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A study was conducted to evaluate the efficacy of RTS, S/AS01E vaccine in preventing malaria in children aged between 5 to 17 months. Results indicated that the RTS, S/AS01E vaccine is quite effective in prevention of malaria.

Introduction Since 2000, artemisinin combination therapies (ACTs) have become widely adopted as first-line treatment policy for uncomplicated P. falciparum malaria in many endemic countries in response to parasite resistance that [...], Background Artemisinin derivatives used in recently introduced combination therapies (ACTs) for Plasmodium falciparum malaria significantly lower patient infectiousness and have the potential to reduce population-level transmission of the parasite. With the increased interest in malaria elimination, understanding the impact on transmission of ACT and other antimalarial drugs with different pharmacodynamics becomes a key issue. This study estimates the reduction in transmission that may be achieved by introducing different types of treatment for symptomatic P. falciparum malaria in endemic areas. Methods and Findings We developed a mathematical model to predict the potential impact on transmission outcomes of introducing ACT as first-line treatment for uncomplicated malaria in six areas of varying transmission intensity in Tanzania. We also estimated the impact that could be achieved by antimalarials with different efficacy, prophylactic time, and gametocytocidal effects. Rates of treatment, asymptomatic infection, and symptomatic infection in the six study areas were estimated using the model together with data from a cross-sectional survey of 5,667 individuals conducted prior to policy change from sulfadoxine-pyrimethamine to ACT. The effects of ACT and other drug types on gametocytaemia and infectiousness to mosquitoes were independently estimated from clinical trial data. Predicted percentage reductions in prevalence of infection and incidence of clinical episodes achieved by ACT were highest in the areas with low initial transmission. A 53% reduction in prevalence of infection was seen if 100% of current treatment was switched to ACT in the area where baseline slide-prevalence of parasitaemia was lowest (3.7%), compared to an 11% reduction in the highest-transmission setting (baseline slide prevalence = 57.1%). Estimated percentage reductions in incidence of clinical episodes were similar. The absolute size of the public health impact, however, was greater in the highest-transmission area, with 54 clinical episodes per 100 persons per year averted compared to five per 100 persons per year in the lowest-transmission area. High coverage was important. Reducing presumptive treatment through improved diagnosis substantially reduced the number of treatment courses required per clinical episode averted in the lower-transmission settings although there was some loss of overall impact on transmission. An efficacious antimalarial regimen with no specific gametocytocidal properties but a long prophylactic time was estimated to be more effective at reducing transmission than a short-acting ACT in the highest-transmission setting. Conclusions Our results suggest that ACTs have the potential for transmission reductions approaching those achieved by insecticide-treated nets in lower-transmission settings. ACT partner drugs and nonartemisinin regimens with longer prophylactic times could result in a larger impact in higher-transmission settings, although their long term benefit must be evaluated in relation to the risk of development of parasite resistance. doi:10.1371/journal.pmed.0050226

Abstract Background Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. Methods Three gametocyte dilutions: 10/μL, 1.0/μL and 0.1/μL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR). Results Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p Conclusions This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

Abstract Background Antibody responses to malaria antigens reflect exposure to parasites, and seroprevalence correlates with malaria transmission intensity. Antibodies are routinely measured in sera or on dried blood spots but a non-invasive method would provide extra utility in sampling general populations. Saliva is already in use in the detection of plasma-derived IgM and IgG to viral infections. In this study, antibodies to Plasmodium falciparum merozoite antigens were compared between blood and saliva samples from the same individuals in unlinked surveys conducted in Tanzania and The Gambia. Methods In Tanzania, 53 individuals provided paired fingerprick blood and saliva sample using two commercially available sampling devices. In the Gambia, archived plasma and saliva samples collected from 200 children in the Farafenni area in a cross-sectional survey were analyzed. IgG antibodies against P. falciparum antigens, Merozoite Surface Protein-1 (MSP-119) and Apical membrane Antigen (AMA-1) were measured by ELISA in paired saliva and blood samples from both sites. Antibody levels were compared as continuous optical density (OD) values and by sero-positivity. Results Significant correlations between saliva and plasma antibody levels were seen in Tanzania for both antigens, AMA-1(r2 range 0.93 to 0.89, p < 0.001) and MSP-119 (r2 range 0.93 to 0.75, p < 0.001), with a weaker correlation for results from The Gambia (r2range 0.64 to 0.63, p < 0.01). When assessed as seropositivity and compared with plasma, sensitivity and specificity were good with saliva antibody levels to both AMA-1 and MSP-119 (sensitivity range 64-77% and specificity range 91-100% & 47-67% and 90-97% respectively) over the different sample sets. Conclusions These data demonstrate anti-malarial antibodies can be detected in saliva and correlate strongly with levels in plasma. This non-invasive relatively simple collection method will be potentially useful for general population surveys, and particularly in migratory populations or those with infrequent contact with health services or opposed to blood withdrawal. Further studies will be needed to optimize collection methods, standardize volumes and content and develop controls.

