Plasmodium falciparum has been widely advocated to save costs and to minimize inappropriate treatment of non-malarial febrile illnesses. HRP2-based RDTs are highly sensitive and stable; however, their specificity is a cause for concern, particularly in areas of intense malaria transmission due to persistence of HRP2 antigens from previous infections.The accuracy of malaria diagnosis has received renewed interest in recent years due to changes in treatment policies in favour of relatively high-cost artemisinin-based combination therapies. The use of rapid diagnostic tests (RDTs) based on histidine-rich protein 2 (HRP2) synthesized by In this study, 78,454 clinically diagnosed malaria patients were tested using HRP2-based RDTs over a period of approximately four years in four highland sites in Kenya and Uganda representing hypoendemic to mesoendemic settings. In addition, the utility of the tests was evaluated in comparison with expert microscopy for disease management in 2,241 subjects in two sites with different endemicity levels over four months.RDT positivity rates varied by season and year, indicating temporal changes in accuracy of clinical diagnosis. Compared to expert microscopy, the sensitivity, specificity, positive predictive value and negative predictive value of the RDTs in a hypoendemic site were 90.0%, 99.9%, 90.0% and 99.9%, respectively. Corresponding measures at a mesoendemic site were 91.0%, 65.0%, 71.6% and 88.1%. Although sensitivities at the two sites were broadly comparable, levels of specificity varied considerably between the sites as well as according to month of test, age of patient, and presence or absence of fever during consultation. Specificity was relatively high in older age groups and increased towards the end of the transmission season, indicating the role played by anti-HRP2 antibodies. Patients with high parasite densities were more likely to test positive with RDTs than those with low density infections.RDTs may be effective when used in low endemicity situations, but high false positive error rates may occur in areas with moderately high transmission. Reports on specificity of RDTs and cost-effectiveness analyses on their use should be interpreted with caution as there may be wide variations in these measurements depending upon endemicity, season and the age group of patients studied. Plasmodium falciparum, but are substantially more expensive than previously used drugs. Currently, ACT antimalarials are made available in many countries through external support, such as that provided by grants from the Global Fund to Fight AIDS, Tuberculosis and Malaria. However, their high cost means that their rational use is essential to ensure sustainability.Most countries in sub-Saharan Africa now recommend first-line treatment of malaria with artemisinin-based combination therapy (ACT). These combinations are highly effective against drug-resistant Malaria treatment in most endemic countries in Africa is based on clinical signs and symptoms due to lack of reliable microscopy in the majority of peripheral health units. The use of rapid diagnostic tests (RDTs) in malaria diagnosis is, therefore, increasing in many countries as the result of their ease of use with minimal training . In the P. falciparum) have been recommended in endemic areas where this species is dominant, due to their relatively low cost, high sensitivity and stability [Plasmodium species. Although HRP2-based tests are generally more sensitive than pLDH-based tests, the relatively low level of specificity in diagnosing clinical malaria of HRP2-based tests is a cause for concern [The diagnostic accuracy of RDTs can vary substantially across different geographical areas making it difficult to compare results from studies conducted under non-standard conditions . RDTs thtability . An alte concern . This re concern . A high There is little information on the epidemiological factors that influence the specificity of HRP2-based tests. In the present study, we assessed the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of RDTs compared with expert microscopy in two different malaria transmission settings. Diagnostic performance was analysed by age group, month of presentation and according to a number of patient-related characteristics and a statistical model was used to investigate the effects of different factors on false positive error rates. Differences in the positivity rates of RDTs across highland sites with varying transmission levels and in different months were also investigated. The implications of the findings are explored in relation to disease management in different areas with varying endemicity. Possible ramifications of the findings in terms of interpretation of cost-effectiveness analyses and accuracy of malaria surveillance data generated from the use of HRP2-based tests are discussed.The results reported in this paper were obtained during the course of two studies undertaken as components of a large epidemic surveillance project conducted in four highland districts of Kenya and Uganda ,8.One of the two studies involved longitudinal monitoring of factors influencing malaria transmission undertaken in four sites at varying altitude where meteorological, entomological, clinical and parasitological data were collected concurrently over a period of approximately four years. The two sentinel sites in Uganda were Bufundi Health Centre  in Kabale District and Kebisoni Health Centre  in Rukungiri District. Bufundi has a cool climate whereas Kebisoni is characterized by a mild climate. Both areas have two rainy seasons: March-April and September-November. Temperatures are highest in February and between June and August. Many inhabitants in Bufundi are subsistence farmers who often also travel to neighbouring districts to work as migrant labourers in large farms. Inhabitants of Kebisoni are relatively sedentary farmers. The two study sites in Kenya were Sengera and Kilibwoni Health Centres, located in Gucha and North Nandi Districts, respectively. Sengera  is the only non-governmental health centre among the four sites and has a mild climate. Kilibwoni  is characterized by a cool and wet climate suitable for growing tea, which is a significant cash crop in the area. In both Kenyan study areas, peak temperatures occur during February and March and the two rainy seasons are April-June and September-December.® test  over approximately four years between November 2002 and September 2006 (except in Gucha where the study terminated in May 2006). The devices were stored at room temperatures within the range recommended by the manufacturer and used within the duration of the recommended shelf life of 24 months. The proper storage and use of the devices were ensured by supervisory staff at each site. The devices were purchased six times during the study period directly from the same manufacturer at the same time for both countries, but no attempt was made to use similar batches in all sites at similar times due to substantial variations in rates of use between sites and due to unpredictable nature of patient numbers. In a few cases, devices stored at sites with lower rates of use were transferred to other sites with higher rates. Clinical diagnosis of malaria was made usually by clinical officers on the basis of presence of fever or history of fever and absence of any other obvious cause of fever. Ethical approval for the study was obtained from relevant authorities in each country and from the London School of Hygiene and Tropical Medicine. Laboratory staff were given training on how to undertake RDT tests and on how to interpret the test results according to the manufacturer's instructions. Patients were asked by laboratory staff about any history of travel during the previous two weeks and about the use of antimalarials prior to the visit. Axillary temperature was measured using a digital thermometer. All patients diagnosed clinically as malaria cases were treated according to national guidelines irrespective of the outcome of the RDTs. The antimalarials used at the time of the study were a chloroquine and sulphadoxine-pyrimethamine (SP) combination in Uganda, and SP and later amodiaquine in Kenya.At each study health centre, patients diagnosed clinically as a case of malaria according to standard national procedures for case management were subsequently tested using the Paracheck PfStudies to determine the sensitivity and specificity of the HRP2-based RDTs in comparison with expert microscopy were carried out at Kebisoni (Uganda) and Kilibwoni (Kenya) between December 2005 and March 2006 (a transmission season in both sites). These sites were selected because of their similarly sedentary populations and the fact that they were both government facilities, while they differed markedly in altitude and malaria endemicity. Finger-prick blood samples were taken from all clinically diagnosed malaria cases for both microscopic examination and RDTs. Verbal consent of patients or guardians was