Plasmodium falciparum, a poor sensitivity of RDTs was reported against Plasmodium vivax based on the adopted antibody against pan-Plasmodium antigen lactate dehydrogenase (pLDH) or aldolase. Levels of pLDH were measured from patient with P. vivax, and the correlations between the levels of pLDH and the sensitivities of RDTs were analysed among Republic of Korea (ROK) isolates.The malaria rapid diagnostic tests (RDTs) are now widely used in the world. Compared to Three RDTs, OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria Pf/Pan antigen test, and the Genedia pLDH antigen ELISA were performed with blood samples from 152 febrile patients and 100 healthy controls.P.vivax by pLDH antigen ELISA test  in comparison to traditional microscopy using Giemsa–stained slides. None of the healthy control tested positive by three RDTs or ELISA, indicating 100% specificity in their respective test. Levels of pLDH among Korean P. vivax isolates ranged between 0 ng/mL and 22,387.2 ng/mL . The lower detection limits of three RDTs were between 25 and 50 ng/mL with artificially diluted samples. The moderate degree of correlation was observed between parasitaemia and concentrations of pLDH .Three malaria RDTs revealed sensitivities between 85.5 (131/152) and 86.8% (132/152) with highest sensitivity for the detection of P. vivax are the main explanation for the variations in the performance of pLDH-based RDTs. Therefore, comparing sensitivities of RDT may need to include targeted biomarker value of patients.The pLDH levels of  Malaria remains to be one of the most difficult infectious diseases to control. Even though many new efforts are being made to eradicate the disease, the incidences and prevalence of malaria does not decrease and is worsening in some incidents . Early dPlasmodium lactate dehydrogenase in Plasmodium falciparum and pan-specific Plasmodium lactate dehydrogenase and aldolase were used for detecting other human Plasmodium species. While sensitivities of currently available malaria assay kits in the market would fluctuate by field conditions, relatively poor sensitivities of RDTs for Plasmodium vivax with reactive antibodies to pan-Plasmodium antigen lactate dehydrogenase (pLDH) or aldolase was reported in comparison to RDTs for P. falciparum[The major target antigens of malaria RDTs were specific histidine-rich protein 2 (PfHRP-2), and alciparum-8. The salciparum. Monitoralciparum.P. vivax parasites, since pLDH usually disappear rapidly within three to five days in the body [P.vivax infected patients and sensitivities of currently available malaria RDTs were not reported. In addition, the association from RDT results between pLDH levels and parasitaemia was not evaluated.Among malaria RDTs, pLDH-based malaria RDTs present better sensitivities, corresponding well to the respective levels of parasitaemia. The levels of pLDH reflect the presence of metabolically viable the body . Hence, the body -14, it sP. vivax in comparison with three simultaneous RDT tests. The results of both RDT and pLDH ELISA assays for P. vivax were analysed and compared against parasitaemia levels from Giemsa-stain microscopy for calculating the detection limit of pLDH-based RDT kits.In the present study, levels of pLDH with pLDH specific ELISA test were measured in diagnosed patients with A total of 252 samples, including 100 healthy controls, were collected between April 2009 and November 2012 at Korea University Hospital, Republic of Korea (ROK). Patients (n = 152) had fever or a recent history of fever within a week of their return from malaria-endemic regions. All patients and control subjects gave their informed written consent to participate in the study. This study was approved by the Ethical Committee of the Korea University Guro Hospital.Thick and thin blood films were prepared for the diagnosis of malaria. The species and density of the plasmodial parasites were blindly determined by two experts in malaria diagnosis, using microscopic examination of Giemsa-stained thick and thin blood films. Parasitaemia was indirectly calculated by counting the parasite numbers per 200 white blood cells (WBC) in blood film, and the WBC counts from the automatic blood cell counter .P. vivax  were amplified as previously described [Genomic DNA was extracted from frozen pellets using a blood genomic DNA extraction kit  and stored at −80°C. Circumsporozoite protein (CSP) genes of P. falciparum and pan-Plasmodium-specific pLDH from P. vivax, as major specific target antigens. The other two tests used HRP-II from P. falciparum and pan-Plasmodium-specific pLDH from P. vivax for the specific target antigens. The assays were performed following each manufacturer’s instructions. All RDTs were read by different technicians, who were blinded against the results from the other diagnostic techniques and RDTs. Detection limit of pLDH and parasitaemia in three RDTs from five individual kits was determined from artificially diluted P. vivax samples with known parasitaemia  and pLDH level .The three RDTs, OptiMAL test, SD BIOLINE Malaria Ag Pf/Pan test and Humasis Malaria Pf/Pan antigen test, were used in this study. Humasis Malaria Pf/Pan antigen test kits (AMAL-7025) were provided by the manufacturer for the evaluation, and the OptiMAL-IT test (710024) and SD BIOLINE Malaria Ag Pf/Pan test (05FK60) kits were purchased from BioRad Inc  and SD Inc , respectively. OptiMAL test incorporated pLDH from Genedia malaria antigen ELISA test  was provided for the evaluation. The ELISA test was based on the quantification of pLDH in a whole blood sample with immobilized pLDH-specific capture antibody on 96-well plates. The ELISA test was performed, as recommended by the manufacturer. Briefly, 50 mL of EDTA whole blood and controls were added to each well with 100 mL of diluent. After one-hour incubation at 37°C, unbound materials were washed away with PBS-Tween 20, and 50 mL peroxidase-conjugated goat anti-pLDH (1:101 dilution) was added to each well. The supplied recombinant pLDH antigen on the kit was diluted serially from 2,500 to 0.02 ng/mL as positive controls and reference standards. After 30 min incubation at 37°C, the excess peroxidase-conjugated antibody was washed away three times with 500 mL PBS/0.05% Tween-20 (PBS/Tween), and the chromogenic signal was developed with addition of 200 mL tetramethylbenzidine-dimethyl sulphoxide (TMB). Following 20-min incubation at 37°C, the reaction was stopped by adding 20 μl of 5 N H2SO4. Absorbance at 450 nm was read using an ELISA reader . Each sample was tested in duplicate, and the optical density (OD) values were averaged. The background value at wavelength at 650 nm was subtracted from the OD values at 450 nm for each sample.The obtained OD values of the standard references were plotted against their concentrations, either on semi-logarithmic graph paper or using an automated method. The concentrations of the samples were read directly from the standard reference curve. Samples with higher concentrations than the highest standard reference were diluted, re-assayed, and calculated according to the dilution factor. For the data analysis of dichotomous groups, cut-off value (CO) for the positive ELISA signals was set at 0.1 plus mean OD of negative control (as recommended by manufacturer). S/CO value was counted as S divided by CO. If S/CO value was greater or less than 1.0 (≥1.0 or <1.0), the samples were interpreted as positive or negative, respectively.P. vivax infected patients was 3,971.5 ± 6120.9 ng/mL with range between 0 and 22,387.2 ng/ml  fell between 500–5,000 ng/mL , and the second frequent level was over 5,000 ng/mL . Thirty seven cases (20.1%) revealed extremely low pLDH levels, less than 50 ng/mL. In healthy controls, pLDH was not detected. The lowest detectable level of pLDH antigen ELISA was 6.3 ng/mL, and lowest parasitaemia level of patients detected by pLDH antigen ELISA was five parasites/μL in clinical samples. Direct correlation was observed between pLDH level and parasitaemia   parasitaemia level was 3954.1 ± 3712.5/μL with range between 2.0 and 19,382.0/μL, and mean (±SD) pLDH level of The diagnostic sensitivity of pLDH antigen ELISA was 95.4% 145/152) among microscopy-positive cases with 100% specificity (0/100), which was superior to three malaria RDTs. OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, and Humasis Malaria Pf/Pan antigen test had sensitivity of 85.5% (131/152), 86.8% (132/152), and 86.8% (132/152) with each 100% specificity (0/100), respectively  showed panel detection score (PDS) of 0% at both 200 and 2000 parasites/μL of P. vivax[P. vivax was 100% with no observed false positivity. As for OptiMAL-IT kit (710024), round 4 report presented PDS of 97.1 and 68.6% at 200 and 2,000 ~ 5,000 parasites/μL of P. vivax, respectively. On the other hand, OptiMAL-IT showed increased clinical sensitivity from 52.7 to 100%, as the P. vivax parasitaemia increased. As for the SD Bioline Malaria Ag P.f/Pan kit (05 F60), round 4 report showed PDS of 97.1 and 100.0% at 200 and 2,000 ~ 5,000 parasites/μL of P. vivax, respectively. However, SD Bioline demonstrated slightly decreased clinical sensitivity of 87.5 ~ 100.0% .pLDH antigen ELISA system showed higher sensitivity (95.4%) in comparison with three RDTs (85.5-86.8%), which might be associated with the fact that ELISA uses 50 μL of blood, while RDTs used only 5–10 μL of blood. RDTs are fast and convenient diagnostic tools. Parasitaemia levels of From three tested RDTs, pLDH levels and parasitaemia levels of three RDTs, positive cases were higher than two RDT positive cases or all RDT negative cases. One positive sample from two RDT tests (n = 3) showed disparity between parasitaemia and pLDH levels, where the pLDH and parasitaemia levels were 15 ng/mL and 2,300/μL, respectively. These levels of parasitaemia most likely produce clinical symptoms. RDTs are fast and convenient diagnostic tools. Using only RDTs, malaria diagnosis might be difficult and patients might be left untreated, leading to severe complications. Since the evaluation of RDTs is currently based on patients’ parasitaemia levels, with little information on targeted biomarkers in clinical isolates, the current study provides additional information for selecting adequate malaria RDT in fields.The authors have declared that they have no competing interests.JWJ carried out the molecular work and RDT test. CHC carried out the immunoassays. ETH & SSA participated in the design of the study and performed the statistical analysis. CSL contributed to the design and coordination of the study, assisted with data entry, analysis and interpretation. All authors read and approved the final manuscript.