Abstract Background Phase III trials of the malaria vaccine, RTS, S, are now underway across multiple sites of varying transmission intensity in Africa. Heterogeneity in exposure, vaccine response and waning of efficacy may bias estimates of vaccine efficacy. Methods Theoretical arguments are used to identify the expected effects of a) heterogeneity in exposure to infectious bites; b) heterogeneity in individual's response to the vaccine; and c) waning efficacy on measures of vaccine efficacy from clinical trials for an infection-blocking vaccine. Results Heterogeneity in exposure and vaccine response leads to a smaller proportion of trial participants becoming infected than one would expect in a homogeneous setting. This causes estimates of vaccine efficacy from clinical trials to be underestimated if transmission heterogeneity is ignored, and overestimated if heterogeneity in vaccine response is ignored. Waning of vaccine efficacy can bias estimates of vaccine efficacy in both directions. Conclusions Failure to account for heterogeneities in exposure and response, and waning of efficacy in clinical trials can lead to biased estimates of malaria vaccine efficacy. Appropriate methods to reduce these biases need to be used to ensure accurate interpretation and comparability between trial sites of results from the upcoming Phase III clinical trials of RTS, S.

Abstract Background Sero-epidemiological methods are being developed as a tool for rapid assessment of malaria transmission intensity. Simple blood collection methods for use in field settings will make this more feasible. This paper describes validation of such a method, by analysing immunoglobulins from blood retained within immunophoretic rapid diagnostic tests (RDTs) for Plasmodium falciparum. RDTs are now widely used for the diagnosis of malaria and estimation of parasite rates, and this method represents a further use for these devices in malaria control. Methods Immunoglobulins eluted from RDTs, designed to detect parasite histidine rich protein-2 (HRP-2), were analysed by indirect ELISA for IgG recognizing the P. falciparum blood stage antigens merozoite surface protein-119 (MSP-119) and apical membrane antigen-1 (AMA-1). Optimal storage conditions for RDTs were evaluated by comparing antibody responses from RDTs stored in dry or humid conditions at 4°C or at ambient temperature (with or without air-conditioning) for 7, 31 or 70 days. Antibody levels estimated using 3,700 RDT samples from attendees at health facilities in North-eastern Tanzania were compared with contemporaneously collected filter paper blood spots (FPBS) and used to estimate seroconversion rates. Results Storage of RDTs at 4°C was optimal for immunoglobulin recovery but short-term storage at ambient temperatures did not substantially affect anti-malarial IgG levels. Results from RDTs were comparable with those from FPBSs, for both antigens. RDT-generated titres tended to be slightly higher than those generated from FPBSs, possibly due to greater recovery of immunoglobulins from RDTs compared to filter paper. Importantly, however, RDT-based seroconversion rates, and hence serological estimates of malaria transmission intensity, agreed closely with those from FPBSs. Conclusion RDTs represent a practical option for collecting blood for sero-epidemiological surveys, with potential cost and logistical advantages over filter paper and other blood collection methods. RDT-based seroepidemiology can be incorporated into routine monitoring of malaria endemicity, providing information to supplement parasite prevalence rates and generating rapid, robust assessment of malaria transmission intensity at minimal extra cost.

Abstract Background The aim of this study was to identify and compare factors associated with Plasmodium falciparum gametocyte carriage in three regions of differing malaria endemicity. Methods Retrospective data from Thailand, The Gambia and Tanzania were used. The data came from large prospective field-based clinical trials, which investigated gametocyte carriage after different anti-malarial drug treatments. Results Gametocytaemia was detected during the observation period in 12% of patients (931 out of 7548) in Thailand, 34% (683 out of 2020) in The Gambia, and 31% (430 out of 1400) in Tanzania (p < 0.001). Approximately one third (33%, 680/2044) of the patients with gametocytaemia during the observation period, already had patent gametocytaemia at enrolment (day 0 or day 1): 35% (318/931) in Thailand, 37% (250/683) in The Gambia, 26% (112/430) in Tanzania. Maximum gametocytaemia was usually observed on or before the seventh day after starting treatment (93% in Thailand, 70% in Tanzania and 78% in The Gambia). Lowest gametocyte carriage rates were observed following treatment with artemisinin derivatives, while sulphadoxine-pyrimethamine (SP) was associated with significantly greater development of gametocytaemia than other drug treatments (p < 0.001). The duration of gametocyte carriage was shorter in Thailand by 86% and Tanzania by 65% than in The Gambia. Gametocyte carriage was 27% longer among people presenting with anaemia, and was shorter in duration among patients who received artemisinin derivatives, by 27% in Thailand and by 71% in Tanzania and The Gambia. Conclusion This study confirms the independent association of gametocytaemia with anaemia, and the significantly lower prevalence and duration of gametocyte carriage following treatment with an artemisinin derivative. The large differences in gametocyte carriage rates between regions with different levels of malaria transmission suggest that drug interventions to prevent transmission will have different effects in different places.