requested before taking blood samples. Thin and thick blood films were prepared and filter paper samples obtained for molecular studies (not reported in the present paper). Giemsa staining was used according to standard procedures. Slides were examined until 200 white blood cells (WBCs) were counted if positive. Slides for which parasites were not detected after counting up to 200 WBCs were examined until 400 WBCs were counted before a slide was considered to be negative. Parasite density per microlitre of blood was estimated by multiplying the counts by 8,000 (the approximate number of WBCs per microlitre) and dividing the result by the WBC counts. Duplicate slides were examined independently by two experienced microscopists who were blinded to the RDT results. In the case of a discrepant result, a third microscopist re-examined both slides. Results from the third microscopist were considered final.Microsoft Access version 2000  was used for data entry. Stata Version 10  was used for data analysis. The sensitivity, specificity, PPV and NPV of the RDTs were compared between the two areas, across different age groups and months covering the transmission period. A logistic regression model was used to study the effects of variations in patient characteristics , month of presentation, and geographical area on the specificity of RDTs. The variable of interest was false positive RDT test results among microscopically confirmed negative tests.P. falciparum malaria. Bufundi and Kilibwoni, both located at relatively high altitude, had lower RDT positivity rates compared to Kebisoni and Sengera  tested positive for ra Table . Positivra Table  except fRDT positivity rates varied by season and year at each site, indicating temporal changes in accuracy of clinical diagnosis of malaria Figure . The absRDT positivity rates increased as the number of RDT-positive cases increased, especially in sites located at lower altitudes. There was a strong correlation between monthly RDT positivity rates and number testing positive with RDTs in all sites, with correlation coefficients varying between 0.64 (Kebisoni) and 0.87 (Sengera). At Kebisoni, both clinical malaria cases and RDT-positive cases increased during the study period, but there was no similar trend in the RDT positivity rate Figure .for P. falciparum by RDT, whereas at the mesoendemic site (Kebisoni), 609/1,237 (49.2%) were positive. The sensitivity, specificity, PPV and NPV of the RDTs at Kilibwoni were 90.0%, 99.9%, 90.0% and 99.9%, respectively, whereas the corresponding figures at Kebisoni were 91.0%, 65.0%, 71.6% and 88.1%, respectively. A significantly higher specificity was observed at Kilibwoni compared to that of the more endemic Kebisoni (p < 0.0001) , but there was no significant difference between the two sites in terms of PPV (p = 0.198). At Kebisoni, 220/628 patients (35%) who tested negative by microscopy tested positive by RDT. At Kilibwoni, only one of the 990 patients who tested negative by microscopy tested positive by RDT. Fifty-five of the 609 patients (9%) confirmed to be positive with microscopy at Kebisoni were declared negative with RDTs. Most of these patients had low mean parasite densities . However, six of the 55 false negative patients at Kebisoni (11%) had parasite densities exceeding 8,000/μl. At Kilibwoni, one patient was false negative by RDT out of a total of 10 who were confirmed positive by microscopy.At the hypoendemic site (Kilibwoni), only 10/1,000 (1.0%) of cases examined microscopically were positive At Kebisoni, true parasite rates (as determined by microscopy) declined during the four months (December 2005 – March 2006) of concurrent collection of blood samples for comparison of RDTs with microscopy. During this period, the specificity of RDTs increased steadily from 56% in December 2005 to 79% in March 2006 Figure . There wThe true parasite rate varied between age groups. The peak parasite rate was observed in children 2–4 years of age and the rate decreased in the older age groups Figure . Specifip = 0.006) but specificity was significantly lower in febrile patients . No significant differences were detected between the two groups in terms of PPV and NPV . At Kebisoni, microscopically confirmed P. falciparum patients with high parasite densities were significantly more likely to be true positive with RDTs than patients with a low parasite density .Sensitivity of RDTs was significantly higher in patients with fever (body temperature of 37.5°C and above) on presentation compared to non-febrile patients , specificity increased as the true parasite rate (as determined by microscopy) decreased at the end of the transmission season. This may have followed from boosting of anti-HRP2 antibodies as a result of infections acquired during the preceding few months. The relatively higher specificity of RDTs at the hypoendemic site compared with the mesoendemic site could, on the other hand, be due to a very low probability of finding patients with recently cleared parasitaemia who sought treatment for non-malarial illnesses.Sensitivity of RDTs was not affected by age of patient or fluctuation in parasite rates during different months. It was, however, affected by parasite density. Patients with high parasite densities were more likely to test positive than those with low parasitaemia. Other studies have also indicated that HRP2-based tests have high sensitivity which increases with parasite density . HoweverIt might be argued that some of the variability observed between the sites and during different parts of the year could have resulted from performance variability of the tests used, especially as climatic conditions affect the stability of the devices. Stability is usually more problematic with pLDH-based tests than with HRP2-based tests. Due to variable rates of use in different sites, the use of similar batches of the tests across all sites and seasons could not be ensured. Nevertheless, the devices were purchased at the same times for both countries, and from the same manufacturer during the entire study period. The devices were also stored and used within the recommended temperature and duration. Due to these and the fact that the sensitivity of the tests in Kenya and Uganda were similar , variability in performance of the devices is unlikely to have played a major role.The use of RDTs is probably cost-effective in many situations. A simulation study has indicated that at a 95% confidence level, RDTs are cost-effective compared to presumptive treatment below 62% parasite prevalence rates  as has bDuring an epidemic caused by flooding in Mozambique, RDTs were shown to have an adequate PPV when combined with clinical diagnosis, although they failed to detect some true malaria cases . Thus, tAlthough the use of RDTs for treatment decisions in epidemic situations may be limited, their use can be helpful in surveillance, for example in the confirmation of reported outbreaks. However, RDT-based longitudinal data should be interpreted with caution due to potential seasonal or annual variations in accuracy resulting from temporal changes in transmission levels as indicated in this study. The study also shows that data generated from clinical diagnosis alone could be useful in epidemic monitoring, in particular in mesoendemic situations. However, in highlands with very low endemicity, the use of clinical data alone without laboratory confirmation can be misleading. This was observed in Bufundi, in Uganda, where an apparent outbreak of clinical malaria was confirmed to be a non-malarial febrile illness .One of the major implications of the findings of the present study is that cost-effectiveness of HRP2-based RDTs is greatly influenced by variations in their sensitivity and specificity between different areas, age groups of patients, and seasons. The study showed that the diagnostic accuracy of HRP2-based RDTs is relatively high in areas or seasons with low transmission, but more area-specific operational studies may be required to evaluate their cost-effective use under different transmission scenarios. For decisions involving the use of these tests, policymakers should take into account the cost implications of treating test negative patients , as wellTAA and JC conceived and designed the study. TAA coordinated the study, trained field staff, compiled data, carried out statistical analyses and wrote the manuscript. MK trained field staff, contributed to planning of the sample collection, assisted in data compilation and participated in revision of the draft. JC and BG revised the draft and facilitated funding of the project. CJ contributed to discussions on manuscript ideas and preliminary exploration of the data, and commented on the draft. JB developed and maintained the database used in the study. JB and DM contributed to revision of the manuscript. MO and BR coordinated the field studies in Uganda and Kenya, respectively. All authors (except BR who sadly passed away) read and approved the final manuscript.


