Understanding the dynamics of malaria transmission in diverse endemic settings is key for designing and implementing locally adapted and sustainable control and elimination strategies. A parasitological and epidemiological survey was conducted in September–October 2012, as a baseline underlying a 3-year population-based longitudinal cohort study. The aim was to characterize malaria transmission patterns in two contrasting ecological rural sites in the Peruvian Amazon, Lupuna (LUP), a riverine environment, and Cahuide (CAH), associated with road-linked deforestation.After a full population census, 1941 individuals 3 years and older  were interviewed, clinically examined and had a blood sample taken for the detection of malaria parasites by microscopy and PCR. Species-specific parasite prevalence was estimated overall and by site. Multivariate logistic regression models assessed risk factors for parasite infection by PCR, while SaTScan detected spatial clusters of PCR-positive individuals within each site. In addition, data from routine malaria surveillance in the period 2009–2012 were obtained.Plasmodium vivax (6.2% vs. 3.9%) and for Plasmodium falciparum (2.6% vs. 1.2%). Among PCR-confirmed infections, asymptomatic (Asy) parasite carriers were always more common than symptomatic (Sy) infections for P. vivax (Asy/Sy ratio: 2/1 in LUP and 3.7/1 in CAH) and for P. falciparum (Asy/Sy ratio: 1.3/1 in LUP and 4/1 in CAH). Sub-patent (Spat) infections also predominated over patent (Pat) infections for both species: P. vivax (Spat/Pat ratio: 2.8/1 in LUP and 3.7/1 in CAH) and P. falciparum malaria (Spat/Pat ratio: 1.9/1 in LUP and 26/0 in CAH). For CAH, age, gender and living in a household without electricity were significantly associated with P. vivax infection, while only age and living in a household with electricity was associated with P. falciparum infection. For LUP, only household overcrowding was associated with P. falciparum infection. The spatial analysis only identified well-defined clusters of P. vivax and P. falciparum infected individuals in CAH. Reported malaria incidence indicated that malaria transmission has long occurred in LUP with primarily seasonal patterns, and confirmed a malaria outbreak in CAH since May 2012.Parasite prevalence by PCR was higher in CAH than in LUP for This parasitological and epidemiological baseline assessment demonstrates that malaria transmission and parasite prevalence is heterogeneous in the Peruvian Amazon, and influenced by local socio-demographics and ecological contexts. Riverine and road construction/deforestation contexts must be taken into account in order to carry out effective anti-malaria control and elimination efforts.The online version of this article (doi:10.1186/s12936-017-1957-y) contains supplementary material, which is available to authorized users. Plasmodium falciparum. It is thought that these interventions contributed to a reduction of microscopically-confirmed malaria cases in Loreto, from 54,291 cases in 2005 to 10,504 and 11,793 cases in 2010 and 2011, respectively [Malaria remains an important public health problem in Peru, especially in the Amazonian department of Loreto where most malaria occurs –4. Folloectively , and to ectively .Anopheles darlingi  [Routine malaria control activities in the Peruvian Amazon are based on passive case detection (PCD) using light microscopy (LM) , 15 on p Amazon) . Collect Amazon) –25.Plasmodium vivax and P. falciparum parasitaemias contribute to the maintenance of malaria hypoendemicity in the Amazon region drives the work of our Amazonia International Center of Excellence in Malaria Research (Amazonia ICEMR), which was established in 2010 as one of ten such centers supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) [P. vivax and P. falciparum prevalence by LM and PCR at the baseline enrollment surveys in September–October 2012 are also discussed and those estimates were related to data from routine malaria surveillance and implemented control efforts to better understand the dynamics of malaria transmission in both sites.The hypothesis that asymptomatic th (NIH) . The Amath (NIH) . This paEthics clearance for the study was obtained from the Ethics Review Board of the Universidad Peruana Cayetano Heredia, Lima, Peru (SIDISI code # 5739) and from the University of California San Diego Human Subjects Protection Program (Project # 100765). Permissions were received from health and local authorities after explaining the purpose and procedures of the study. Signed informed consent was obtained prior the study enrollment to participation and blood sampling by all adults and the parents of all participating children <18 years. In addition to parental/guardian consent, children older than 7 years provided a signed informed assent. All the methods were carried out in accordance with approved guidelines.A census and an epidemiological survey were conducted as baseline activities