Abstract Background Malaria in The Gambia is highly seasonal, with transmission occurring as Anopheles gambiae s.l. populations expand during and immediately after a single annual rainy season that lasts from June to October. There has been very limited investigation of the ecology of vectors during the dry season, when numbers are very limited and distributions may be restricted. Methods Weekly adult mosquito collections (pyrethrum spray, light trap, and search collections from rooms, as well as light trap collections from animal shelters, abandoned wells and grain stores), and artificial sentinel breeding site surveys were performed in four villages near the upper tidal and partially saline part of the Gambia River in the last four months of an annual dry season (March to June). Mosquito species were identified by morphological and DNA analysis, and ELISA assays were performed to test for Plasmodium falciparum sporozoites and human blood meal components. Results Adults of An. gambiae s.l. were collected throughout the period, numbers increasing towards the end of the dry season when humidity was increasing. Adult collections were dominated by An. melas (86%), with An. gambiae s.s. (10%) and An. arabiensis (3%) also present throughout. Most females collected in room search and spray collections contained blood meals, but most from light traps were unfed. None of the females tested (n = 1709) contained sporozoites. Larvae (mostly An. gambiae s.s.) were recovered from artificial sentinel breeding sites in the two villages that had freshwater pools. These two villages had the highest proportions of An. gambiae s.s. adults, and experienced the most substantial increase in proportions of An. gambiae s.s. after the onset of rains. Conclusion During the dry season population minimum, An. melas was the predominant vector species, but differences among villages in availability of fresh-water breeding sites correlate with egg laying activity and relative numbers of An. gambiae s.s. adults, and with the increase in this species immediately after the beginning of the rains. Local variation in dry season vector persistence is thus likely to influence spatial heterogeneity of transmission intensity in the early part of the rainy season.

Abstract Background Artemisinin combination therapies (ACT), which are increasingly being introduced for treatment of Plasmodium falciparum malaria, are more effective against sexual stage parasites (gametocytes) than previous first-line antimalarials and therefore have the potential to reduce parasite transmission. The size of this effect is estimated in symptomatic P. falciparum infections. Methods Data on 3,174 patients were pooled from six antimalarial trials conducted in The Gambia and Kenya. Multivariable regression was used to investigate the role of ACT versus non-artemisinin antimalarial treatment, treatment failure, presence of pre-treatment gametocytes and submicroscopic gametocytaemia on transmission to mosquitoes and the area under the curve (AUC) of gametocyte density during the 28 days of follow up. Results ACT treatment was associated with a significant reduction in the probability of being gametocytaemic on the day of transmission experiments (OR 0.20 95% CI 0.16–0.26), transmission to mosquitoes by slide-positive gametocyte carriers (OR mosquito infection 0.49 95% CI 0.33–0.73) and AUC of gametocyte density (ratio of means 0.35 95% CI 0.31–0.41). Parasitological treatment failure did not account for the difference between ACT and non-artemisinin impact. The presence of slide-positive gametocytaemia prior to treatment significantly reduced ACT impact on gametocytaemia (p < 0.001). Taking account of submicroscopic gametocytaemia reduced estimates of ACT impact in a high transmission setting in Kenya, but not in a lower transmission setting in the Gambia. Conclusion Treatment with ACT significantly reduces infectiousness of individual patients with uncomplicated falciparum malaria compared to previous first line treatments. Rapid treatment of cases before gametocytaemia is well developed may enhance the impact of ACT on transmission.