African countries are scaling up malaria interventions, especially insecticide treated nets (ITN) and indoor residual spraying (IRS), for which ambitious coverage targets have been set. In spite of these efforts infection prevalence remains high in many parts of the continent. This study investigated risk factors for malaria infection in children using three malaria indicator surveys from Zambezia province, Mozambique. The impact of IRS and ITNs, the effects of keeping farm animals and of the construction material of roofs of houses and other potential risk factors associated with malaria infection in children were assessed.Cross-sectional community-based surveys were conducted in October of 2006, 2007 and 2008. A total of 8338 children (ages 1–15 years) from 2748 households were included in the study. All children were screened for malaria by rapid diagnostic tests. Caregiver interviews were used to assess household demographic and wealth characteristics and ITN and IRS coverage. Associations between malaria infection, vector control interventions and potential risk factors were assessed.Overall, the prevalence of malaria infection was 47.8% (95%CI: 38.7%–57.1%) in children 1–15 years of age, less than a quarter of children  were sleeping under ITN and almost two thirds were living in IRS treated houses . Protective factors that were independently associated with malaria infection were: sleeping in an IRS house without sleeping under ITN (Odds Ratio (OR)  = 0.6; 95%CI: 0.4–0.9); additional protection due to sleeping under ITN in an IRS treated house  versus sleeping in an unsprayed house without a ITN; and parental education  versus parents with no education. Increased risk of infection was associated with: current fever  versus no fever; pig keeping  versus not keeping pigs; living in houses with a grass roof  versus other roofing materials and bigger household size  versus small households (1–4 persons).Malaria infection among children under 15 years of age in Zambezia remained high but conventional malaria vector control methods, in particular IRS, provided effective means of protection. Household ownership of farm animals, particularly pigs, and living in houses with a grass roof were independently associated with increased risk of infection, even after allowing for household wealth. To reduce the burden of malaria, national control programs need to ensure high coverage of effective IRS and promote the use of ITNs, particularly in households with elevated risks of infection, such as those keeping farm animals, and those with grass roofs. Plasmodium falciparum, remains one of the most important causes of morbidity and early mortality in endemic regions of sub-Saharan Africa Malaria, especially that caused by Many of the risk factors for malaria are related to access to interventions and inversely related to a household's socio-economic status (SES) with increasing vulnerability of the poorest The African Summit on Roll Back Malaria, held in Abuja, Nigeria in 2000, specified that 60% of population at risk of malaria should use ITNs by 2005, a target that was raised to 80% by 2010 During the past decade, IRS has been implemented successfully in Mozambique, Eritrea, Ethiopia, Equatorial Guinea, Island of Principe and Madagascar P falciparum. Transmission is perennial with peaks during and after rainy seasons. The most important malaria vectors belong to the Anopheles gambiae complex and the An funestus group In Mozambique malaria is endemic, affecting the entire population of around 23 million people Mozambique is currently promoting ITNs and IRS for malaria prevention. Long lasting insecticidal nets (LLIN), are distributed free to pregnant women and children under five years old. IRS campaign for malaria control has been conducted in Mozambique using various insecticides. Before 2000, pyrethroids were used for IRS but the detection of resistance In this study, the effects on malarial infection of vector control interventions, house construction, ownership of farm animals, household wealth and other potential risk factors were investigated using malaria indicator survey data from Zambezia province, Mozambique.2, much of it drained by the Zambezi River Zambezia province is situated in central Mozambique, has an estimated population of 3,794,509 and covers a total area of 103,127 kmAn. funestus was first reported in southern Mozambique in 2001 In Zambezia province, IRS with DDT was re-introduced in 2006 through the Mozambique National Malaria Control Program (MNMCP), supported by the US Presidents Malaria Initiative The ITN used are mostly long lasting types: the Olyset® (active ingredient: 2% permethrin) and PermaNets® (a.i: 50–55 mg/m2 deltamethrin) brands that were distributed in Zambezia since 2004 Malaria surveys were conducted annually in October in 2006, 2007 and 2008 in 19 sentinel sites  establisst quartile being the poorest. Houses were categorized into those covered by a grass roof and those covered with roof materials other than grass. The number of people in the household was grouped into 1–4 people, 5–7 people and 8–15 people.History of fever was defined as parents reporting children who experienced fever in the past 4 weeks. Current fever was defined as body temperature ≥37.5°C taken during the survey. Head of household's education was categorised into those without formal education, those with primary/secondary education and those with tertiary education. Knowledge of malaria prevention was classified as adequate if the respondent was aware of malaria preventive measures in addition to IRS and ITN . During the interview, respondent provided information on whether the net was treated or not but the ITN status was not verified by the interviewer. Given both IRS and ITN are malaria interventions applied in Zambezia, a composite variable constituting the 4 combinations of vector control interventions was used to develop the risk model as: 1) children in households without IRS and not sleeping under ITN, 2) children in houses with IRS and sleeping under ITN, 3) those protected by IRS but not sleeping under an ITN, and 4) those in unsprayed houses but sleeping under ITN. Socio economic status (SES) for each household was calculated from an index which combined ownership of assets  and house construction materials using principal component analysis The following risk factors for malaria infection were investigated: whether the house had been sprayed (IRS) in the past 12 months, whether the child slept under a ITN the previous night, the child's sex and age, head of household's level of education, SES quartile, ownership of farm animal, house construction, knowledge of other malaria prevention practices, history of fever and current fever, and number of people in the house. Point estimates, confidence intervals, survey chi-square tests and estimates for odds ratio were derived taking account of the two-stage sample design by using the survey commands in Stata 11 Crude odds ratio for the association between malaria infection and each explanatory variable were assessed one at a time using logistic regression. Variables with association (p<0.025) in the univariate analysis were considered for inclusion in a multivariable logistic regression model. The contribution of each covariate in the risk model was assessed by Wald tests and was included if p<0.05 after adjusting for confounders.Sex and age were used as a priori potential confounders following practice adopted elsewhere Ethics approval for the study was given by the Ministry of Health of Mozambique (Reg: 3622/IMS-2/DNS/06). Written informed consent was obtained from heads of households or responsible adults in each of the participating households.A total of 2,748 households in 19 villages were surveyed in 2006, 2007 and 2008, with an average of 144 houses per sentinel site [range 99 to 175]. A total of 8,338 children from 1 to below 15 years of age participated, giving an average of 439 children/site.The majority of children belonged to households whose head was not educated (42.4%) or had primary/secondary education (41.7%). The majority of children lived in houses covered with grass roof (77.6%) . The proThe proportion of children who slept under any type of net the previous night increased from 29.8% (95%CI: 26.9%–32.8%) in 2006, to 34.3% (95%CI: 31.4%–37.4%) in 2008. Overall, 32.6% (95%CI: 30.9%–34.4%) of children were reported sleeping under any net