of a 3-year population-based, longitudinal cohort study in the Lupuna (LUP) and Cahuide (CAH) sites in the northeastern Peruvian Amazon Department of Loreto. The study population included individuals aged 3 years and older who resided in study sites and agreed to participate in the cohort providing a written signed consent.Anopheles darlingi is the primary malaria vector in the area [LUP site is located on the outskirts of Iquitos district, 10 km from downtown (latitude 03°44.591′S longitude 73°19.615′W)  for 3 days , plus primaquine (PQ) for 7 days (0.5 mg/kg/day); P. falciparum malaria was treated with mefloquine (MQ) (12.5 mg/kg/day for 2 days) plus artesunate (AS) (4 mg/kg/day for 3 days), according to Peruvian national policy.ire year . As in mA complete census of the population in each village was conducted in July–August 2012, collecting individual data on socio-demographics , and on previous malaria episodes. Each house was identified with a unique number and geo-referenced using a handheld Global Positioning System (GPS) device , and household characteristics collected . Each individual subject was given a seven-digit unique code number combining the village, household and individual code.Individuals aged 3 years and older available at the time of the baseline survey (September–October 2012) were enrolled in the study area after consenting. Whenever selected individuals were absent, the household was revisited up to four times in a period of 30 days to maximize subject participation. Each participant had the axillary temperature taken, history of fever or any other malaria symptoms were recorded, and a finger-prick blood sample collected for immediate microscopy (thick and thin blood smears) and on filter paper  for later molecular tests. Filter paper dried blood samples were individually stored at 4 °C with desiccant until processed. Subjects with microscopically-confirmed infections were referred to health posts for treatment according to Peruvian national guidelines.Additionally, routine surveillance data, namely reported malaria episodes by species in Iquitos and San Juan district from 2009 to 2012 were obtained from the Regional Directorate of Health, Loreto-Ministry of Health in Iquitos. Peruvian policy mandates weekly notification of confirmed malaria cases (by standard LM) at health facilities and data aggregation at district and department level. Since malaria surveillance data could be reliably disaggregated only to the district level but not to the village level, a retrospective review of all registered individuals detected by PCD between January 2009 and December 2012 was carried out in all health facilities near the study sites. Any other surveillance in addition to routine PCD conducted in CAH and LUP in the 12 months before the survey was recorded.Thick and thin smears were stained for 10 min with a 10% Giemsa solution, and parasite density was calculated by counting the number of asexual parasites for 200 white blood cells (WBC) in the thick smear, assuming a concentration of 8000 WBCs/μl. Slides were read on site, then a day later by an expert at microscopy at the reference laboratory in Iquitos. A slide was declared negative if no malaria parasite was found after examining 100 fields . Quality2 pieces for DNA extraction using QIAamp DNA Microkit of QIAGEN following the manufacturer instructions. A quantitative real-time polymerase chain reaction (qPCR) method targeting the 18s rRNA gene region was used for molecular diagnosis, following the protocol reported by Mangold et al. [P. vivax from P. falciparum.Filter paper blood spots were cut into ~6 mmd et al.  Primers Census and survey data were doubly entered and cross-checked in Access . Data analysis was performed using R v.2.15 software . Baseline characteristics between sites were compared using the Chi squared test. Species-specific parasite prevalence by LM and by PCR were estimated overall and by site, and their two-sided 95% confidence intervals (CI) were calculated using the Wilson score method.Plasmodium spp.) infection was an individual with a positive PCR result regardless of symptoms. Cases were further classified as patent (detectable by LM) or sub-patent (not detectable by LM). A symptomatic infection was defined as an individual with confirmation of parasites in the blood sample at any level who also had fever or history of fever, headache, chills or general discomfort in the previous 7 days. Uni- and multivariate mixed-effects logistic regression models, with fixed effects and a random intercept that accounted for individual clustering at household, were used to determine risk factors for species-specific malaria infection in each site The following potential risk factors were assessed: age, gender, time living in the village, education level, forest-related job as main economical activity (including the intensity of these activities during the week), trip in the past month to