Abstract Background Anaemic status is determined by haemoglobin using the HemoCue system or haematocrit measurements, and a threefold conversion is commonly used to equate the two measures (haemoglobin = haematocrit/3). The validity of this conversion in malaria endemic settings was assessed. Methods Concurrent measures of haemoglobin and centrifuged haematocrit in children aged 6–59 months were compared by modelling the difference between the two measures against their average. A random effects linear regression of the difference of the measures on their average was used to describe the line of best agreement and 95% limits of agreement for these two measures over a range of values after adjusting for statistically significant covariates. Results There was a consistent bias between the two measures, with haemoglobin less than haematocrit/3 in 87% (899/1,030) of observations. This difference was non-uniform, decreasing with the average measure, i.e. less difference at higher haemoglobin and haematocrit values. In these studies, use of haematocrit would have underestimated the prevalence of anaemia by misclassifying 10% (89/920) of individuals with haemoglobin < 11 g/dl, 66% (252/380) of individuals with haemoglobin < 8 g/dl and 100% (23/23) of individuals with haemoglobin < 5 g/dl. The mean difference between the measures was greater in males than females, increased with age between 6–59 months, and was greater in the wet than dry season suggesting that the relationship between haemoglobin and haematocrit may be modified by exposure to malaria. Conclusion The regression model indicated that the standard threefold conversion from haematocrit to haemoglobin underestimates the prevalence of haemoglobin < 11 g/dl in children under five years of age in malaria endemic settings. This bias was more acute for more severe anaemia defined by haemoglobin < 8 g/dl and haemoglobin < 5 g/dl. This has important implications for the comparability of studies using these different measures. Direct determination of haemoglobin should be the measurement of choice for assessing anaemia outcomes in malaria intervention trials and surveys.

Abstract Background African malaria vectors bite predominantly indoors at night so sleeping under an Insecticide-Treated Net (ITN) can greatly reduce malaria risk. Behavioural adaptation by mosquitoes to increasing ITN coverage could allow vector mosquitoes to bite outside of peak sleeping hours and undermine efficacy of this key malaria prevention measure. Methods High coverage with largely untreated nets has been achieved in the Kilombero Valley, southern Tanzania through social marketing programmes. Direct surveys of nightly biting activity by An. gambiae Giles were conducted in the area before (1997) and after (2004) implementation of ITN promotion. A novel analytical model was applied to estimate the effective protection provided by an ITN, based on published experimental hut trials combined with questionnaire surveys of human sleeping behaviour and recorded mosquito biting patterns. Results An. gambiae was predominantly endophagic and nocturnal in both surveys: Approximately 90% and 80% of exposure occurred indoors and during peak sleeping hours, respectively. ITNs consistently conferred >70% protection against exposure to malaria transmission for users relative to non-users. Conclusion As ITN coverage increases, behavioural adaptation by mosquitoes remains a future possibility. The approach described allows comparison of mosquito biting patterns and ITN efficacy at multiple study sites and times. Initial results indicate ITNs remain highly effective and should remain a top-priority intervention. Combined with recently developed transmission models, this approach allows rapid, informative and cost-effective preliminary comparison of diverse control strategies in terms of protection against exposure before more costly and intensive clinical trials.

Abstract Background Mutations in the haemoglobin beta-globin (HbB) and glucose-6-phosphate dehydrogenase (G6PD) genes cause widespread human genetic disorders such as sickle cell diseases and G6PD deficiency. In sub-Saharan Africa, a few predominant polymorphic variants of each gene account for a majority of these deficiencies. Examining at a larger scale the clinical importance of these independent genetic disorders, their possible association with malaria pathogenesis and innate resistance, and their relevance for antimalarial drug treatment, would be easier if an accurate screening method with limited costs was available. Methods A simple and rapid technique was developed to detect the most prominent single nucleotide polymorphisms (SNPs) in the HbB and G6PD genes. The method is able to detect the different haemoglobin polymorphisms A, S, C and E, as well as G6PD polymorphisms B, A and A- based on PCR-amplification followed by a hybridization step using sequence-specific oligonucleotide probes (SSOPs) specific for the SNP variants and quantified by ELISA. Results The SSOP-ELISA method was found to be specific, and compared well to the commonly used PCR-RFLP technique. Identical results were obtained in 98% (haemoglobin) and 95% (G6PD) of the tested 90 field samples from a high-transmission area in Tanzania, which were used to validate the new technique. Conclusion The simplicity and accuracy of the new methodology makes it suitable for application in settings where resources are limited. It would serve as a valuable tool for research purposes by monitoring genotype frequencies in relation to disease epidemiology.