Plasmodium falciparum, have failed in part due to the emergence of drug resistant parasites and mosquitoes resistant to insecticides. Using an artemisinin-based combination therapy (ACT) that kills parasites quickly, a strategy was designed to eliminate the source of transmission by mass treatment of human populations in malaria-endemic areas Cambodia.Previous efforts to eradicate malaria parasites, particularly A combination drug of artemisinin and piperaquine given with low doses of primaquine was used to eliminate all stages of parasites from human carriers.P. falciparum rate in children decreased from 37.0% to 1.4%, reaching 0% in eight of 17 villages. In a second field study, that included one additional mass treatment of artemisinin-piperaquine, the P. falciparum rate in children was reduced from 20.8% to 0% within six months. No major adverse effects were observed.In a pilot study, mass administration of artemisinin-piperaquine  and primaquine  was carried out in 17 villages . Parasite rates were dramatically reduced from 52.3% to 2.6% after three years. The Mass administration of artemisinin-piperaquine and low doses of primaquine can be an effective, safe, and affordable strategy for efficiently eliminating malaria parasites in human carriers and interrupting parasite transmission. This study provides important information for future strategies for the eradication of malaria. Malaria has been eliminated from some formerly endemic regions of the world, mainly in more temperate zones including countries in Europe, North America, some of the former Soviet Republics, and some island nations. Improvement in public health, efforts in treating malaria patients, and mosquito control measures were some key factors for the success of malaria elimination. Unfortunately, malaria control programmes have been less successful in many developing countries in the tropics and subtropics. Lack of resources for disease management and the emergence of drug resistant parasites and insecticide resistant mosquitoes contributed to the failure of many malaria eradication programmes during the era of the Global Malaria Eradication Programme initiated by the World Health Organization in 1955 [Plasmodium vivax malaria in endemic areas along the Yangtze River [Plasmodium falciparum malaria has been eliminated in many endemic regions in China, the parasite is still present in Hainan and Yunnan provinces in Southern China after more than 50 years of disease control efforts [Similarly, the goals of the1998 Action Plan to Roll Back Malaria have not been fulfilled in many countries . In Chinze River . Althoug efforts .To reduce or totally eliminate malaria parasite infections from a population, interruption of parasite transmission is critical. One strategy for achieving the goal is to interrupt parasite transmission through mosquito vector control such as insecticide spraying and large-scale distribution of insecticide-impregnated bed nets . AlthougRapid-acting artemisinin combination therapy (ACT) provides a potential tool for malaria control and eradication by removing the source of malaria transmission through eliminating parasites in the human populations. This strategy has been shown to be effective previously, particularly when combined with anti-vector methods -11. ThisTo investigate the feasibility of eradicating malaria parasites from endemic regions through eliminating the sources of host transmission, two pilot studies were conducted in Cambodia during December 2003 to December 2006. Here results are presented from the studies testing a hypothesis that mass treatment with an ACT followed by primaquine to block transmission is an effective method for reducing and/or arresting malaria transmission in endemic area.The population for the first study consisted of 3,653 individuals from 17 villages in Kampong Speu province, Cambodia  at 0 and 24 hours. For children aged 6-10 years, one tablet was given at each dose; and for children aged 11-15 years, one and a half tablets were used. For children under 6 years, a complete two-dose artemisinin-piperaquine Granules (artemisinin 24 mg and piperaquine 144 mg/sachet) was administered: two sachets each dose for children aged 5-6 years; 1.5 sachets for children aged 3-4 years; and one sachet for children aged 1-2 years. At the same time, low-dose primaquine tablet (9 mg) for adults was given at 10-day intervals for six consecutive months without obtaining individual G6PD information, to prevent transmission to mosquitoes from infected subjects. The first dose of primaquine was taken with artemisinin-piperaquine. The doses of primaquine for children were: 3/4 tablet for children aged 11-15 years; 1/2 tablet for children aged 7-10 years; 1/3 for children aged 3-6 years; and 1/4 for children aged 1-2 years.Village Malaria Volunteers (VMV) were recruited to distribute drugs and monitor drug administration. Anti-malarial drugs were given to every individual simultaneously in the villages. A complete two-dose therapy of artemisinin-piperaquine was given to the whole population without obtaining individual infection information. Adults (≥16) were given two tablets of a co-formulated artemisinin and piperaquine  were the main indices used to monitor malaria prevalence and treatment effectiveness for the studies.After the mass treatment programme was started, periodic surveys monitoring parasite positive rates in villages with initial parasite rates of ≥ 20% were carried out every 6 months. Parasite rates in 50 adults and 50 children were determined randomly in every village. In a village with a population less than 100, the number of adults and children checked was not less than 70% of the population. When the population parasite rate in a village was decreased to zero, all negative blood smears of that village were rechecked by another senior microscopist, appointed by both Chinese and Cambodian investigators, to confirm the results. One skilled microscopist was responsible for microscopic examination of blood smears throughout the study so that errors due to variation of diagnostic skill would be minimized.Children parasite positive rates in three of the nine villages in the second study were surveyed by malaria microscopy on Days 7, 14, 28, 35, 42, 60, 90, 120, 150, and 180.Severe adverse events are passively reported by the VMV. No active monitoring takes place.P < 0.05.The chi-square test (SPSS 10.0) was used for comparisons of parasite rates with two-tailed significance tested at P. falciparum, P. vivax, and Plasmodium malariae, were found in the villages.Human populations were surveyed in 27 villages in Kampong Speu province for malaria parasites before the programme was started. Three different malaria species, t-test) ; P. vivax was the second most prevalent (23.8% and 38.7%) followed by P. malariae (7.5% and 3.2%). Interestingly, there appeared to be no correlation between parasite rates in adults and children among the villages. That is, villages having the highest rates in children were not necessarily those with the highest parasite rates in adults. Similarly, the ratios of P. falciparum over P. vivax and P. malariae in children were often quite different from those in adults in each village.Among the 27 villages, 17 villages had parasite rates ≥ 20%, six villages had parasite rates 10-19%, and four villages had parasite rates 6-9%. The 17 villages with high parasite rates had 3,653 residents, with parasite rates among children ranging form 19.1% to 81.0% Figure . In adul) Figure . Among tA total of 252 villagers (only one from every family) were screened for G6PD deficiency using G-6-PDH Kit Visual Colour, Trinity biotech. The G6PD deficiency rate is 17.1% (43/252); male 18.6% and female 13.3%.P. falciparum rate in children decreased from 37.0% to 10.8% (a reduction of 70.8%) in the first year, to 2.3% (93.8% reduction) in second years and to 1.4% (96.2% reduction) by the third year. The combined P. vivax and P. malariae rate decreased from 18.8% to 4.9% (73.9% reduction) in the first year and to 3.0% (84.0% reduction) in the second year, and 1.4% (92.6% reduction) in the third year  after three years; the combined P. vivax and P. malariae rate decreased from 12.3% to 0.5% (95.9%). It should be noted that the P. falciparum positive rate in children in eight of 17 villages decreased from an average of 46.5% to 0% 18 months after the start of the mass administration programme.Reductions in parasite rates for adults were similar to those for children Table . ParasitAlthough the mass treatment worked well in most of the villages, not all villages had similar reductions in parasite rates after one year Figure . VillageP. falciparum  1.5 years after treatment. This could be mainly due to the interference from a nearby village (SL), a highly endemic village in a neighbouring province that was not included in the programme. The village was only three kilometres away from village 9 and the parasite rate in children in the nearby village was 51.2%, 77.3% of which was m Figure . TherefoPlasmodium falciparum gametocyte rates dropped significantly  through mass administration of artemisinin, piperaquine, and primaquine was effective. Safety monitoring was not performed systematically as would be done in a controlled clinical trial, but there was no indication of clinically significant adverse drug reactions. Higher doses of primaquine can cause haemolytic anaemia in individuals with G6PD deficiency, but the lower doses used in this programme are thought to be safe, based on extensive experience in malaria elimination campaigns in past decades. The cost of this approach is also acceptable since the prices of artemisinin-piperaquine and primaquine are relative low ($2/treatment/person) and payments for local VMVs is affordable.Although this three-year mass drug administration project had dramatically reduced parasite rates to very low levels, including some villages with no detectable parasites, residual parasites may act as parasite source of future outbreaks. Another round of treatment is believed to eliminate the residual parasites from the villages. Another potential issue is that parasite carriers in nearby villages may reintroduce the parasites into villages that were clear of parasites. This concern can be resolved if a national wide mass treatment campaign is conducted. A third concern is that mass treatment may lead to the emergence of drug resistant parasites. The mass drug administration programme used three different drugs at the same time, which should greatly reduce the chance of encountering drug resistant parasites.Mass drug administration with artemisinin-piperaquine and primaquine can be considered as an alternative strategy for malaria control, and, in combination with other anti-malarial measures, may provide a tool for malaria elimination and eradication.The authors declare that they have no competing interests.JS organized the programmes and draft the manuscript; DS participated in and supervised the programmes; BT participated in the projects and analyzed the data. PD participated in the Kampong Spur project; CD did the field work and analyzed the data; PS participated in Kampot project; S Sokunthea, FO, HJ checked the blood smear; S Seila did the field work; and GL conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


However, it also causes an excessive mortality amongst the offspring due to an increase in homozygosis of recessive lethal alleles. The aim of this study was to explore the overall effects of inbreeding on the fitness of a population infested with malaria.The practice of consanguineous marriages is widespread in countries with endemic malaria. In these regions, consanguinity increases the prevalence of α+-thalassemia genotypes were obtained from the odds of severe forms of disease from a field study. Survivals were further estimated for different values of mortality from malaria.In a stochastic computer model of population growth, the sizes of inbred and outbred populations were compared. The model has been previously validated producing results for inbred populations that have agreed with analytical predictions. Survival likelihoods for different α+-thalassemia allele and the loss of life due to homozygosis of recessive lethal alleles; both are proportional to the coefficient of inbreeding and the frequency of alleles in population. Inbreeding-mediated decrease in mortality from malaria  mitigates inbreeding-related increases in fatality . When the death rate due to malaria is high, the net effect of inbreeding is a reduction in the overall mortality of the population.Inbreeding increases the frequency of αConsanguineous marriages may increase the overall fitness of populations with endemic malaria. Plasmodium falciparum in the population  and their survival is always smaller than when F = 0 [p and q = 1 - p). With an increase in mortality due to malaria, the ratio of the differential survival increases to 1.0  reported over the last half century [As the death rate due to malaria increases, the relative excess of the frequency of α6 Figure , conspic century ,14.+-thalassemia is low (0.03), the onset of inbreeding elevation is delayed until the selection increases the frequency of α+-thalassemia  [When the initial frequency of αa Figure  and 1c. 5 Figure ; then ths low 0.0, the onst times) ,16.+-thalassemia) after it experiences an inbreeding depression , marries an outsider- the spouse is likely to be a non-carrier. On an average, 20% more children from consanguineous unions would survive malaria  increases the relative fitness against malaria. When mortality from malaria is high, this increase in fitness could offset the loss of life resulting from inbreeding. Therefore, consanguinity augments the fitness of a population with endemic malaria through its effect on α+-thalassemia allele.Human inbreeding enhances the speed of fixation of recessive and codominant alleles. Consequently, the elimination of recessive lethal alleles is increased by an excessive mortality of children in consanguineous populations. However, an enhanced speed of selection of the codominant αThe authors declare that they have no competing interests.SD designed and obtained funds for the study, conducted the experiment, analyzed results and drafted the manuscript. NN analyzed results, provided analytical conformation of experimental results, and revised intellectual content of manuscript. MMA revised intellectual content and redrafted the manuscript. All authors contributed to writing and read and approved the final manuscript.


