared to 2002, with 76.1% (95% CI 69.9–82.2) of children under five years old and 52.4% (95% CI 38.2–66.6) of pregnant women sleeping under ITNs. The ITN distribution programme resulted in a gross increase in ITN use among children and pregnant women of 68.3% and 48% respectively.Eritrea has exceeded the Abuja targets of 60% coverage for ITN household possession and use among children under five years old within two malarious provinces. Results point to several important potential sources of bias that must be considered when interpreting data for ITN coverage over time, including: disparate survey universes and target populations that may include non-malarious areas; poor date recall of bednet procurement and treatment; and differences in timing of surveys with respect to malaria season. Insecticide-treated bednets (ITNs) have been shown to significantly reduce malaria transmission and concomitant malaria related morbidity and all-cause child mortality across a range of transmission settings, even with sustained use -6. ITN uEfforts are currently underway to roll-out ITNs to populations across sub-Saharan Africa with increased funding from the Global Fund to Fight AIDS, Tuberculosis and Malaria, the World Bank Global Strategy and Booster Program, the President's Malaria Initiative (PMI) and the Bill and Melinda Gates Foundation among others. Accordingly, there is an unprecedented need for data to measure the effectiveness of ITN distribution programmes in terms of population coverage . The AbuTwo household surveys implemented 18 months apart that measured ITN coverage within two malarious zobas (zones) of Eritrea are compared. Issues of interpreting cross-sectional survey data for measuring core RBM coverage indicators of ITN possession and use among children less than five years old and pregnant women are explored. Additionally, we demonstrate how an evaluation of a national programme is possible using these data from two cross sectional surveys, and present gross programme effects useful for managers and public health strategists as they focus on moving forward with the global agenda to protect populations most at risk of malaria.zobas. Malaria transmission is seasonal, concentrated primarily in the low-lying areas of three zobas, Anseba, Gash-Barka and Debub from August through October .These results demonstrate that the NMCP ITN distribution programme was effective in increasing ITN household possession and use among children under five and pregnant women in Anseba and Gash Barka. However, better estimates of the net programme effect are required to determine the exact level of the ITN coverage increase that can be attributed directly to the distribution programme. Unfortunately, multilevel analyses that account for non-programme effects on the outcomes of ITN coverage between the two surveys were not possible due to the limitations of the cross-sectional pre-post evaluation design and lack of a suitable control group due to the nature of the full coverage programme. While the two surveys were relatively comparable over time, results point to several important factors that must be considered when interpreting household survey data for assessing progress towards global targets.It is expected that at least some of the gross programme effect observed between these surveys can be attributed to the fact that individuals would have been more likely to use ITNs during the Bednet Survey which was implemented during the rainy season when mosquito nuisance would have been more common as compared to the EDHS that was implemented in the dry season -22. ThisAs global targets are intended to be measured among those at risk for malaria, inclusion of populations not at risk for malaria, such as those within heavily urban areas, highland areas or within extremely arid regions, in the numerator and denominator will likely yield biased estimates of RBM ITN coverage indicators . The twoRecall bias of respondents within both the EDHS and Bednet Survey resulted in substantial date heaping at 12 months, or "one year", for the time since a bednet was procured and treated. This is best illustrated by the extent of date heaping on the Bednet Survey where of the 1439 households with a bednet that reported the number of months since it was procured, nearly half (47.5%) reported "one year" or 12 months, with no households stating 11 or 13 months. Accordingly, date heaping at 12 months had a significant effect on differentiating between a bednet and an ITN as defined by the RBM indicators [TE helped conceive this research, performed the statistical analysis and led the drafting of this manuscript. KM conceived this research and helped with drafting of this manuscript; JY helped with statistical analysis and drafting of the manuscript; TG helped conceive this research and reviewed the manuscript. All authors read and approved the final manuscript.The work presented here was funded through two projects: 1) the American Schools of Public Health/Centers for Disease Control and Prevention cooperative agreement (S1942-21/21), Enhancing National Malaria Control in Eritrea; and 2) the Environmental Health Project Contract # HRN-1-00-99-00011-00. Support also came through the Ministry of Health, Eritrea and Tulane University.