Abstract Background The relationship between the burden of uncomplicated malaria and transmission intensity is unclear and a better understanding of this relationship is important for the implementation of intervention programmes. Methods A 6-month longitudinal study monitoring risk factors for anaemia and febrile malaria episodes was conducted among individuals aged below 20 years, residing in three villages of different altitude in areas of high, moderate and low malaria transmission intensity in North-Eastern Tanzania. Results The burden of anaemia and malarial fever fell mainly on the youngest children and was highest in the village with high transmission intensity. Although a considerable percentage of individuals in all villages carried intestinal worms, logistic regression models indicated that Plasmodium falciparum was the only significant parasitic determinant of anaemia. Interestingly, children who carried low-density parasitaemia at the start of the study had a lower risk of contracting a febrile malaria episode but a higher risk of anaemia during the study period, than children who were slide negative at this point in time. Conclusion Young children living in the high transmission village carried a very high anaemia burden, which could be attributed to malaria. The overall incidence of febrile malaria was also highest in the high transmission village particularly among those under five years of age. These data suggest that in rolling back malaria, available resources in prevention programmes should primarily be focussed on young children, particularly those residing in areas of high malaria transmission.

Abstract Background Studies on Plasmodium falciparum gametocyte development and dynamics have almost exclusively focused on patients treated with antimalarial drugs, while the majority of parasite carriers in endemic areas are asymptomatic. This study identified factors that influence gametocytaemia in asymptomatic children in the absence and presence of pyrimethamine-sulphadoxine (SP) antimalarial treatment. Methods A cohort of 526 children (6 months – 16 years) from western Kenya was screened for asexual parasites and gametocytes and followed weekly up to four weeks. Children with an estimated parasitaemia of ≥1,000 parasites/μl were treated with SP according to national guidelines. Factors associated with gametocyte development and persistence were determined in untreated and SP-treated children with P. falciparum mono-infection. Results Gametocyte prevalence at enrolment was 33.8% in children below five years of age and decreased with age. In the absence of treatment 18.6% of the children developed gametocytaemia during follow-up; in SP-treated children this proportion was 29.8%. Age, high asexual parasite density and gametocyte presence at enrolment were predictive factors for gametocytaemia. The estimated mean duration of gametocytaemia for children below five, children from five to nine and children ten years and above was 9.4, 7.8 and 4.1 days, respectively. Conclusion This study shows that a large proportion of asymptomatic untreated children develop gametocytaemia. Gametocytaemia was particularly common in children below five years who harbor gametocytes for a longer period of time. The age-dependent duration of gametocytaemia has not been previously shown and could increase the importance of this age group for the infectious reservoir.

Introduction Over the past five years, dramatic declines in malaria disease caused by Plasmodium falciparum have been reported across a range of settings within sub-Saharan Africa. These declines are associated [...], Background: Over the past decade malaria intervention coverage has been scaled up across Africa. However, it remains unclear what overall reduction in transmission is achievable using currently available tools. Methods and Findings: We developed an individual-based simulation model for Plasmodium falciparum transmission in an African context incorporating the three major vector species (Anopheles gambiae s.s., An. arabiensis, and An. funestus) with parameters obtained by fitting to parasite prevalence data from 34 transmission settings across Africa. We incorporated the effect of the switch to artemisinin-combination therapy (ACT) and increasing coverage of long-lasting insecticide treated nets (LLINs) from the year 2000 onwards. We then explored the impact on transmission of continued roll-out of LLINs, additional rounds of indoor residual spraying (IRS), mass screening and treatment (MSAT), and a future RTS, S/AS01 vaccine in six representative settings with varying transmission intensity (as summarized by the annual entomological inoculation rate, EIR: 1 setting with low, 3 with moderate, and 2 with high EIRs), vector-species combinations, and patterns of seasonality. In all settings we considered a realistic target of 80% coverage of interventions. In the low-transmission setting (EIR~3 ibppy [infectious bites per person per year]), LLINs have the potential to reduce malaria transmission to low levels (90%) or novel tools and/or substantial social improvements will be required, although considerable reductions in prevalence can be achieved with existing tools and realistic coverage levels. Conclusions: Interventions using current tools can result in major reductions in P. falciparum malaria transmission and the associated disease burden in Africa. Reduction to the 1% parasite prevalence threshold is possible in low- to moderate-transmission settings when vectors are primarily endophilic (indoor-resting), provided a comprehensive and sustained intervention program is achieved through roll-out of interventions. In high-transmission settings and those in which vectors are mainly exophilic (outdoor-resting), additional new tools that target exophagic (outdoor-biting), exophilic, and partly zoophagic mosquitoes will be required. Please see later in the article for the Editors' Summary. 10.1371/journal.pmed.1000324