Surrogate markers of protective immunity to malaria in humans are needed to rationalize malaria vaccine discovery and development. In an effort to identify such markers, and thereby provide a clue to the complex equation malaria vaccine development is facing, we investigated the relationship between protection acquired through exposure in the field with naturally occurring immune responses  to molecules that are considered as valuable vaccine candidates.-up study of this village. Of the 30 immune responses measured, only one, antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3), was strongly associated with clinical protection against malaria in all age groups, i.e., independently of age. This immunological parameter had a higher statistical significance than the sickle cell trait, the strongest factor of protection known against Plasmodium falciparum. A single determination of antibody was significantly associated with the clinical outcome over six consecutive years in children submitted to massive natural parasite challenges by mosquitoes (over three parasite inoculations per week). Finally, the target epitopes of these antibodies were found to be fully conserved.We analyzed, under comparative conditions, the antibody responses of each of six isotypes to five leading malaria vaccine candidates in relation to protection acquired by exposure to natural challenges in 217 of the 247 inhabitants of the African village of Dielmo, Senegal (96 children and 121 older adolescents and adults). The status of susceptibility or resistance to malaria was determined by active case detection performed daily by medical doctors over 6 y from a unique followP. falciparum infection in young children, our results provide the encouraging indication that these antibodies should be possible to elicit by vaccination early in life. Since these antibodies have been found to achieve parasite killing under in vitro and in vivo conditions, and since they can be readily elicited by immunisation in naïve volunteers, our immunoepidemiological findings support the further development of MSP3-based vaccine formulations.Since anti-MSP3 IgG3 antibodies can naturally develop along with protection against  Using data from malaria cases in Senegal, Pierre Druilhe and colleagues found that antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3) were strongly predictive of clinical outcome. Plasmodium falciparum, a parasite transmitted to people through the bites of infected mosquitoes. In the human body, the parasites replicate in liver cells before changing into so-called “merozoites.” These infect red blood cells, where they replicate rapidly before bursting out and infecting more red blood cells. This massive increase in the number of parasites in the body causes a fever and can also damage vital organs. Although individuals can protect themselves against being bitten by mosquitoes, a vaccine is urgently needed to reduce the global burden of malaria. Vaccines help the immune system fight infectious diseases. When a disease-causing organism (pathogen) enters the human body, the immune system produces antibodies. These are proteins that recognize molecules (antigens) on the pathogen and that enlist other parts of the immune system to kill the invader. This process is often slow the first time around, so people can be ill for a while. However, the immune system “remembers” the experience and responds much quicker to subsequent attacks by the same pathogen. Vaccines, which contain antigens from pathogens, prepare the immune system so that it responds quickly and effectively to a pathogen's first attack.Malaria kills about one million people—mainly children—every year. Most of these deaths are caused by An effective vaccine against merozoites would limit the severity of malaria and prevent many deaths, but scientists do not know which of the antigens on merozoites stimulate a protective immune response. Each candidate therefore has to be tested in long, expensive field trials. Young children who live where malaria is endemic  have frequent attacks of malaria but gradually develop natural immunity to the disease so that after the age 10 y, even though there are always parasites in their blood, they rarely become ill. If researchers knew which malaria antigens and which parts of the immune response are involved in natural immunity, they could use this information in vaccine design. In this study, the researchers have investigated which type of antibody  and which of five potential malaria antigens is associated with protection against clinical malaria.In 1990, the researchers started a unique study in Dielmo, Senegal, West Africa, a small village where malaria is transmission is very high all year round. For 7 y  medical staff visited every villager daily to look for clinical signs of malaria. Diagnoses of malaria were checked by looking for parasites in blood samples. One year into the study, the researchers tested blood from each participant for antibodies that recognized the candidate malaria antigens. When they analyzed their data, they found that only one immune response—the production of antibodies of the IgG3 isotype directed against an antigen called merozoite surface protein 3 (MSP3)—was strongly associated with clinical protection against malaria in all age groups. The researchers also found that some of the children developed protection against malaria when they were very young. Furthermore, in children, production of anti-MSP3 IgG3 in 1991 was associated with protection against malaria for the next 6 y.These findings show that the production of IgG3 antibodies that recognize MSP3 is strongly associated with the development of long-lasting natural protection against malaria. They also show that both protection and these antibodies can develop in very young children. Malaria transmission is unusually high in Dielmo, so these findings may not necessarily be relevant to regions where transmission is lower. In addition, although other research has shown that antibodies of this type can kill parasites in test tubes and in animals, it is possible that these antibodies are markers of, rather than causes of, protective immunity against malaria. Nevertheless, these findings support the continued development of MSP3-based vaccines, particularly since they suggest that early vaccination has the potential to protect infants against life-threatening attacks of malaria.http://dx.doi.org/10.1371/journal.pmed.0040320.Please access these Web sites via the online version of this summary at malaria and on vaccination (in English and Spanish)The MedlinePlus encyclopedia contains pages on malaria Information is available from the World Health Organization on malaria and on immune responses to malaria (in English and Spanish)The US Centers for Disease Control and Prevention provide information on malaria and malaria vaccine developmentInformation is available from the Malaria Vaccine Initiative on  A malaria vaccine is urgently needed ,2; howevP. falciparum , p = 0.183; and for anti-MSP2 (3D7), 0.71 [0.05–9.14], p = 0.785).In contrast, in the children group, a significant association of IgG3 anti-MSP-3 antibodies with clinical protection was observed in the following 12, 24, and 36 mo of follow-up  and remained marked by year 4 of the study . The differential number of attacks was still significant by year 6 of the follow-up  when the model was compared with and without the antibody response. Hence, the two methods of analysis provided convergent conclusions indicative of the long-term clinical significance of a single IgG3 anti-MSP3b determination, in terms of the resistance of children to malaria attacks.The above results were confirmed using a nonlinear model fitted to the variables demonstrated to be significant in the previous analysis, i.e., age, haemoglobin AS phenotype, and anti-MSP-3 IgG3. The maximum difference between the mean number of malaria attacks identified in children with anti-MSP-3 IgG3, and children without such a response (4.3-fold), was detected 3 y after sampling , which revealed 16 nucleotide substitutions in the same 45 strains (ten within the Th2R and six within the Th3R regions), all resulting in amino acid substitutions, in agreement with previous reports [Since MSP3 appeared at this point to be a strong surrogate marker of clinical protection, we deemed it important to investigate whether this antigen exhibits the polymorphisms reported for many malarial vaccine candidates –50. The  reports .Anopheles mosquitoes [A detailed analysis of the malarial clinical status, based on daily medical records actively collected from each inhabitant of Dielmo, Senegal, demonstrated a highly significant relationship between clinical protection acquired through natural exposure and the production of cytophilic anti-MSP-3 antibodies, notably IgG3. The stronger relationship of this marker with protection than that conferred by sickle cell trait  indicates that its contribution to the control of parasites is highly significant and therefore demonstrates its promise as a vaccine candidate. Above all, the clinical value of this surrogate marker, i.e., its association with protection, persisted out to six years in individuals who are exposed to approximately 260 inoculations per year