the previous night . Sixty fAs ITN and IRS campaigns were rolled out in Zambezia province, the proportion of children living in houses with IRS and sleeping under ITNs increased from 12.4% (95%CI: 7.6%–19.7%) in 2006 to 19.0% (95%CI: 13.4%–26.4%) in 2008 . OverallThe proportion of children whose parents reported knowledge of other malaria preventive measures in addition to IRS or ITN was 67.0% (95%CI: 62.4%–71.4%), increasing from 61.4% (95%CI: 55.5%–67.1%) in 2006, to 70.5% (95%CI: 65.3%–75.2%) in 2008 .P. falciparum malaria infection in children (1–15 years) was 47.8% (95%CI: 38.7%–57.1%), of whom 15.8% (95%CI: 12.1%–20.3%) were co-infected with other plasmodial species. Malaria prevalence therefore refers to infections by P. falciparum alone or P. falciparum with other plasmodia. Malaria infection prevalence among children varied between the 19 villages ranging from 22.1% at Mocuba to 87.2% at 25-de-Junho. The proportion of infected children varied between years of survey from 51.9% in 2006, 60.3% in 2007, to 31.8% in 2008.Overall prevalence of There was no evidence of difference in prevalence of malaria infection by sex (p = 0.27) but some evidence that it differed by household size (p = 0.065) and age group (p<0.012) ranging from 43.6% (95%CI: 41.2%–45.9%) in the 1–3 years age group to 51.7% (95%CI: 49.7%–54.1%) in the 7–9 years age group. More children presenting with current fever were infected with malaria  compared with those without fever .Malaria infections among children living in IRS houses and not sleeping under ITN were lower  than those in unsprayed (non IRS) houses and without sleeping under ITN. Children living in IRS houses and sleeping under ITN were additionally protected from malaria infection  relative to those not protected by either method. There was no evidence of a lower odds of malaria infection among children sleeping under ITN in unsprayed houses relative to those not protected by either methods  .In univariate analysis, pig-keeping was associated with increased risk of malaria infection for children , even after excluding 25 de Junho, the village with highest malaria infection prevalence (87.2%) and highest number of children living in households keeping pigs , there was still strong association between pig-keeping and malaria infection . In the analysis including all villages, sheep keeping was associated with reduced risk of malaria infection  , but theA majority of children (77.6%) were living in houses with a grass roof and these children were at elevated risk of malaria infection compared to those living in houses without a grass roof  . ChildreChildren of educated parents  compared to uneducated parents, and those from high SES   compared to low SES (poorest quartile) were associated with lower odds of malaria infection .The multivariable logistic regression model was fitted sequentially starting with variables having an association with malaria infection (p<0.025) . Age of In the final model, the risk of malaria infection was much lower among children living in an IRS household and sleeping under an ITN  and those living in IRS household and not sleeping under ITN  relative to those living in households without either of the two interventions. There was no evidence that sleeping under ITN in an unsprayed house was protective against malaria infection . Children of educated parents were at lower risk of malaria infection  relative to those with no education.On the other hand, pig-keeping  was associated with increased risk of malaria infection in children compared to those living in households without pigs. Malaria infection was also associated with children living in houses with a grass roof  relative to children living in houses with a non-grass roof. Overall the risk of malaria infection increased for children living in large households  relative to those living in small household (1 to 4 people). Children with current fever were more likely to be infected  with malaria than those without fever.There was no evidence that knowledge of prevention measures other than IRS and ITN was independently associated with risk of infection. The odds of malaria infection for sheep keeping  was in the same direction as pig keeping  when sheep keeping was included in the model.In Zambezia province, malaria prevalence is high (47.8%) in children of all age groups below 15 years confirming that malaria remains a major cause of illness during childhood. Likewise, a survey of malaria across Mozambique reported overall malaria prevalence of 49% and relatively high age-specific malaria infection ranging from 39% for 7–10 years to 55% for children 1–2 years An. funestusChildren living in IRS treated houses and sleeping under an ITN  were at lower risk of malaria infection than children living in IRS houses and not sleeping under ITN , p = 0.028. The combined protective effect of IRS and ITN has been reported previously Knowledge of malaria preventive measures other than IRS and ITN , did not appear to have any protective effect on children, emphasising that conventional methods such as IRS and use of ITNs should be encouraged for public heath use of malaria control.Anopheles arabiensis, attracted to animals and displaying opportunistic feeding behaviour An. gambiae s.s and An. funestus. Although the latter are inherently endophilic, feeding mainly on human hosts An. funestus being not necessarily entirely anthropophagic Keeping farm animals, in particular pigs and to lesser extent sheep was associated with increased risk of malaria infection even after adjusting for confounders such as household wealth, suggesting a possibility that certain farm animals may attract malaria vectors to the houses. Several studies have evaluated the relationship between malaria infection and keeping animals with conflicting results Malaria risk can vary widely between villages or even households Education and smaller household size were associated with lower odds of malaria infection. Educated parents are more likely to encourage their children to sleep under a net because they are better informed and living in smaller household can provide adequate space to hang nets.The likelihood of a child presenting with fever to be infected with malaria infection was high, with 56.7% of fever cases having malaria infection compared to 46.7% in none fever cases. Other studies have confirmed that malaria infections remain a major cause of febrile illness during childhood In this study several limitations should be noted. Firstly, the surveys were conducted in October coinciding with the onset of the rainy season with still low mosquito densities and malaria transmission. Also the study does not provide all information on factors determining the risk of malaria infection throughout the year. Secondly, survey sites are mainly located in the southern part of Zambezia province therefore the results are not necessarily representative of the whole province or the country. Thirdly, the malaria rapid tests are subject to limitations in sensitivity and specificity which could have lead to an underestimation or overestimation of infection prevalence since no blood slides were taken for validation purposes In conclusion, the malaria burden among children 1 to under 15 years of age is high in Zambezia province. Consequently, health education and treatment should not only target vulnerable groups (children under 5 and pregnant women), but all the age groups. Since almost half (43%) of fever cases were not infected with malarial parasites, this has implications on policy of fever treatment, particularly in rural areas where all febrile cases may be wrongly treated as clinical malaria. Children living in IRS sprayed houses, or living in a sprayed house and sleeping under an ITN were at much lower odds of malaria infection than children living in unsprayed houses and not sleeping under ITN. The risk of malaria infection was associated with keeping farm animals, particularly pigs and living in houses with grass roofs. In addition to ensuring high coverage of IRS, which should include the spraying of grass roofs, and promotion of the use of ITNs, malaria control programs should consider advising owners of farm animals to ensure a reasonable separation between animal sheds and sleeping areas for humans. Households with increased risk of infection, such as those keeping animals, and those with grass roofs should be particularly targeted for ITN use.