Plasmodium falciparum can be detected by microscopy, histidine-rich-protein-2 (HRP2) capture test or PCR but the respective clinical relevance of the thereby diagnosed infections in pregnant women is not well established.falciparum was screened for in both, peripheral and placental blood samples, and associations with maternal anaemia, low birth weight (LBW) and preterm delivery (PD) were analysed.In a cross-sectional, year-round study among 839 delivering women in Agogo, Ghana, P. falciparum was observed in 19%, 34%, and 53% by microscopy, HRP2 test, and PCR, respectively. For placental samples, these figures were 35%, 41%, and 59%. Irrespective of diagnostic tool, P. falciparum infection increased the risk of anaemia. Positive peripheral blood results of microscopy and PCR were not associated with LBW or PD. In contrast, the HRP2 test performed well in identifying women at increased risk of poor pregnancy outcome, particularly in case of a negative peripheral blood film. Adjusting for age, parity, and antenatal visits, placental HRP2 was the only marker of infection associated with LBW (adjusted odds ratio (aOR), 1.5 ) and, at borderline statistical significance, PD ) in addition to anaemia ). Likewise, HRP2 in peripheral blood of seemingly aparasitaemic women was associated with PD ) and anaemia ).In peripheral blood, P. Peripheral blood film microscopy not only underestimates placental malaria. In this highly endemic setting, it also fails to identify malaria as a cause of foetal impairment. Sub-microscopic infections detected by a HRP2 test in seemingly aparasitaemic women increase the risks of anaemia and PD. These findings indicate that the burden of malaria in pregnancy may be even larger than thought and accentuate the need for effective anti-malarial interventions in pregnancy. Plasmodium falciparum infection, malaria and related consequences [P. falciparum Erythrocyte Membrane Protein-1, parasite strains adhere to the syncytiotrophoblast, i.e. the surface lining the placental intervillous space. As immune recognition of and, thus, response to these parasites are low in primigravidae susceptibility and clinical manifestation are increased. With subsequent pregnancies, protective immune mechanisms gradually develop and expand, and manifestation of infection declines [Malaria in pregnancy continues to be a major public health problem in sub-Saharan Africa. Pregnant women are a specific risk group for equences . This indeclines -4.P. falciparum results in the accumulation of parasitized erythrocytes in the intervillous space, infiltration by inflammatory cells and release of pro-inflammatory mediators [The placental sequestration of ediators . Clinicaediators ,6-9. In ediators . Becauseediators ,12.P. falciparum infections, i.e. below the threshold of peripheral blood microscopy, is not well established [P. falciparum infections detected in peripheral and placental blood by microscopy, histidine-rich-protein-2 (HRP2) capture test, and PCR among women delivering at a district hospital in rural southern Ghana. The performances of the diagnostic tests in a subset of 596 women have been reported previously [As with malaria in children, the prevalence and manifestation of malaria in pregnancy varies with transmission intensity, access to treatment, coverage and quality of antenatal services, and drug resistance, among others. The role of sub-microscopic ablished ,11,13-16eviously . Here, rP. falciparum among women attending antenatal care at Agogo District Hospital was detected in 32% and 63% by microscopy and PCR assays of peripheral blood samples, respectively [P. falciparum mutations conferring PYR resistance are frequent in the area [Agogo is a community of some 30,000 inhabitants located in the forested hills of Ashanti Akim North District. Subsistence farming, trade and mining are the main income sources in that region, and malaria is hyper- to holoenedemic . In 1998ectively . Then anthe area . From JaP. falciparum was screened for in both placental and peripheral blood samples by microscopy, HRP2 test, and PCR: malaria parasites were counted microscopically on Giemsa-stained thick blood films per 500 white blood cells for peripheral samples and per 100 high-power fields for placental samples in which the presence of leukocyte-associated haemozoin was also recorded. Based on placental thick blood film microscopy, the stage of placental infection was categorized as [P. falciparum-specific PCR assays were performed [All women were clinically examined, socio-demographic data were documented, and venous peripheral blood collected into EDTA. Fever was defined as an axillary temperature >37.4°C. Following expulsion and after a small incision had been made into the maternal surface of the placenta, blood from the intervillous space was collected with a syringe containing EDTA. erformed . Plasma erformed  with limerformed .P. falciparum infection and anaemia, LBW, and PD were identified by χ2 test, odds ratios (ORs) calculated, and adjusted for confounders by logistic regression models.Geometric mean parasite densities (GMPDs) and 95% confidence intervals (95%CIs) were calculated. Continuous variables were compared between groups by t-test, analysis of variance, Mann-Whitney U test, and Kruskal-Wallis test as applicable. Associations between From January 2000 to January 2001, 889 delivering and consenting women were recruited and 922 babies were born , of whom 22 (2.4%) were born dead or died within 24 hours after delivery. The characteristics of the 839 women with live-singleton deliveries are shown in Table P. falciparum was detected in 19.0%, 34.1%, and 52.9% by microscopy, HRP2 test, and PCR, respectively. For placental samples, these figures were 34.9%, 40.8%, and 59.4%; placental haemozoin was seen in 30.9%. As reported before [P. falciparum declined in prevalence with parity as did placental parasite density  and not related to parity.In all 839 peripheral samples, d before , sensitiP = 0.09). PYR in plasma was associated with a slight reduction in the prevalence of haemozoin only . In primiparae, this difference was pronounced and statistically significant .Placental haemozoin was the clinically most relevant marker of infection and factors influencing its presence were analysed. In multivariate analysis, primiparity, low age, and delivery in the rainy season were independently associated with haemozoin Table . In womeP. falciparum infection increased the risk of maternal anaemia (Table n = 160), the proportions of LBW (15%) and PD (14%) were similar to the figures in de facto non-infected women . Still, the prevalence of anaemia was increased .Irrespective of diagnostic tool, ia Table . A positia Table . Importa10 parasite density, r = -0.18, P = 0.02) and gestational week  but not with birth weight . Placental parasite density  did not show such correlations.Peripheral blood parasite densities  correlated negatively with Hb (logvs. 