of parasites by squitoes .Except for one study in which two antigens were investigated in parallel , most imIn such epidemiological investigations, age is a confounding factor, since protection builds up over time with exposure, and hence with age ,54. ThisFollowing the report that the isotype distribution of antimalarial antibodies was a critical parameter of malaria immunity , variousThe long-term clinical effect of a single anti-MSP3 antibody determination over a number of years in participants that are receiving malaria inoculations by mosquitoes every two days is impressive, particularly as it remained significant over six consecutive years. This finding has not been reported for any other antigen, to our knowledge. The conceptual importance and implications for vaccine development merit further investigation in other settings. The longer-term association in children than in adults is likely related to two factors: (i) children can be divided into two groups, a small subset acquiring protection early in life who remain protected in the ensuing years (this is associated with our surrogate marker) and the majority who do not, and (ii) most adults are continuously protected against malaria, but some may briefly lose this protection and reacquire it after a short, self-resolving attack. A long-term effect cannot be expected in these occasional situations of brief loss of existing protection.http://www.who.int/vaccine_research/documents/en/malaria_table.pdf) and a concomitant increase in development costs. The second reason is that the elucidation of the malaria genome, although welcome, nonetheless generates knowledge about ∼5,300 proteins—500-fold more molecules than was previously known. These potential antigens must now be assessed for their suitability for inclusion in a malaria vaccine, a task that is not achievable through individual vaccine efficacy trials with each one.Malaria vaccine discovery, which has relied for several decades on preclinical in vitro and in vivo animal models, is now increasingly reliant on clinical trials in humans. For two major reasons this is driving vaccine development towards an ever more unmanageable situation. One reason is that since none of the preclinical models is recognised as being of undisputable predictive value, each candidate must presently be taken into lengthy, time-consuming and costly clinical trials. From an initial number of 12 vaccine candidates, this translates, with various antigen presentation systems, into 58 planned clinical trials  to various pre-erythrocytic antigens among 217 inhabitants from Dielmo  was measured by ELISA against LSA1-, LSA3-, and SALSA-derived peptides, two recombinant proteins (R32 LR and R32Tet), and a peptide (NANP4) derived from the CS protein.(6.9 MB EPS)Click here for additional data file.Figure S2For each antigen, the median value of antibody responses was determined. Individuals with antibody responses above the median (>median) were considered high responders (closed circles). Individuals with antibody responses below the median (<median) were considered low responders (open circles). Following a single determination of antibodies to each antigen in blood samples, the mean number of malaria attacks recorded in Dielmo over 6 y was calculated for each year of the study and for each group of children (either high or low responders to each antigen). The two subgroups had the same mean age, so that there was no age imbalance between children with and without malaria attacks. The mean cumulative numbers of malaria attacks are illustrated for anti-MSP3b (A), anti-MSP1 (B), anti-AMA1 (C) and anti-MSP2-3D7 (D) IgG3 responses. Error bars indicate SD.(1.1 MB EPS)Click here for additional data file.Table S1ORs and 95% CIs were calculated to evaluate the relationship between two binary variables . The results are given as an indication of effect size with ORs greater than 1 indicating that the no malaria attack condition was more likely to happen in the group with antibody responses specific for the antigen tested. C:NC indicates the cytophilic to noncytophilic ratios ; NA (not available) indicates situations where ORs could not be calculated.(1.2 MB EPS)Click here for additional data file.Table S2A subgroup of Dielmo inhabitants present during 6 y of survey after blood sampling was identified. Presence or absence of anti-MSP3 IgG3 responses was tested with regard to the cumulative number of malaria attacks identified each year. The indications in favour of a potential association between anti-MSP3 IgG3 responses and resistance to malaria attacks recorded during 6 mo to 6 y following blood sampling are given as age-adjusted ORs and 95% CIs determined for children and adults separately.(482 KB EPS)Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number AF024624.The primer positions in the MSP3seqence are based on GenBank (


Antigen-specific antibody-mediated immune responses play an important role in natural protection against clinical malaria, but conflicting estimates of this association have emerged from immuno-epidemiological studies in different geographical settings. This study was aimed at assessing in a standardized manner the relationship between the antibody responses to four malaria vaccine candidate antigens and protection from clinical malaria, in a cohort of Ghanaian children.19), Merozoite Surface Protein 3 (MSP3) and Glutamate Rich Protein (GLURP) antigens in plasma samples from 352 Ghanaian children, aged three to 10 years with subsequent malaria surveillance for nine months. This is one of a series of studies in different epidemiological settings using the same standardized ELISA protocols to permit comparisons of results from different laboratories.Standardized ELISA protocols were used to measure isotype and IgG subclass levels to Apical Membrane Antigen 1 (AMA1), Merozoite Surface Protein 1–19 , p = 0.018)] and IgM  to MSP119 were independently associated with protection from malaria.The incidence rate of malaria was 0.35 episodes per child per year. Isotype and IgG subclasses for all antigens investigated increased with age, while the risk of malaria decreased with age. After adjusting for age, higher levels of IgG to GLURP, MSP119 in reducing the risk of clinical malaria in Ghanaian children, thus substantiating its potential as a malaria vaccine candidate.Using standardized procedures, the study has confirmed the importance of antibodies to MSP1 But the estimates of rate ratios for other variables in the model were unchanged when this variable was included in the model suggesting treatment effects did not cause a bias in the estimation of the effects of immunological variables.19, MSP3, AMA1 and GLURP) with the incidence of clinical malaria in different epidemiological settings. Previous results have been difficult to interpret due to different study protocols and analytical methods having been used [This study in Ghanaian children is one of a series of studies designed to assess, using standardized methods, the association of antibody levels to four leading asexual blood-stage malaria antigens  [E. coli, which had been used in the previous study. Although antibodies to MSP119 have been shown to be associated with both exposure and protection from disease, the fine specificities of such responses may contribute to protection [19, and other antigens, that are produced in different expression systems, in order to select the most appropriate antigen/expression system for malaria vaccine development. In another study in this series, in Burkina Faso, the same antigens, reagents, ELISA procedure and analytical methods were used; none of the isotypes and subclasses to MSP119 was associated with the incidence of clinical malaria. Since the same laboratory methods were used, the different outcomes of these two studies may be attributed to differences in malaria transmission or to the age of the children [19 were independently associated with the incidence of clinical malaria, which may indicate the importance of MSP119 in malaria vaccine development. Parasite growth inhibition assays would be required to confirm if this association reflects a functional role of MSP119 in immunity.The IgG subclasses, IgG1, IgG2 and IgG3 for MSP3, MSP1children ,47,54 Inncidence . This maS---FCS)  comparedotection . The antotection ,55,56, wchildren . In Burkchildren . Althoug19 antibodies (IgG1 and IgM) have been shown to be the most strongly correlated with reduced risk of clinical malaria among the four malaria vaccine candidates tested. The standardized AIA ELISA developed for this project could be used to validate malaria vaccine candidate antigens, provide useful baseline information for clinical trials, and contribute to quality assured laboratory capacity in Africa.In conclusion, using standardized AIA ELISA, anti-MSPDD carried out field studies, developed assays and drafted the manuscript. AA performed the ELISA, compiled data and assisted in the manuscript writing. KAK and HL assisted with the ELISA. MT assisted with the assay development and manuscript writing, while PM, SB and ER wrote the analysis plan, performed the data analysis and together with RC, BDA and YDO contributed to the writing of the manuscript. All authors read and approved the final manuscript.