another endemic area, the history of malaria in the past year, household overcrowding , low household bed net coverage (<80% of beds covered with a net in a household), housing structure , and the availability of electricity. Factors with p values <0.1 for the Wald test in the univariate analysis were considered for inclusion in the multivariate model. Using manual backward, final models retained all factors that were significantly associated with malaria infection . Interactions were systematically checked for up to order two. Likelihood ratio tests (LRTs) were used to assess statistical differences between nested models.The case definition for malaria parasite  was identified as the most likely cluster. The relative risk (RR) reported for each identified cluster was the estimated risk within the cluster divided by the estimated risk outside the cluster [The QGIS software QGIS v.2.16  was used to map all surveyed households and to classify them according to the number of household members with malaria infections identified by PCR. The SaTScan software v.9.3  was used to identify spatial clustering of households with malaria infections in each site, using the following characteristics: pure spatial analysis, Poisson probability model , latitud cluster .A total of 487 households (HHs) and 2447 individuals were identified during the census in July–August 2012, distributed over the two sites as follows: LUP  and CAH . In addition, the distribution by village was: LT , SR  and SP  for LUP, and CT , LH  and DA  for CAH (Table p = 0.008). Males slightly outnumbered females  without differences between sites. The proportion of participants aged >10 years old who have resided in the site ten or more years was significantly higher in LUP (85.0%) than in CAH (41.3%) (p < 0.001). Among adults 18 years and older, the proportion of individuals who had completed secondary education level was higher in LUP (47.0%) than in CAH (32.8%) (p < 0.001). The most commonly reported occupation among adults was farmers, followed by housewives, indoor laborers/employees and traders with similar proportions in both sites; security guards (p = 0.002) and loggers (p < 0.001) were more frequent in CAH than in LUP. Participants reporting having experienced malaria in their lifetime were more common in LUP (73.4%) than in CAH (67.0%) (p = 0.003). However, this was not the case when only recent episodes were considered, with more participants reporting have had malaria episodes within the past 12 months in CAH (48.9%) than in LUP (14.1%) (p < 0.001).The cohort study enrolled 1941 of the 2447 censused inhabitants (79.3%) in 448 HH (92.0%) available at the time of the baseline survey. Of these 1941 individuals, 829 (42.7%) lived in LUP and 1117 (57.3%) in CAH. Socio-demographic characteristics and history of past malaria episodes by site and by village are presented in Table p < 0.001). In both sites, most individuals lived in houses with wooden walls and roofs composed of palm leaf; concrete (brick and cement) and tin were also common building materials, especially in the LT village of LUP (p < 0.001) . Most participants had electricity at home in CAH (55.7%), primarily those living along the road and in the DA village (p < 0.001); however this electrical service mainly exclusively benefited participants living in LT village in LUP (p < 0.001). The use of potable water for drinking and food preparation was reported by only a small proportion of individuals in LUP (15.6%) and CAH (30.7%), while firewood was reported as the main fuel used for cooking by most participants (>80%) in both sites. Moreover, most participants in both sites reported having precarious or lack of sanitation facilities, as well as lack of trash disposal systems. The majority (97.0%) of participants in CAH reported that their houses were sprayed indoors with insecticide in the past 3 months, while only 12.4% reported this in LUP (p < 0.001). Household bed net coverage was high in both sites; the predominant material of bed nets reported by participants varied across sites. In all villages of LUP, most bed nets (about 90%) were LLINs. In CAH, about half of subjects reported having LLINs and half reported using non-impregnated insecticide nets made of tocuyo (non-processed cotton) or nylon.Table P. vivax, 2 P. falciparum infections), and by PCR 7.2%  (Table (p < 0.05). Of the total 38 P. vivax infections by LM, 30 were found in CAH (16 with parasitaemia <100 parasites/μl), and only 8 in LUP (one with parasitaemia <100 parasites/μl); while the two microscopically confirmed P. falciparum infections were found in LUP (one with parasitaemia <100 parasites/μl). The presence of gametocytes was recorded in 15 (50.0%) and 3 (37.5%) of the total microscopically confirmed P. vivax infections in CAH and LUP, respectively. Gametocytes were not observed in microscopically confirmed P. falciparum infections. Parasite prevalence by PCR was higher in CAH