3000 g (1420–4500), P < 0.0001) which was smaller in case of a positive HRP2 test (2880 g (1420–4160) vs. 3000 (1280–4500), P = 0.0009) or a positive PCR result (2900 (1280–4350) vs. 3000 g (1500–4500), P = 0.04), and smallest if placental parasites were seen by microscopy (2900 g (1280–4160) vs. 2970 g (1420–4500), P = 0.02). Likewise, any diagnosis of placental P. falciparum was associated with significantly reduced Hb levels, but the reduction was largest in the presence of haemozoin  vs. 11.7 g/dL (4.7–16.8), P < 0.0001). The week of gestation was not associated with infection, irrespective of diagnostic mean applied (data not shown).On the basis of placental sampling, again, microscopic and PCR detection of parasitaemia were not associated with LBW or PD whereas a positive HRP2 result was. Nevertheless, haemozoin was the marker of infection most strongly associated with all clinical consequences Table . LikewisP. falciparum infection and clinical manifestation for the confounders age, parity, and number of antenatal care visits did not substantially change the findings, however, statistical significance in terms of LBW and PD was reduced  or PCR (97%) in detecting microscopically proven placental parasitaemia in peripheral blood were substantially higher than the one of peripheral blood film microscopy (50%). Improving performances of the peripheral blood tests with increasing placental parasite density have been described previously . Because of 100% ,33. Neve of 100% ,35.P. falciparum as identified by peripheral blood microscopy or PCR in Burkina Faso was not associated with LBW or birth weight whereas the latter was reduced in case of a positive peripheral blood HRP2 dipstick [P. falciparum positive by PCR exclusively exhibited no increased risk of LBW in Malawi [P. falciparum infection in pregnancy, e.g. abortive, chronic or progressive, is unknown. Considering the common fluctuations of parasite density [Poor pregnancy outcome, e.g. LBW or PD, in this cross-sectional study at delivery was neither associated with a positive peripheral blood result of microscopy nor of PCR. This may be explained by the low sensitivity of the former, and the very high one of the latter. The HRP2 test with in-between sensitivity appears to be a practical compromise between sensitivity on the one hand and detecting clinically relevant infection on the other. Importantly, in seemingly aparasitaemic women, i.e. in those with a negative peripheral blood film, a positive HRP2 test was associated with anaemia, LBW and PD. Infections detected beyond that exclusively by PCR had no discernible effect on pregnancy outcome but were still associated with reduced maternal Hb. Previous studies on that topic produced conflicting results: in Ghana but not in Sudan, Mozambique, and Cameroon, sub-microscopic peripheral blood infections as detected by PCR were associated with low Hb levels or anaemia ,13,15,16dipstick . Women fn Malawi . One stun Malawi . Differi density , the tem density  and the  density .The detection of haemozoin and placental HRP2 to similar extents were univariately associated with anaemia, LBW and PD but the reductions of Hb and birth weight were most pronounced in women with placental pigment. The association between placental haemozoin and LBW is well documented, e.g. in Senegal . In contP. falciparum, irrespective of diagnostic tool. However, in terms of placental diagnosis, only HRP2 was associated with LBW and PD, the latter at borderline statistical significance. Also, seemingly aparasitaemic women with HRP2 in peripheral blood exhibited an increased risk of PD. Infection detected by microscopy or PCR were not correlated with birth outcome. This unexpected but not uncommon finding [via maternal anaemia [After adjusting for age, parity, and antenatal visits, the risk of maternal anaemia remained to be increased in the presence of  finding ,7,42 may finding -45 but t finding . Still,  anaemia ,47.P. falciparum isolated from pregnant women in Agogo exhibited a dihydrofolate reductase resistance core mutation and the high resistance triple mutation, respectively [The effects of antenatal care and of PYR in plasma were modest in that infection was not consistently prevented by either factor. Reasons for the limited impact of PYR chemoprophylaxis likely include poor compliance (only 35% of the women had detectable PYR) and drug resistance. Already in 1998, 81% and 36% of ectively . From 20P. falciparum infections associated with maternal and foetal morbidity. This is particularly true for women with a negative peripheral blood film. These findings are of importance for the implementation and evaluation of preventive measures for pregnant women in endemic areas and likely also for the assessment of response to treatment. The incorporation of sensitive rapid test devices such as HRP2 tests into antenatal services would be desirable. So far, the costs of IPT with SP are lower than the price of a dipstick of €1 ore more. However, cost-benefit considerations may change once SP has to be replaced because of intensifying drug resistance by more expensive drugs. In an operational sense, a dipstick in the second and third trimester may identify women in whom antimalarial interventions failed and who are at increased risk of maternal and foetal morbidity.In conclusion, placental malaria commonly is missed by standard microscopy of peripheral blood films. Whereas positive results of PCR seem not to be related to poor pregnancy outcome, detection of HRP2 is capable of identifying FPM, GBA, and UB designed the study. CvG, RB, KF, IH, FK, MS, UU, FPM and PAA were responsible for patient recruitment, and clinical and parasitological examinations. ED and FPM did the statistical analyses; TAE measured drug concentrations. FPM wrote the paper with major contributions of the other authors.