Should the Kalimbe  be banned, based on its association with an increased risk of malaria? Studies on malaria conducted on Amerindian children in the Oyapock region, French Guiana suggest that there is an argument for replacing the Kalimbe with a modern alternative. However, the wider issue of how the positive (risk reduction and related benefits) and negative effects (exacerbation of acculturation processes and associated consequences) should be assessed needs to be considered before suggesting a change in ancestral behaviour for medical purposes. A multidisciplinary approach is needed, together with caution and humility from epidemiologists. The preservation of cultural practices is a fundamental, valid and attainable goal. However, whether ancestral customs should still be practised is brought into question when they go against hygiene education and disease prevention. Indeed, the introduction of hygiene and disease prevention measures is major factor contributing to the process of acculturation. In most cases, traditional customs are considered undesirable. For example, the prevention of neonatal tetanus requires umbilical cord asepsis, which is incompatible with Angolan traditional plasters made out of charcoal, palm oil and plant leaves . HoweverIn the Amazon, a region of endemic malaria, leishmaniasis and Chagas disease, people wearing a Kalimbe  on the eastern border of French Guiana (Oyapock region). It was based on medical, environmental and behavioural predictive factors for malaria, using the Kaplan Meier method and Cox modelling [Cox modelling identified five variables linked to a first attack of malaria table . ClearinIt is advisable to clear vegetation around homes to reduce the risk of malaria in children. However, in an unchanging Amazonian environment, recommending the removal of carbets  away from the river is debateable, because the river represents a source of life, hygiene, transportation and entertainment. Furthermore, it is problematic to blame the Kalimbe for malaria at a time when Amerindian populations are faced with the loss of cultural identity with undeniably negative consequences. Loss of ancestral values  without constructive alternatives is even more regrettable when some of these values have protected against established negative effects of behaviours such as alcoholism, consumerism, delinquency and rural exodus without the appropriate skills for living in the city.Studies on clothing and exposure to vector-borne diseases have so far been limited to those involving military uniforms and clothing impregnated with insecticide ,4). HoweIn general, changing ancestral behaviour because of links to risk factors should only be suggested after careful thought and justification, taking into account the potential negative consequences and risk of acculturation. The following points should be considered:1) There should be no bias in the analysis model and a valid causal relationship should be demonstrated before questioning traditions.2) Is the change for medical purposes only ?3) Are there also economic aspects involved (amount of money saved)?4) Can the benefits of programmed change be evaluated in terms of medical and economic factors?5) What is the ratio of expected benefit to actual gain?We thus need to consider not only the means used, but also the potential negative effects of the changes to be introduced and on the strength of tradition. Although the means can be quantified, the potential negative effects, and in particular, the strength of tradition, are more difficult to evaluate. Is it possible to place a value on ancestral traditions?The socio-cultural and philosophical dimensions of this issue are complex; and taking these factors into account does not simplify the methods of evaluation. Cultural globalisation seems to have its limits.The way forward undoubtedly requires a multidisciplinary approach involving work in applied public health that requires cooperation with social and behavioural scientists and both caution and humility from epidemiologists.The author declares that they have no competing interests.


Intermittent preventive treatment for malaria in children (IPTc) involves the administration of a full course of an anti-malarial treatment to children under 5 years old at specified time points regardless of whether or not they are known to be infected, in areas where malaria transmission is seasonal. It is important to determine the costs associated with IPTc delivery via community based volunteers and also the potential savings to health care providers and caretakers due to malaria episodes averted as a consequence of IPTc.Two thousand four hundred and fifty-one children aged 3–59 months were randomly allocated to four groups to receive: three days of artesunate plus amodiaquine (AS+AQ) monthly, three days of AS+AQ bimonthly, one dose of sulphadoxine-pyrimethamine (SP) bi-monthly or placebo. This paper focuses on incremental cost effectiveness ratios (ICERs) of the three IPTc drug regimens as delivered by community based volunteers (CBV) in Hohoe, Ghana compared to current practice, i.e. case management in the absence of IPTc. Financial and economic costs from the publicly funded health system perspective are presented. Treatment costs borne by patients and their caretakers are also estimated to present societal costs. The costs and effects of IPTc during the intervention period were considered with and without a one year follow up. Probabilistic sensitivity analysis was undertaken to account for uncertainty.Economic costs per child receiving at least the first dose of each course of IPTc show SP bimonthly, at US$8.19, is the cheapest to deliver, followed by AS+AQ bimonthly at US$10.67 and then by AS+AQ monthly at US$14.79. Training, drug delivery and supervision accounted for approximately 20–30% each of total unit costs. During the intervention period AS & AQ monthly was the most cost effective IPTc drug regimen at US$67.77  per malaria case averted based on intervention costs only, US$64.93  per malaria case averted once the provider cost savings are included and US$61.00  when direct household cost savings are also taken into account. SP bimonthly was US$105.35  and AS & AQ bimonthly US$211.80  per malaria case averted based on intervention costs only. The incidence of malaria in the post intervention period was higher in children who were <1 year old when they received AS+AQ monthly compared to the placebo group leading to higher cost effectiveness ratios when one year follow up is included. The cost per child enrolled fell considerably when modelled to district level as compared to those encountered under trial conditions.We demonstrate how cost-effective IPTc is using three different drug regimens and the possibilities for reducing costs further if the intervention was to be scaled up to the district level. The need for effective training, drug delivery channels and supervision to support a strong network of community based volunteers is emphasised. It is estimated that 300–500 million malaria episodes are recorded each year, 90% of which occur in sub Saharan Africa, causing approximately 800,000 deaths Intermittent preventive treatment for malaria in children (IPTc) is a promising new approach to malaria control in areas where malaria transmission is seasonalIn addition to showing the efficacy and effectiveness of IPTc, it is important to determine the costs associated with IPTc in terms of the total expenditure needed to provide IPTc and also the potential savings to providers and caretakers achieved by reducing the number of children who present at health facilities for inpatient or outpatient care.The findings in this paper are based on a randomised, placebo controlled trial designed to evaluate the effectiveness of IPTc in reducing anaemia and malaria in an area with up to 6 months of transmission in Ghana. Two thousand four hundred and fifty-one children aged 3–59 months were randomly allocated to four groups to receive: three treatments with artesunate plus amodiaquine (AS+AQ) monthly, three with AS+AQ bimonthly, one dose of SP given bi-monthly or placebo. Compared to placebo, trial results showed that monthly AS+AQ reduced the incidence of malaria by 69%  and anaemia by 45% , bimonthly AS+AQ reduced the incidence of malaria by 17%  and anaemia by 32%  and bimonthly SP reduced the incidence of malaria by 24%  and anaemia by 30% The study was approved by ethical committees of the Ghana Health Service/Ministry of Health (GHS/MOH) and the London School of Hygiene & Tropical Medicine. After obtaining written, informed consent, data on malaria treatment costs were collected from the families of children involved in the study. Verbal consent was sought from the health care professionals before they were asked, or observed, in an attempt to assess the resource use and associated costs of treating children with malaria. Verbal consent was considered sufficient for health care workers as the socio economic analysis came at the end of the wider efficacy study and thus facility staff had a history of working with those in the study and were already sensitized to the aims and objectives of the cost effectiveness sub study. The ethics committees in Ghana and London were made aware of the use of both verbal and written consent.This study was carried out in Hohoe district, Ghana. A full description of the study area has been published elsewhere The study was a randomized, placebo-controlled trial of IPTc conducted in children aged 3–59 months who resided in the study district. The trial took place during the six months of the high malaria transmission season. From April to September 2005, 2451 children were randomised to receive one of the following three IPTc regimens under direct supervision of the CBVs every 28 days (placebo or active drug): (i) a single dose of SP every two months, (ii) a three-day course of AS+AQ every two months and (iii) a three-day course of AS+AQ every month. All single and multiple doses of SP and AS+AQ respectively were administered by the CBVs. Children were followed for one year after stopping IPTc to monitor the possibility of a rebound in malaria morbidity.Field workers visited study children once a week during the period of drug administration to enquire about their health and completed a morbidity form if a child had any illness. A passive surveillance