Bojang and colleagues report a randomized trial showing that delivery of intermittent preventive treatment for malaria in children by village health workers is more effective than delivery by reproductive and child health trekking clinics. The Expanded Programme on Immunisation (EPI) provides an effective way of delivering intermittent preventive treatment for malaria (IPT) to infants. However, it is uncertain how IPT can be delivered most effectively to older children. Therefore, we have compared two approaches to the delivery of IPT to Gambian children: distribution by village health workers (VHWs) or through reproductive and child health (RCH) trekking teams. In rural areas, RCH trekking teams provide most of the health care to children under the age of 5 years in the Infant Welfare Clinic, and provide antenatal care for pregnant women.During the 2006 malaria transmission season, the catchment populations of 26 RCH trekking clinics in The Gambia, each with 400–500 children 6 years of age and under, were randomly allocated to receive IPT from an RCH trekking team or from a VHW. Treatment with a single dose of sulfadoxine pyrimethamine (SP) plus three doses of amodiaquine (AQ) were given at monthly intervals during the malaria transmission season. Morbidity from malaria was monitored passively throughout the malaria transmission season in all children, and a random sample of study children from each cluster was examined at the end of the malaria transmission season. The primary study endpoint was the incidence of malaria. Secondary endpoints included coverage of IPTc, mean haemoglobin (Hb) concentration, and the prevalence of asexual malaria parasitaemia at the end of malaria transmission period. Financial and economic costs associated with the two delivery strategies were collected and incremental cost and effects were compared. A nested case-control study was used to estimate efficacy of IPT treatment courses.Treatment with SP plus AQ was safe and well tolerated. There were 49 cases of malaria with parasitaemia above 5,000/µl in the areas where IPT was delivered through RCH clinics and 21 cases in the areas where IPT was delivered by VHWs,  −0.24 to 3.5]). Delivery through VHWs achieved a substantially higher coverage level of three courses of IPT than delivery by RCH trekking teams . For both methods of delivery, coverage was unrelated to indices of wealth, with similar coverage being achieved in the poorest and wealthiest groups. The prevalence of anaemia was low in both arms of the trial at the end of the transmission season. Efficacy of IPTc against malaria during the month after each treatment course was 87% (95% CI 54%–96%). Delivery of IPTc by VHWs was less costly in both economic and financial terms than delivery through RCH trekking teams, resulting in incremental savings of US$872 and US$1,244 respectively. The annual economic cost of delivering at least the first dose of each course of IPTc was US$3.47 and US$1.63 per child using trekking team and VHWs respectively.In this setting in The Gambia, delivery of IPTc to children 6 years of age and under by VHWs is more effective and less costly than delivery through RCH trekking clinics.NCT00376155ClinicalTrials.gov Please see later in the article for the Editors' Summary In sub-Saharan Africa, malaria kills 800,000 people, the majority of whom are children, every year. Intermittent preventive treatment (IPT) of malaria is an effective malaria control strategy. IPT involves administration of antimalarial drugs at defined time intervals to individuals regardless of whether they are known to be infected with malaria to prevent morbidity and mortality from the infection. IPT was initially recommended for pregnant women (IPTp) who are given at least two doses of suphadoxine pyrimethamine (SP) during antenatal visits after the first trimester of pregnancy. IPT is also effective in infants (IPTi) and recently IPTi has been rolled out with the administration of three doses of an antimalarial drug during the expanded program of immunization visits. Clinical studies have also shown that IPT is effective at reducing malaria incidence in children (IPTc) by administering SP alone, or in combination with artesunate (AS) or amodiaquine  over three intervals during the peak malarial season.The inclusion of IPTp in antenatal visits and IPTi in the expanded program of immunization has effectively scaled up these interventions to the population level. So far, IPTc has only been administered to children within the confines of clinical trials—there is currently no established system for delivery of IPTc. For the scale-up of IPTc to be successful, there needs to be an appropriate point of entry and the roll out of a delivery system that can be generalized to most settings in sub-Saharan Africa. In order to address this issue, the researchers conducted a randomized trial to compare the effectiveness of IPTc delivery to children up to 6 y of age by village health workers (VHW) or by reproductive and child health (RCH) trekking teams  in rural areas of The Gambia.During the 2006 malaria transmission season, the researchers randomly allocated the catchment populations of 26 RCH clinics, each with 400–500 children 6 y of age and under, to receive IPT from an RCH trekking team or from a VHW. Before the trial started, the researchers, accompanied by the district health team, visited all villages in the study area to explain the purpose and methods of the study and to obtain consent from the elders of all participating villages. Eligible children were treated with a single dose of SP plus three doses of AQ given at monthly intervals during the malaria transmission season. The researchers passively monitored malaria incidence throughout the transmission season and at the end of the malaria season, examined a random sample of 40 children from each cluster to measure their temperature, height, and weight and to take a finger-prick blood sample to measure blood hemoglobin and parasite levels (by microscopy of thick blood smears). The researchers recorded the financial costs associated with each delivery strategy .There were 49 cases of clinical malaria in the areas where IPT was delivered through RCH clinics and 21 cases in the areas where IPT was delivered by VHWs. In addition, VHW delivery of IPTc achieved a higher coverage level of three courses of IPT than delivery by RCH trekking teams (74% versus 48%). The prevalence of anemia was low in both arms at the end of the transmission season. Delivery of IPTc by VHWs was cheaper than delivery through RCH trekking teams, resulting in incremental savings of US$872 and US$1,244, respectively. The annual economic cost of delivering at least the first dose of each course of IPTc using the RCH trekking team was US$3.47 per child and with VHWs was US$1.63 per child.The results of this study show that in rural areas of The Gambia, delivery of IPTc by VHWs is more effective and less costly than delivery by RCH trekking teams through RCH clinics. Delivering IPTc through community-based VHWs versus monthly visits by the RCH team has several advantages: VHWs are resident in the community, making drug administration easy and flexible (as children were able to receive their medication on any day of the month), and they can remind mothers/guardians to attend for treatment. Therefore, operationally, VHW delivery is less restrictive and more convenient for parents and guardians.http://dx.doi.org/10.1371/journal.pmed.1000409.Please access these Web sites via the online version of this summary at PLoS Medicine research articles by Dicko et al. and Konat et al., and a PLoS Medicine Perspective by BeesonThis topic is further discussed in two The GambiaWHO provides information about health workforce, including the role of village health workersWHO also provides information about the intervention strategiesRoll Back Malaria has information about malaria in children, including malaria in childrenUnicef also provides comprehensive information about  Trials conducted in a number of sub-Saharan African countries have shown that antimalarial chemoprophylaxis reduces malaria morbidity and mortality in children and school absenteeism The IPT strategy was explored first in pregnant women (IPTp) IPT in children (IPTc) with SP, given alone or in combination with artesunate (AS) or amodiaquine (AQ), reduced the incidence of malaria in children under the age of 5 y in Mali In rural areas of The Gambia, there are two possible routes through which IPT might be given to older children: reproductive and child health (RCH) trekking teams and community (village) health workers (VHWs). We have, therefore, undertaken a randomised trial in which we compared the effectiveness of delivery of IPTc to Gambian children 6 y of age and under by VHWs or by RCH trekking teams.Study participants were recruited from villages and hamlets on the south bank of Upper River Region (URR), The Gambia. The study was conducted between September 2006 and June 2007. In this area, malaria transmission is highly seasonal, occurring mostly during and immediately after the rainy season (July–November) The study was designed as an open-label, cluster randomised trial with the unit of randomisation being the catchment population of RCH trekking teams. There were 33 trekking clinics in the study area on the south bank of URR of which 27 were in rural areas and visited by trekking teams from health centres at Gambisara, Basse, or Fatoto. 26 rural trekking clinics were selected for inclusion in the study . The catchment populations of these clinics were grouped into three strata on the basis of the number of children under 5 y of age in each catchment area, and the average distance from the nearest health centre. It was intended that only children under five would be enrolled. However at the time of enrolment about 16% of the children enrolled were more than 5 years. Hence the trial and the analysis were expanded to include children up to and including the age of 6.Within each stratum, clinic catchment areas were randomised, using permuted blocks using the random number generator in Stata version 10 (Stata Corporation), to receive IPTc delivered either by a VHW or through the RCH trekking team. The randomisation was well balanced with respect to distance from the nearest health centre, population of children up to 6 y of age, proximity to a main road, and quality of the road, factors that might influence access to the intervention and to malaria diagnosis and treatment. For all the villages in the study area, the nearest health centre could be reached in less than 4 h by local transport or was within a few hours walking distance.Following discussions with the National Malaria Control Programme (NMCP) and the URR district level health authorities, members of the study team, accompanied by the district health team, visited all villages in the study area, explained the purpose and methods of the study, and answered questions during meetings open to all villagers. During these meetings, consent was obtained from the elders of all villages in the study area to participate in the trial. Before the start of the study, field workers and investigators carried out a census in each of the 172 villages in the study area that agreed to participate in the trial. A list of all children