system to monitor malaria and anaemia in study children throughout the study period was set up in the district hospital and in 21 health centres in the study area. If a child who presented at one of these facilities had fever or any features suggestive of malaria, a finger prick blood sample was collected for malaria parasite examination before treatment was given. Blood slides were read at the respective health facility to decide on treatment and read again in a central laboratory to confirm the diagnosis. Children with proven or presumptive malaria were treated with oral quinine according to the Ministry of Health (MOH) treatment guidelines.Three main areas of costing were determined - the cost of delivering IPTc, the cost of malaria case management from the provider perspective (inpatient and outpatient visits to a government facility) and the costs incurred by the caretakers of the children visiting the inpatient and outpatient facilities. Care was taken to exclude resources related to research activities. Financial and economic total costs are presented. Financial costs reflect the additional resources required to deliver IPTc in terms of the actual expenditures incurred. For example the payment of resources such as IPTc drugs, incentives, materials and supplies. Straight line depreciation of capital costs is used in the financial cost estimates. The economic costs capture the opportunity cost of all resources used to provide IPTc, whether or not they incur a financial cost. For example, the time health personnel are involved in IPTc delivery represents an economic cost to the programme, because although they are already receiving a salary from the Ministry of Health, they could have spent their time in other activities. An annualisation and discount rate of 3% is used to calculate economic costsThe cost of delivering  and administering IPTc during the six months of the intervention was identified using components of the trial budget and data collected on resource use. Costs categories included those of IPTc drugs, training of health personnel and CBVs, health personnel staff time, utilities , supplies, transport supervision and incentives. Children aged 3–5 months received a quarter of a tablet, those aged 6–11 months half a tablet, those aged 12–23 months three quarters of a tablet and those aged 24 months and above received one tablet each of SP, co-formulated AS+AQ or placebo. The full tablet was costed across all age groups to allow for wastage.The volunteers who had been selected by the caretakers of the children in each community to administer the IPTc drugs were paid an allowance of approximately US$10 a month. This was based on the amount paid to volunteers who administer vaccines during poliomyelitis, measles and neonatal tetanus campaigns (US$3 per day for three days).The training of volunteers took place at the Hohoe District Health Directorate for five days. The volunteers were trained on how to identify drugs packaged for each child, administer drugs, complete drug administration and morbidity forms and when to refer patients to the health facility. Two field supervisors visited the volunteers to provide support and supervise the administration of every first dose of drug administration. They also visited the each volunteer every week to collect adverse event and morbidity forms. They used the forms to follow-up the adverse event cases.Health personnel costs associated with IPTc delivery were obtained from the 2005 records of salaries and allowances and 2006 consolidated salaries paid at the Hohoe district hospital and district health centres. Although not collected in Hohoe, the cost to the caretaker of accessing IPTc was explored in a similar study in The Gambia and found to be negligible (unpublished data).Having undertaken a detailed costing of delivering IPTc to the study population, the costs of scaling up the intervention to Hohoe district were modelled. This involved increasing the potential number of children receiving IPTc from 2451  to 33,000 . Resources used and their associated costs were based on information from the study budget, other community based programmes such as poliomyelitis, measles and neonatal tetanus campaigns underway in the Hohoe district and estimates form the principal investigator involved in the original IPTc clinical trial.Provider costs were based on detailed retrospective cost data obtained from government facilities. Costs were identified using records supplied by the Ministry of Health and the Ministry of Finance, together with components of the study budget and patient folders in the study site in Hohoe. An ingredients approach was used and costs included personnel, materials and supplies, transport, utilities and buildings Staff time required for the management of children with clinical malaria and/or anaemia was estimated by direct observation and interviews undertaken with staff at the hospital and health centres. Once verbal consent was obtained from the health workers and parent/guardian of patient, children were tracked from the time of arrival at the out-patients department and time spent with each health worker was recorded. More general costs were apportioned based on the number of under 5 year old malaria  cases as a proportion of the total number of outpatient and inpatient visits recorded at the facilities.Having given informed consent, structured interviews with primary caretakers of children were conducted to determine any costs related to malaria treatment incurred at the household level. All children in the IPTc study who visited the outpatient department during the intervention period with clinical malaria or malaria with anaemia were visited at home up to 12 months after the intervention. Only 207 out of the 448 (46%) children were available at the time of the survey. Ten out of 44 (25%) of those admitted to the hospital with a diagnosis of malaria or malaria with severe anaemia were also interviewed at home. Families of patients incurred both indirect and direct costs. Direct costs included out-of-pocket expenses on items such as formal hospital fees , informal hospital fees , medication, food and transport costs. Due to the long recall period, once the mode of transport used by the career and the sick infant was identified a standard fare was applied to the number of journeys reported (based on reported Ghana private road and transport union standard taxi and bus fares). Similarly the market rate for an adult meal was used as a proxy for the direct cost of food. Drug costs were calculated using the records of study patients who had received inpatient or outpatient care respectively. The drug costs borne by the household were cross referenced with costs identified at the district hospital pharmacy, the district health directorate store and published market drug prices Care was taken to avoid double counting costs when combining both provider and household direct costs as certain costs were considered cross subsidisations, for example the admission fees paid by the household was a transfer of funds to subsidise certain provider costs. Using the total costs  and lateCosts and incremental cost effectiveness ratios (ICERs) are expressed in US$ 2008. Expenses were converted into the local Ghanaian Cedis. An average value of the Cedi was taken over the time of the analysis. This translated into costs spanning from March 2005 until November 2005, with an average of Cedis 9074 =  1US$ (ranging from Cedis 8617 to Cedis 9150) ICERs were calculated as probability distributions rather than as point estimates; 10,000 iterations were runThe total costs of giving SP bimonthly to 613 children, AS+AQ monthly to 626 children and AS+AQ bi-monthly to 562 study children are presented in The unit costs of an inpatient or outpatient visit for malaria with or without anaemia are presented in The acceptability curves of the three IPTc drug regimens are presented in The costs of delivering IPTc presented here are based on a well funded, small scale research study. In an attempt to better understand the costs of operationalising this intervention on a district wide scale, a model was develop to scale up costs (both fixed and variable) and explore potential savings from economies of scale. Costs reflect the amount required by the health care system to implement IPTc in the whole district with an estimated total of 33,000 children aged less than five years. To try and calculate a realistic level of IPTc coverage the total number of children in the district under five is multiplied by 74% to reflect the coverage rate reported for Ivermectin delivery  are reported to be US$1.46–4.00 Twenty years ago in The Gambia, mass chemoprophylaxis with Maloprim administered over several years by primary health care workers, with the support of village volunteers, to children aged 3–59 months reduced both malaria mortality and morbidity. When inflated to US$ 2008, the cost per child protected per season was US$ 4.75; the cost per childhood death averted was $239The high IPTc intervention costs in Hohoe are in part due to the small scale and vertical nature of the study. If IPTc were to be implemented routinely there is a greater likelihood of increased shared costs as costs associated with IPTc specific supervision and drug delivery could be expanded to include other non IPTc activities that would help establish a CBV network As reported previously This economic evaluation shows the importance of including societal cost savings when deciding on the value of an intervention. Based on intervention costs alone IPTc may appear costly, however, once the savings to the health system and to households are included IPTc appears more favourable. The cost savings would have been more pronounced had there been more of an impact on severe episodes as has been observed in larger studies conducted in Burkina Faso and MaliCentral to the success of IPTc is the sustainability of the community health volunteer network. This study has shown that this network can be sustained for a six month study but challenges may appear if this network of people is to work effectively year on year and on a larger scale. The challenges of scaling up a community – based health planning and services (CHPS) initiative in Ghana from an experimental project to a national program have been discussed elsewhere, and if IPTc is to be launched on a larger scale in Ghana, those responsible for its implementation could learn from some of the constraints to scaling up identified in the CHPS study Increasing attention is being given to the role of community health workers and community health volunteers in public health programmes The recognition that the CBV needs to be compensated was central to the success of this study and this has been reported as an important factor in other successful community health interventions The literature on the costs of scaling up health interventions is scarce and difficult to compare across studies This study is one of the few conducted in Ghana that has investigated the costs of treating malaria from both the provider and household perspective and the only one to use primary cost data to calculate the costs of treating severe malaria and anemia. The provider costs of malaria treatment in this study are comparable to the costs presented for WHO Choice Ghanaian inpatient visits and slightly lower for outpatient visits  IPTc has proved to be a safe and effective approach to reduction of the burden of malaria in Hohoe, an area of Ghana with a prolonged, intense malaria transmission season


Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment.Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included.Ab responses to 13 pre-erythrocytic The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure.The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era. Malaria is a major threat in tropical and sub-tropical regions, with nearly 50% of the world population exposed to different degrees, and an estimated 250 million people suffer annually from the disease . DespiteP. falciparum  carbodiimide hydrochloride  and N-hydroxysulfosuccinimide , vortexed and incubated at room temperature for 20 min in the dark . Activated beads were washed twice with 400 μl of coupling buffer (50 mM of 2-[N-morpholino] ethanesulfonic acid (MES) monohydrate pH 5, Sigma-Aldrich) and resuspended in 400 μl of coupling buffer prior to peptide addition. For optimization purposes, three different concentrations of peptides  were added to the beads. Beads and peptides were vortexed, sonicated and then incubated for 2.5 h in the dark, with shaking at room temperature. Coupled beads were blocked for 30 min with shaking in the dark using 500 μl of PBS-TBN  buffer. Coupled beads were washed with 1 ml of PBS-TBN buffer, resuspended in 200 μl of the same buffer and stored at 4°C in the dark. Four aliquots of 5 × 106 beads were prepared for each peptide and mixed for homogenous coupling.Carboxylated Luminex beads with different fluorescences  were covalently coupled with BSA only or 15 peptide-BSA complexes using a modification of the protocol from Luminex Corporation  as follows. Beads  diluted 1:500 (1 μg/ml), was added to each well. After 30 min of incubation in the dark at room temperature with shaking, plates were washed as described previously. Beads were resuspended in 100 μl of a solution of 5% BSA-PBS, pH 7.4 and finally analysed on Luminex system. The system was set to read a minimum of 100 beads per spectral address, and results were expressed as the median fluorescent intensity (MFI).Ag-coated beads were thoroughly resuspended by vortexing and sonication, and were diluted in equal volumes of PBS and MFIA (Multiplexed Fluorescence ImmunoAssay) diluents  at a final concentration of 80 beads/μl per peptide. The 1.2-μm filter-bottom 96-well microtitre plates  were pre-wetted with washing buffer  using a vacuum manifold (Millipore). Equal volumes of beads and sera  were added to the wells. Plates were incubated at room temperature in the dark for 1 h with shaking at 500 rpm. After incubation, plates were washed eight times with 200 μl of washing buffer, then 100 μl of the secondary Ab (R-phycoerythrin F(ab')i.e. from the four groups : 8 travellers, 8 adults from Diama, 5 adults from Ndiop and 9 adults from Dielmo) were also tested against the same antigens by conventional ELISA using a method previously described [i.e. using peptide-BSA complexes that were linked to the beads and BSA-coated wells as control wells, i.e. without peptide.A random sub-sample of 30 sera representative of those used for the bead-based assays  of synthetic peptides were coupled to 5 × 10As shown by the distribution of Ab MFI in figure With regard to serum dilutions, the highest MFI values were observed with the lowest 1:50 dilution  were compared to those detected by monoplex assay (each Ag tested in a separate assay). As shown in Figure 2 = 0.994. Therefore, StarpR-coupled beads had to be used within 1 month after preparation, whereas all other Ags-coupled beads can be stored for longer periods before use.The stability of Ag-coupled beads after 1, 2 and 3 months storage at 4°C was examined by testing against sera from the exposed and unexposed adults; results are shown in Figures A. gambiae Saliv1 Ag  and young people (from 10 to 20 years old) within the three groups living in endemic areas was also compared. The comparison showed that the proportion increased significantly with age and according to the level of malaria exposure for the following Ags: Lsa1-41, Lsa1J, Lsa3NR2, Glurp, GlurpP3, Salsa1, Salsa2, StarpR, CSP, SR11.1 and Saliv1 (p ≤ 0.001).Ag/MFIBSA). The median specific Ab intensity increased significantly with the malaria exposure level for Lsa1-41 (p < 0.02), Glurp (p < 0.002), and CSP (p = 0.002) Ags. Moreover, a similar trend was observed for some of the remaining Ags, as shown in Figure The mean of MFI values of seropositive adults are presented in Figure P. falciparum Ags for which adults within the groups were seropositive. The higher the exposure level to malaria, the higher the mean number of P. falciparum Ags that produced seropositivity. In the travellers group, the median number of P. falciparum Ags for which individuals were seropositive was one, whereas it was three for individuals from Diama, six for individuals from Ndiop and seven for individuals from Dielmo. The difference between groups was significant (p < 0.0001).The number of Ags in the different groups that produced seropositive results was investigated. Figure There were significant correlations of the evaluations of the Ab responses obtained by bead-based assay and conventional ELISA assay with the peptides Lsa1-41, Lsa1-J, Lsa3-NR2, Glurp, Glurp P3, Salsa1, Salsa2, Trap1, Starp-R, CSP and Saliv1  and may on occasion give unexpected cross-reactivities. However, the peptides chosen for the present study had been previously compared to recombinant proteins in ELISA essays and were shown to give consistent results. A high prevalence of Ab response to pre-erythrocytic Ags has been found in individuals living in malaria endemic areas [P. falciparum correlates with prevalence rates for those Ags [Thirteen pre-erythrocytic peptides from ic areas ,26,27, ahose Ags , and unpWhen evaluating the distribution of the mean number of peptides against which individuals were seropositive, the value was found related to the level of malaria endemicity to which they were exposed. Figure P. falciparum Ags, the assay also included two peptides derived from one A. gambiae salivary Ag. As previously shown, the evaluation of immune responses to mosquito salivary Ags can indicate exposure to vector bites. Studies have demonstrated that children living in malaria endemic regions develop Abs against the vector's salivary proteins [A. gambiae salivary Ags represent an immunological marker of exposure to malaria vector [Anopheles species. It was reported to be potentially antigenic in travellers briefly exposed to Anopheles bites and was more recently confirmed as antigenic in Senegalese children [In addition to pre-erythrocytic proteins . Thus, Aa vector . One of a vector . The gSGchildren .Anopheles bites, short-term exposure in travellers and long-term exposure in people living in endemic countries. Furthermore, immune responses against such peptides are difficult to detect in briefly exposed travellers, though a response was observed when the Abs were tested directly against mosquito saliva [The two peptides derived from gSG6 protein included in the assay  were antigenic, but the intensity of their IgG responses was peptide-dependent, where Saliv1 showed the higher response . In the o saliva .Anopheles bites, notably in Senegalese children ≥2 years of age [Anopheles bites at a lower rate compared to Senegalese children due shorter exposure times and systematic use of anti-vectorial devices . This factor, together with the different assay used - multiplex at a 1:100 dilution of sera in our case and ELISA at a 1:20 dilution of sera in the study performed by Poinsignon et al [It has been reported recently that the gSG6-P1 peptide  can be used to evaluate low-level exposures to s of age . Unfortuon et al  - could P. falciparum and A. gambiae salivary Ags in large populations, allowing efficient identification of putative protective Abs. Furthermore, the association of Ab responses to malaria exposure levels provides a way to monitor exposure in differentially exposed indigenous people, as well as in travellers not immune to the parasite, allowing the assessment of P. falciparum transmission and malaria risk.The multiplex technology developed in the present study is a reliable and fast method to test immune responses to The authors declare that they have no competing interests.EA carried out statistical analysis and interpretation of the data, drafted and revised the manuscript. CD developed the techniques, carried out all the immunological investigations, carried out initial statistical analysis and revised the manuscript. AP carried out the ELISA analysis. ATB, AT, CS, J-FT, EO-P, FR and JBS collected data and sera. TF, FR, AP, EO-P and PD contributed to the design of the method and to the choice of the antigens and peptides. KP produced the peptides. CR was the principal investigator of the study, conceived the research programme, participated in the development of the technique, statistical analysis, interpretation of the data and revision of the manuscript. All authors read and approved the final manuscriptCorrelation between Luminex and ELISA results obtained with 30 sera. The Spearman correlation coefficient and the Pearson correlation coefficient between Luminex and ELISA results have been estimated on 30 sera.Click here for file