living in these villages who would be aged between 6 and 72 mo at the time of the first treatment in September 2006 was obtained from this database to ensure that all the children in the right age group were considered for inclusion in the study. Families of prospective study participants were visited at home, where more information was provided about the trial and individual, written informed consent for a child to participate in the trial was obtained from parents or guardians. Exclusion criteria included known allergy to any antimalarial drug or the presence of acute or chronic, clinically significant pulmonary, cardiovascular, hepatic, or renal disease.A reporter employed by the project was based in each village and asked to keep records of all deaths, birth, immigrations, and emigrations that occurred during the study period. Village reporters were visited fortnightly by project field workers and their records collected and checked. Deaths that occurred at home were investigated with a standardised verbal-autopsy questionnaire and cause of death established whenever possible Drugs were delivered by either members of RCH trekking teams or by VHWs. Study participants were observed for approximately 30 min after dosing and, if vomiting occurred within this period, it was recommended that the child take another dose. RCH trekking teams visited each of the study villages approximately once a month to administer routine EPI vaccines and vitamin A. During the course of the intervention each trekking team in the RCH arm of the trial comprised an average of ten staff members, four of whom were provided by the study team to assist with the intervention. Trekking teams comprised staff with varying levels of training ranging from state enrolled nurses to volunteers. Each member of the team employed by the Ministry of Health received a salary supplement of US$28 per month (US$ 2006)  during the 3 mo of the intervention as an incentive for the extra hours of work that administration of IPTc incurred. Trekking team drivers received a supplement of US$14 per month.All villages in The Gambia with a population of approximately 400 or more have provision for a VHW who receives limited training from the Ministry of Health on the recognition and treatment of common illnesses including malaria. VHWs are meant to be supported by their community and do not receive any payment from the Ministry of Health. VHWs retain a small stock of essential drugs, including antimalarials, but as these must be purchased from a government pharmacy they frequently run out of stock. Thus, for the period of the study, all VHWs in the study were supplied with antimalarials by the project for the period of the study. VHWs received US$11 per month (US$ 2006) during the period of drug administration and a driver with a supplement of US$14 per month to deliver drugs to the VHWs. VHW supervisors did not receive any additional payment. Before the start of the study, VHWs were trained for 5 d on how to distribute monthly IPTc using drug treatment charts, recognise signs and symptoms of malaria and their relation to severity of illness, and maintain a compliance ledger. Study participants from villages within the IPTc arm of the study but without a resident VHW received IPTc from the nearest village with a VHW.A system for recording drug administration that could be used by a VHW was devised, which consisted of a treatment card held by the mother or guardian of each child and an IPT register held by the VHW or RCH trekking team. The treatment card and the IPT register were labelled with the child's, mother's, and village's names and compound, and with demographic surveillance and study numbers. A set of three sticky labels were preprinted with the child's identifiers and placed in an envelope that was stapled onto the treatment card. The dosage of trial medication for each study participant was indicated on a largely pictorial treatment card, employed because of the low levels of literacy of some VHWs, using coloured circles and semicircles . To ensure that a child did not receive more doses of IPTc than they should, blue-coloured treatment cards were issued to children in villages where IPTc was to be delivered by VHWs and pink-coloured cards to those allocated to receive medication from the RCH trekking clinic. When a child presented to the VHW or RCH trekking clinic for their monthly treatment, one sticky label was removed from the envelope and attached to the IPTc register, the child's identity checked and trial medication SP (tablets containing 500 mg sulfadoxine/25 mg pyrimethamine) (Microlabs) and AQ (200 mg base tablets) (Microlabs) were given. A single dose of SP was given; children aged 3–11 mo received one half tablet and a whole tablet was given if a child was aged 1–6 y. AQ was given daily for 3 d: one quarter of a tablet if a child was aged 3–11 mo, one half of a tablet if aged 1–2 y, and a whole tablet if aged 3–6 y. SP and the first dose of AQ were given under direct observation by the VHW or member of the trekking team. The mother or carer was given tablets for the second and third dose of AQ in a small plastic bag and was asked to administer these on the following 2 d at home. Children were given their tablets crushed, suspended in water, and administered with a spoon. The solubility and drug content of the SP and AQ tablets was confirmed by analysis with high performance liquid chromatography undertaken at the London School of Hygiene & Tropical Medicine.To determine the prevalence of adverse events following drug administration, 15 children in each cluster selected from the participant database by simple random sampling were visited at home within a week of the start of each round of drug administration by a trained field worker who completed an adverse event questionnaire. Parents and guardians of study children were encouraged to take their child to the study nurse based in the village or to the Medical Research Council (MRC) clinic at Basse if the child became unwell between visits. Any adverse reaction was graded by trained field workers as mild (grade 1) if it was easily tolerated, moderate (grade 2) if it interfered with normal activity, and severe (grade 3) if it prevented normal activity and required treatment. In addition, records were kept of all deaths and hospital admissions among study children during the period of the trial. A serious adverse experience was defined as any event that was fatal, life threatening, disabling or incapacitating or resulted in hospitalisation, prolonged a hospital stay, or was associated with overdose . A decision was made by the study physician as to whether a serious adverse event was likely to be drug related.Plasmodium falciparum histidine-rich protein 2 antigen, which is sensitive and specific for detection of P. falciparum in whole blood samples, was used to diagnose malaria if fever (axillary temperature ≥37.5°C) or a history of fever within the previous 48 h was present. In such cases, a thick blood smear was also collected for subsequent confirmation of the diagnosis and estimation of parasite density. Study participants with documented fever or history of recent fever and malaria parasitaemia were treated with Coartem. Treatment of study participants seen at the health centres for other conditions was carried out in accordance with national guidelines.Passive surveillance for malaria was maintained throughout the malaria transmission season (September to December). In both arms of the trial, study children had 24-h access to medical treatment provided by VHWs based in key villages or by study nurses based in the health centres within the study area. In both arms of the study, VHWs were asked to refer study participants who presented to them to the nearest health facility for evaluation. Each time a study patient presented to a health facility, axillary temperature was recorded using a digital thermometer and haemoglobin (Hb) concentration was measured using a portable haemoglobinometer (HemoCue AB). The Core Malaria Pf test (CORE Diagnostics), based on detection of circulating A cross-sectional survey was undertaken in all 26 clusters at the end of the malaria transmission season. Children in each cluster were selected for review by simple random sampling from the list of study participants. The families of children selected for review were visited at home, and follow-up visits made to find children who were not present at the first visit. The first 40 children to be located in each cluster were included in the survey. Children included in the survey were examined, their axillary temperature measured, and their height and weight recorded. A finger-prick blood sample was taken for preparation of thick smears and measurement of Hb concentration. The dates of IPT treatments received were recorded from the child's identification (ID) card, and the mother or career was asked about reasons for missed treatments, about the acceptability of the most recent round of treatment, and about compliance with the unsupervised doses. The families of the same children were visited 2–3 wk later and asked questions about household assets, level of education and sources of income of the child's primary care giver, the care giver's perception of the relative wealth of the household, bednet use by the study child, and asked to inspect the place where the child usually slept and to inspect the condition of their net if a net was being used.In order to estimate efficacy of IPTc, a nested case-control approach was used. Cases were children who presented to one of the study health centres with an axillary temperature ≥37.5°C or a history of fever in the previous 48 h and who had malaria parasitaemia with a parasite density ≥5,000/µl on initial reading. Controls  were selected from among children who presented to the same health centre with a febrile illness in the same week that the case was detected, who had a negative blood film, and whose village of residence was in the same delivery arm (VHW or RCH) as the case child. Cases and controls were visited at home to inspect the place where the child slept, to ask the child's mother or carer about bednet use at the time that the case child became sick, and about household assets. Dates of IPT treatment courses were obtained from the child's ID card and cross-checked against study registers.10 densities recorded was more than 1.5, slides were read by a third technician. Agreement was reached among the three technicians after the slides had been rechecked. Discrepancies occurred mainly in smears with very low parasite densities. Hb concentration was measured during morbidity surveillance and at the survey at the end of the malaria transmission season using a portable haemoglobinometer (Hemocue AB).Thick smears were stained with Giemsa stain and 200 high power fields (HPFs) were examined before a smear was declared negative. Parasite density was expressed per microlitre with the assumption that one parasite per HPF equals a density of 500 parasites per microlitre n = 390). Fieldworkers were asked to stagger their interviews over the course of the day to ensure that care givers accessing IPTc at various times could be included.A standard ingredients approach was used to cost the value of each unit of input needed to deliver IPTc and to treat inpatient or outpatient malaria episodes To reflect potential health system savings associated with fewer malaria episodes, economic costs of treating malaria were based on detailed retrospective cost data from health facilities across primary, secondary, and tertiary level health facilities in the study area between 2006 and 2007. Resource use associated with personnel, materials and supplies, equipment, transport, utilities, and buildings were recorded. Costs were identified using information found in patient folders, facility stock records, activity data collected by the district medical team, discussions with health facility personnel , and components of the study budget. Potential savings to households (direct and indirect), such as reduced expenditure on transport and other out-of-pocket expenses and potential savings in time lost from seeking treatment were collected from the families of all study children who were admitted to a hospital or health centre and from the families of a subsample of 100 children treated as outpatients. Costs are reported in 2008 US$.In order to calculate the incremental cost-effectiveness ratios, the costs of delivering IPTc via RCH trekking teams were compared with the costs of delivery by VHWs and this difference in costs was then compared to the following additional effects: (i) number of malaria episodes averted; (ii) number of children receiving the first dose of all three treatments; and (iii) number of children who received the first dose of at least one treatment. For ethical reasons, no placebo group was included in the current study so it was not possible to compare cost-effectiveness of these two strategies to current practice. Net cost effectiveness was calculated for each delivery system by subtracting resources saved from the total programme compared to the relevant outcome measure.Data from participants were recorded on forms and were checked by field supervisors, the study physician, and the data manager for consistency and accuracy. All data were entered into an SQL database using MS Access front-end software. The accuracy of data input was checked and validated using customized validation programmes. In addition, source data verification was done by the data manager. The cleaned data were converted to Stata version 10 file (Stata Corporation) prior to analysis.The primary study endpoint was the incidence of malaria  observed during the study period. Secondary endpoints were the incidence of a febrile illness with parasitaemia at any density, coverage of three IPTc treatment courses, the proportion of children who received no IPTc, the incidence of adverse events reported after IPTc administration, the mean Hb concentration, and the prevalence of asexual parasitaemia in the cross-sectional survey undertaken at the end of the malaria transmission season. The study was designed to determine whether delivery of IPTc through RCH trekking clinics would be at least as effective in preventing malaria as delivery by VHWs, and the sample size was calculated on this basis. On the basis of previous studies carried out in The Gambia, we assumed that 75% coverage with three IPT treatment courses would be achieved in both arms, that the efficacy of IPT would be 90%, and that the incidence rate of malaria by passive case detection would be 0.2 per child per transmission season without IPT. The predicted cumulative incidence of malaria would therefore be 6.5%, and we assumed that malaria incidence in each cluster would range from 0% to 20%. With 26 clusters and a cluster size of 500 children per cluster, the study was designed to have 90% power to show  that the difference in cumulative incidence between the delivery methods was not more than 3.5%. Similar calculations were done for coverage of IPTc, using a noninferiority margin of 10% to 15%, and for mean Hb at the end of the transmission season, using a margin of 0.5 g/dl. An analysis plan was agreed before the data were analysed.The primary analysis included all children in the correct age range who were present in the study area at the initial census and were issued with a malaria card, regardless of the number of treatments received.Characteristics of all children enrolled in the study were tabulated by study arm, overall estimates (not adjusting for clustering) are presented. For the primary outcome of malaria incidence during the study period, analysis was by intention to treat. Time at risk was calculated from September 11 to December 8 (the surveillance period) or until date of death or emigration. All episodes of malaria were included in the calculations; only two children had two episodes of malaria. The incidence of malaria in each study arm was calculated as the total number of cases divided by the total time at risk. The point estimate of the rate difference between the two arms was the difference between these rates and a 95% CI adjusting for clustering, and strata was calculated from the variances of the stratified ratio estimates For the analysis of the efficacy of IPTc against clinical malaria using the case-control approach, since some cases had parasitaemias <5,000/µl on definitive reading, two case definitions of malaria were used: a febrile illness accompanied by malaria parasitaemia of any density or by malaria parasitaemia at a density ≥5,000 per µl. The efficacy of an IPTc course given within the previous 28 d in preventing malaria was estimated as 100× (1−1/OR) in which OR is the odds ratio estimated by conditional logistic regression relating case/control status to IPTc. Bednet usage , age, ethnicity, self-reported wealth, and distance to the nearest health centre were considered as potential confounders. In a secondary analysis, interaction with bednet usage was assessed using a likelihood ratio test, to determine whether there was any difference in the benefit of IPTc in children using bednets compared to children not using nets. Interactions with delivery method and socioeconomic variables were also assessed.Coverage with IPTc was evaluated in the cross-sectional sample survey conducted at the end of the study. The proportions of children who had received each monthly treatment course, those who received three treatment courses, and those who received no treatment were tabulated. Differences in coverage estimates between trial arms were estimated by calculating the arithmetic mean of coverage proportions in each cluster, and conducting a two-way analysis of variance, allowing for stratification.To determine whether one delivery method was better at reaching particular subgroups of the population, the coverage indicators were tabulated, stratified by wealth, and by age of each child. Wealth quintiles were calculated as quintiles of the scores on the first component from principal components analysis of household asset ratings (based on household ownership of ten items), as recorded in the cross-sectional survey.Numbers of adverse events were tabulated by study arm, and risk differences with 95% CIs were calculated adjusting for clustering and strata using analysis of variance on the arithmetic cluster means, as described above. Outcomes collected at the December cross-sectional survey were also analysed using the analysis of variance method described above.The study was approved by the London School of Hygiene & Tropical Medicine ethics committee and by the joint MRC/Gambia Government ethics committee. The conduct of the trial was guided by a Data Safety and Monitoring Board. The trial was registered on the NCHS clinical trials database (number NCT00376155).12,326 children aged 6 mo to 6 y were enrolled into the study, of whom 6,076 were in the RCH arm and 6,250 in the VHW arm . BaselinOn the basis of data collected during the cross-sectional survey undertaken at the end of the surveillance period, 92% and 86% of participants issued with a malaria card at time of the census reported for enrolment and received the first IPTc treatment in the VHW and RCH arms, respectively. More children in the VHW arm received three treatment courses compared to the RCH arm . Coverage was higher in the VHW arm than in the RCH arm for each monthly treatment round . In the 18 study children died during the surveillance period, nine in each study arm. In the VHW group, five children died of severe pneumonia, two children died of acute gastroenteritis, one child died of cerebral malaria, and one from the complications of severe malnutrition. In the RCH arm, three children died of severe pneumonia, two died of malnutrition, two of acute gastroenteritis, and two of severe malaria. 68 study children were admitted to one of the health facilities in the study area during the follow-up period; 30 and 36 in the RCH and VHW groups, respectively. The main causes of admissions were malaria and pneumonia. 24 children were diagnosed with severe malaria, 14 in the RCH group and 10 in the VHW group. 30 children were diagnosed with pneumonia, 13 and 17 in the RCH and VHW groups, respectively. None of the deaths or hospital admissions was considered to be related to drug administration. There were no deaths or hospital admissions associated with severe skin lesions and no deaths suggestive of a blood dyscrasia.Field workers visited 838 study participants within the first week after the start of IPT treatment to document the incidence of adverse reactions. The treatment regimen was generally well tolerated. The most frequently solicited adverse events reported after treatment were fever, vomiting, diarrhoea, and abdominal pain, which were reported by 24%, 12%, 13%, and 5% of children, respectively. Seven children reported a rash but in no child was this severe.VHWs in the study area presumptively treated 288 study participants for malaria ; these were mainly those whose parents/guardians were unable or were unwilling to travel to health centre. There were 49 cases of malaria with parasitaemia >5,000/µl in the communities where IPTc was delivered through RCH clinics and 21 cases in the communities where IPT was delivered by VHWs, , and 144 cases with any parasitaemia in the RCH areas compared to 81 in the VHW areas (rate difference 3.7 [95% C1 −0.7 to 8.1) .1,096 children were seen during the December 2006 survey at the end of the malaria transmission season . Very fe81 cases of malaria and 89 controls were recruited into the case-control study (73 cases had one control and eight cases had two controls). 56 case-control sets came from villages in the RCH study area and 25 from villages in the VHW study area. 18 cases had parasitaemia <5,000/µl on definitive reading of a blood film. 20 (32%) of the 63 cases with parasitaemia ≥5,000/µl had received an IPT course in the 28 d prior to the date the case was detected, compared to 64% (45/70) among controls. All 65 cases and controls who received an IPT course in the 28 d prior to case detection reported full compliance with the additional home-administered AQ doses. The crude estimate of protective efficacy was 82%, and after adjustment for potential confounding effects of bednet use, age, ethnicity, self-reported wealth, and distance to nearest health centre, the efficacy of any IPTc treatment received within the previous 28 d against a febrile illness accompanied by malaria parasitaemia ≥5,000/µl was 87% (95% CI 54%–96%). When cases with parasitaemia <5,000/µl were included, adjusted efficacy was 80% (95% CI 42%–93%). There was no evidence of an interaction between the efficacy of IPTc and bednet use.In terms of cost-effectiveness, this incremental analysis shows that the VHW strategy is both more effective and less costly that the RCH approach. Comparing VHWs against the RCH trekking team there are three positive incremental effects: a reduction in the number of malaria episodes averted by 63, an improvement in the number of fully adherent children by 1,429, and an improvement in the number of children who received at least one treatment by 251. The VHW strategy is also less costly in both economic and financial terms than the RCH approach, resulting in incremental saving of US$872 and US$1,244, respectively, on the basis of intervention costs alone. When we include both intervention costs (the costs of delivering IPTc) and health system savings  savings increase to US$1,070 and US$1,443 in economic and financial terms, respectively. Finally, when household savings due to the reduction in malaria treatment costs are also added, by selecting the VHW approach over the RCH approach, the incremental savings increase further to US$1,148 and US$1,521 in economic and financial terms, respectively.The efficacy results of this trial are consistent with findings from previous studies of IPTc carried out in other West African countries, which have shown significant levels of protection against clinical attacks of malaria during the malaria transmission season In The Gambia delivery of IPTc by VHWs proved to be more effective than delivery by RCH trekking teams as judged by the level of coverage achieved. This finding is in agreement with the findings of a small-scale study that investigated different options for the delivery of IPTc in Ghana. This study also found that the proportion of children who received at least the first dose of three or more courses of IPTc was slightly higher in the community-based arm compared to delivery by health workers at health centres or at EPI outreach clinics (facility based) Delivering IPTc through community-based VHWs has several advantages. VHWs are resident in the community, making drug administration easy, and they can remind mothers/guardians if they do not come for treatment. Children were able to receive their medication on any day of the month. In contrast, children in the RCH arm were able to receive their treatment only once a month during the monthly visit of the trekking team, which may explain the lower coverage observed in the RCH arm of the study in October and November. Thus, operationally, VHW delivery is less restrictive and more convenient for parents and guardians. A previous study carried out in The Gambia showed that that VHWs were able to achieve a high level of coverage of antimalarial chemoprophylaxis among children and that in about 50% of the study villages, an acceptable level of compliance was sustained for a period of 5 y, despite minimal outside supervision and support for the programme A potential weakness of the trial was that it was not a placebo controlled. It was not considered appropriate to include a placebo group in this study because of the clear evidence from previous studies in neighbouring Senegal and elsewhere that IPTc is beneficial. Efficacy of the intervention was estimated using a nested case-control study and, although this is not as rigorous as a randomised trial, the estimates of efficacy were similar to those obtained in previous trials. A further possible limitation is that, although care was taken to ensure balanced randomisation, we did not have prior data on malaria incidence in the study villages, and an imbalance cannot be ruled out. However, the difference in incidence of malaria between the study arms is consistent with the difference in coverage of IPT and the observed efficacy of IPT doses, and so these may not have been important sources of bias. It has to be noted, however, that the CI for the difference in malaria incidence is wide . A further potential source of bias in this study is that VHWs were involved in IPTc delivery and this may have influenced their treatment or referral of suspected malaria patients.No serious adverse event attributable to study medication was observed following administration of about 30,000 courses of SP and AQ, adding to the increasing body of evidence that this drug combination is safe when used for IPTc in African children. No serious skin lesions were observed and there were no clinical cases suggestive of a blood dyscrasia, although, as white cell counts were not done routinely, the possibility that a blood dyscrasia contributed to one of the fatal infections that occurred among study children cannot be completely excluded. The overall mortality rate among the children in the study was approximately 1.5 per 1,000 during the 4-mo period of observation, which included, the period when most childhood deaths occur in The Gambia, a figure well below that expected on the basis of previous studies conducted in URR We selected SP plus AQ as the combination to be used for IPTc in this study because a trial undertaken in Senegal, which compared four different treatment regimens, showed that the best results were obtained with this regimen At between US$3.47 (using trekking teams) and US$1.63 (using VHWs), the annual economic cost of delivering at least the first dose of each treatment of IPTc under trial conditions are comparable to the range of the costs associated with delivering other malaria interventions. For example, the costs per year of protection in US$ 2008 are reported to be US$1.46–US$4.00 for ITNs This study has shown the influence of scale on delivery of IPTc. A smaller study of IPTc delivery gave unit costs of village-based delivery more than three times higher than those presented here There are many potential ways in which VHWs can contribute to the health of the communities in which they are based. These ways include treatment of common infections, recognition and referral of seriously ill patients, provision of advice on hygiene and nutrition, and encouraging women to attend antenatal clinics and to immunize their children. Community volunteers are also being used increasingly as a route of delivery for mass treatment campaigns against infections such as onchcocerciasis, intestinal helminth infections, shistosomiasis, filariasis, and trachoma. In the case of malaria, VHWs can provide treatment for clinical attacks of malaria  (HMM) either presumptively or following the use of an RDT. They can encourage the use of ITNs and, as we have shown in this study, effectively administer IPTc. In countries, such as The Gambia, where malaria transmission is seasonal, this activity in required for only a few months of the year. However, although the incidence of malaria in The Gambia, and countries with a similar epidemiology, is concentrated within just a few months of the year, occasional cases do occur at other times of the year and require treatment. Thus, a logical approach is to ask VHWs to combine the role of HMM throughout the year with the administration of IPTc during the high transmission season. This approach has recently been explored in The Gambia Sustainability is a major concern for all interventions, especially those that depend upon volunteers. Given the renewed focus on VHWs and their ever-expanding repertoire of services Whether volunteers should be offered incentives to encourage sustainability is debated. Some mass drug delivery systems have been successful without incentives whilst others have employed financial incentives of some kind. In The Gambia, the tradition has been that VHWs should be supported by their community, an approach that has not been particularly successful over the years in many villages, leading to some VHWs becoming disillusioned due to lack of financial and nonfinancial support. In Gambia VHW can be paid US$7.50 per day for participation in mass drug administration, polio, or vitamin A campaigns. In this study, VHWs received an incentive of $11 per month. It is highly likely this financial payment and the strengthened drug supply contributed to the trials success. However, even with a small financial payment the cost effectiveness of IPTc is still comparable to that of other malaria interventions. Any incentives must reach the VHW in a timely and efficient manner to avoid demotivation. Longer term experience will show whether the high level of coverage obtained in this study can be sustained but the results of this trial suggest that community volunteers can achieve high level coverage with this very effective malaria control intervention if they are supported to do so.Text S1Study protocol.(0.18 MB PDF)Click here for additional data file.Text S2CONSORT checklist.(0.03 MB PDF)Click here for additional data file.


Plasmodium falciparum or Plasmodium vivax, up to 45%. Given the high rates of infection observed both in mother and placenta, a first exploratory study was proposed in order to characterize the effect on the inflammation status, tissue damage and hypoxia in Plasmodium spp. infected placentas.A large-scale study was set up in order to study the epidemiology, clinical aspects, and immunopathology of gestational and placental malaria in north-west Colombia. In this region, recent reports using a qPCR technique, confirmed frequencies of infection, by A descriptive, prospective, cross-sectional design was applied to pregnant women with (PM+) and without (PM-) placental malaria. Messenger RNA expression of Fas, FasL; COX-1, COX-2, HIF, VEGF, and the cytokines IL-2, IL-4, IL-10, IFN-γ and TNF, were measured in peripheral and placental blood using a quantitative PCR. The percentage of apoptotic cells was determined with a TUNEL assay.Plasmodium infection. Expression of IL-4 and IL-10 was observed high in placental tissue of PM+, while IL-2 was high in peripheral blood of the same group. Expression of TNF and IFNγ in peripheral blood of the PM + group was high. Similarly, the apoptotic index and Fas expression were significantly high in PM+. However, FasL expression was observed low in PM + compared to PM-. Inflammation markers  and hypoxia markers  were high in the PM + group.In total 50 placentas were studied: 25 were positive for submicroscopic infection and 25 were negative for During placental malaria expression of some pro-inflammatory cytokines is up-regulated and markers of hypoxia and tissue damage are increased in cases of submicroscopic infection. Plasmodium falciparum and Plasmodium vivax can cause adverse pregnancy outcomes, including maternal anaemia and low birth weight, albeit different mechanisms appear to be involved depending on the infecting species [P. falciparum, adherence of infected erythrocytes to chondroitin sulfate A (CSA), present on the trophoblast surface, is crucial in the pathophysiology of the disease [Globally, an estimated 125 million pregnancies are at risk of malaria each year [ species . In the  disease , since pWomen inhabiting areas with different degrees of endemicity experience variable exposure to infection and this affects the course of disease . In areaP. falciparum, specifically the molecular aspects of these infections, the associated damage to the placenta, and the consequences on the mother, the placenta and the foetus [P. falciparum placental malaria might be determined by changes in the regulation of apoptosis, via Fas /FasL [P. vivax infection during pregnancy, these phenomena remain to be studied.Recent extensive reviews have focused on the pathogenesis and immunity of gestational and placental malaria caused by e foetus . A signie foetus . Some stas /FasL , and incas /FasL ,11. In aas /FasL ,13, althas /FasL . As for On a routine basis, detection of parasites in placenta requires examination of histological sections of fixed placental tissue or microscopy of placental blood . HoweverP. falciparum is highly endemic. In Colombia, where P. vivax represents 60-70% of the total reported cases, malaria in pregnancy constitutes a public health problem [Most knowledge gathered about pregnancy-associated malaria resulted from studies in areas of Africa where  problem ,21 with  problem ,15. Duri problem ,22. To fWomen were recruited from January 2007 to April 2011, at the hospital obstetric facilities in the municipalities of Monteria , and Puerto Libertador  of the Uraba-Sinu-San Jorge-Bajo Cauca region of Colombia. This region has an estimated area of 43,506 km2, a population of 2.5 million at risk of malaria, with a mean annual parasite index of 35.8 cases/1,000 inhabitants . The regPlasmodium spp. infection. The status of infection was defined by thick smear and real-time PCR (qPCR) of placental blood. Positive infected units were those with a negative diagnosis by microscopy and a positive qPCR result; negative units were negative by both tests.The study was part of a larger project aimed at exploring the epidemiology, clinical aspects and immunopathology of gestational and placental malaria in north-west Colombia, from which partial results have been reported elsewhere ,24. A toInclusion criteria were voluntary acceptance to participate in the study, signature of the informed consent, permanent residency (>1 year) in the malaria endemic region, negative history of pre-eclampsia and negative HIV and TORCH tests. The exclusion criterion was withdrawal of consent.3 sections of placenta were removed from the maternal side of the organ and blood was collected by aspiration into a tube containing EDTA. From mothers, 3 ml of peripheral blood was obtained and collected into EDTA containing vials. Thick smears were prepared from placental and mother’s blood and blood spots (approximately 100 μl of blood) were placed onto Whatman 3 mm filter paper for molecular diagnosis. One tissue fragment was preserved in RNA later® before processing for expression of markers of apoptosis, hypoxia and inflammation, and the remaining tissue fragment was fixed with 10% buffered formalin (pH 7.0-7.4), before embedding in paraffin, for the TUNEL assay.After enrolment, a questionnaire was completed which recorded data including age, number of pregnancies, number of malaria episodes during the ongoing pregnancy  and antimalarial treatment administered. In addition, labour/delivery and infant outcomes data, as well as maternal haemoglobin value, were obtained from the delivery chart. Placentas were processed immediately after delivery and maternal peripheral blood was obtained within 24 hours. After cleaning with saline, two ~1 cmPlasmodium infection was evaluated in mother’s peripheral and placental blood by microscopy of Field-stained thick films and qPCR. An experienced microscopist based at the field laboratory determined the presence of infection by counting the number of parasites per 200 leukocytes, based on a mean count of 8,000 leukocytes per microliter of blood. Samples were negative when no parasites were detected in 200 high power (100x) fields. For the qPCR, DNA was extracted from a 6 mm2 fragment of placenta using the Chelex method described by Plowe et al.[Plasmodium using a genus-specific set of primers and probe. The reaction was performed in a final volume of 25 μl containing 5 μl of DNA, 12.5 μl of TaqMan universal master mix (Applied Biosystems), 200 nM of each primer (Plasmo1 and Plasmo2), 50 nM of Plasprobe on the ABI 7500 FAST platform, under universal cycling conditions as published. Samples with a cycle threshold (Ct) value under 45 were tested in a duplex species-specific real-time PCR reaction for P. falciparum and P. vivax[we et al.. A qPCR we et al.. Briefly P. vivax.Cytokines were measured in mRNA isolated from maternal whole blood and placental tissue, while markers of tissue damage, hypoxia and inflammation were measured in mRNA isolated from placental tissue. Relative quantitation for expression analysis was performed using a reverse-transcription real time PCR assay (RT-PCR). Total RNA was extracted using QIAamp RNA Blood Mini® (QIAGEN) and cDNA was synthesized using First Strand cDNA Synthesis® (Fermentas). The reaction was set up in a Roche LightCycler® or ABI 7500. Table Apoptosis in placentas was assessed by a TUNEL assay as described earlier . PlacentData were analysed using Excel and SPSS 19. Significance was set at p < 0.05. Non-parametric tests were used for data analysis and the U Mann–Whitney test and the Kruskal-Wallis test were used for correlations. According to the data distribution, the variable number of DNA copies/μl of template was grouped by convenience as follows: Group 1 (0 copies-no infection); Group 2 (0.01 to 1.99 copies/μl), Group 3 (2.00 to 100 copies/μl), and Group 4 (> 100 copies/μl) .Pregnant women or guardians (in case of <18 years of age) signed a voluntary consent form. The study involved a minor risk and approval was granted by the Comite de Etica of Instituto de Investigaciones Medicas, Facultad de Medicina, Universidad de Antioquia .Plasmodium spp. by microscopy, and 25 of them were positive for infection by qPCR: 16 P. falciparum, six P. vivax and three mixed infection. The characteristics of the women from which placentas were collected are summarized in Table Plasmodium species infecting the placenta.All fifty placentas were negative for Maternal peripheral blood and placental tissue were evaluated for the expression (mRNA) of IFN-γ, TNF, IL-2, IL-4 and IL-10. In both compartments, expression of IFN-γ, TNF, and IL-10 was significantly higher in PM + when compared to PM-. Meanwhile, expression of IL-4 was high in placentas in PM+, and IL-2 was high in peripheral blood of the same group Table . In periIn general, the differences observed in expression of cytokines in peripheral blood and placenta between PM + and PM-, persisted when analysis was performed taking into account the infecting species Table .vs. 3.42). However, FasL expression was higher in PM- than in PM +   ; Group 2 (0.01 to 1.99 copies/μl), Group 3 (2.00 to 100 copies/μl), and Group 4 (> 100 copies/μl) . In genePlasmodium spp in this endemic region. Such mechanisms are important to a healthy pregnancy since they contribute to implantation and promote placental development [Although the effects of malaria infection on the mother and the foetus depend upon multiple factors, the host immune response and the histopathology of the placenta are specifically associated with complications observed before birth and around the neonatal period . This stelopment .P. falciparum and P. vivax are endemic and is the first study to report on apoptosis and tissue injury during submicroscopic placental infection. All studied groups came from the same malaria endemic region as to control for any socio-epidemiology associated bias. According to a recent report [This is a first exploratory study on the subject in women from a region where both t report , most plvs. haematoxylin-eosin staining.Changes in apoptosis have been scarcely studied in placental malaria. In other pathologies such as preeclampsia, the high rate of apoptosis has been directly linked with intrauterine growth restriction . Importain vitro studies with cell models and placental tissue explants stimulated with pro-inflammatory cytokines, the expression of Fas and FasL was high and similar to the results reported here [Fas and FasL expression have been proposed as markers of apoptosis, and this is considered an important mechanism by which cytokines act locally and may influence critical signaling processes. In ted here . Taken tIn the current study, increased apoptosis in infected placentas was also associated with high expression of the pro-inflammatory cytokines IFN-γ and TNF. Similar observations with increased expression of Th1 cytokines such as IL-2, TNF, IFN-γ and decreased Th2 cytokines such as IL-6 and TGF-β have been reported by others , and haevs. uninfected placentas. The increased expression of this cytokine in cases of submicroscopic infection is a novel and promising finding.Other authors, based on high levels detected in serum of women with gestational malaria, have proposed IL-10 as biomarker of placental infection  and a dePlasmodium[Hypoxia has been associated with complications from placental malaria infection . Howeverlasmodium. Similarlasmodium.et al., increased expression of COX-2 and IL-10 in chronic placental malaria was detected, and COX-2 was associated with maternal anaemia, placental macrophage infiltration and haemozoin deposition [Cyclooxygenase (COX) and lipoxygenase (LOX) transform fatty acids into prostaglandins and leukotrienes, which play important roles in pregnancy and foetal development. In addition, COX-2 has been proposed as a marker of preeclampsia and recovery after infectious conditions . In a stposition . These rP. vivax infected placentas compared with those infected with P. falciparum. Some complications of P. vivax infection might be explained by the production of pro-inflammatory cytokines, which alter the balance at the foetal-maternal interface. Based on this, it is hypothesized that P. vivax is as pathogenic as P. falciparum, regardless of the presence of sequestering parasites.Interestingly, no differences were observed between the changes in the markers studied in P. falciparum infection. The current results demonstrate that P. vivax might also be equivalent in the pathophysiology of placental damage. These results need to be complemented with a study of a large series of P. vivax infected placentas. In addition, the role of naturally acquired specific humoral immunity should complement these studies in order to clarify its implication on clinical status. Finally, studies of the consequences of P. vivax placental infection in Asia and America in the context of a pregnancy vaccine and intermittent preventive therapy should be a priority.The knowledge gathered so far on the pathophysiology of placental malaria mainly describes cases of Plasmodium spp. infected placentas.In conclusion, submicroscopic placental infection by malaria parasites failed to induce major clinical effects on mother or foetus but associated with a pro-inflammatory cytokine profile, regardless of the infecting species, and this was more evident in the placental tissue. Finally, increased apoptosis was common in The authors affirm that they have no commercial or other association that might pose a conflict of interest.OA, EA BA designed and performed the analysis in blood samples. OA, JC-F and AM conceived the project and designed the experiments, supervised overall design and development and wrote the manuscript. SKY designed the experiments. All authors read and approved the final manuscript.