than in LUP for P. vivax  and for P. falciparum . Considering PCR-confirmed infections, asymptomatic (Asy) parasite carriers were always more common than symptomatic (Sy) infections for P. vivax (Asy/Sy ratio: 2/1 in LUP and 3.7/1 in CAH) and for P. falciparum (Asy/Sy ratio: 1.3/1 in LUP and 4/1 in CAH). Similarly, sub-patent (Spat) infections also predominated over patent (Pat) infections for both species: P. vivax (Spat/Pat ratio: 2.8/1 in LUP and 3.7/1 in CAH) and P. falciparum malaria (Spat/Pat ratio: 1.9/1 in LUP and 26/0 in CAH).All 1941 individuals enrolled in the cohort had samples obtained for malaria diagnosis by LM at the time of the baseline survey; most of these  had available sample for PCR analysis. By LM, the overall parasite prevalence was 2.1% . No significant factors were found for P. vivax infection . Moreover, the lack of electricity increased the odds of infection in household members by a factor of 1.9  compared with individuals living in households that had available this public service. Considering P. falciparum, the odds of infection confirmed by PCR significantly increased at younger age (test for trend p < 0.001), with individuals <15 years being four times more likely to have P. falciparum infections than adults >40 years . In this case, the availability of electricity was associated with highest risk of P. falciparum in household members  compared with individuals living in households without electricity. No association was found between P. vivax and P. falciparum infection with regard to time of residence in the village, education level, occupation, trip in the past month to another endemic area, the history of malaria in the past year, and housing characteristics of study participants.Given the relatively small number of infections detected by LM, uni- and multivariate mixed-effects logistic regression analyses were carried out only for confirmed infections by PCR for each site. In final models the estimates of the random-effects coefficients confirmed the influence of clustered sampled data at household level. In LUP, household overcrowding  was the only factor significantly associated with I 1.0–24.). No sigP. vivax and P. falciparum infections were identified using SaTScan  was in the southern part of CT village . For P. falciparum, the most likely cluster adjusted for age and electricity availability was the closest area to the fluvial port in CT . With a radius of 99 meters, the cluster included 61 individuals  in 14 households   without adjustment for covariates.Figures can Fig. , mainly age Fig. a, includal) Fig. b. This cP. vivax, 7 P. falciparum) represented about 15% of the total 880 reported cases in Iquitos district. Regarding malaria intervention efforts, deliveries of LLINs by GFATM’s PAMAFRO malaria project in 2008 and 2010 were the main malaria control intervention in LUP during the evaluated period. On the other hand, malaria cases in CAH between 2009 and 2011 occurred with stochastic patterns , and did not follow the same trends of the malaria incidence than San Juan district during that period  from CAH villages accounted for about 35% of the total 3617 reported cases in San Juan district in 2012.The retrospective analysis of reported malaria cases between 2009 and 2012 showed that the malaria incidence in LUP varied across years, with some periods (mainly in year 2009) not following the same trends of malaria incidence compared to the Iquitos district Fig. a. Despitiod Fig. b. The raP. falciparum and P. vivax.The baseline analysis presented here of a population-based, longitudinal malaria cohort study in two contrasting ecological villages in the Peruvian Amazon indicates substantial prevalence of malaria parasitaemia in the region and is an important benchmark for ongoing longitudinal studies. The riverine rural site, Lupuna (LUP), is home to a fairly stable population . Here, the combined analysis of survey findings and routine reported malaria incidence suggests that malaria transmission has long occurred with primarily seasonal patterns. In contrast, Cahuide (CAH), a rural site with road-driven deforestation and a more transient population (about 60%), has experienced a notable and concerning increase of cases since May 2012 that compelled the regional government to carry out intensive outbreak control measures until August 2012 (1 month before the survey). Low parasite rates were observed in both sites during the survey, with high predominance of asymptomatic and sub-patent infections over symptomatic and patent infections. Moreover, the site-specific analysis of malaria prevalence and reported incidence suggested a high degree of heterogeneity at the micro-geographic level and over time for both Community surveys for determining malaria parasite carriage are commonly used to estimate malaria transmission in endemic areas , 33. BecP. vivax and P. falciparum. This was observed here to have occurred despite the intensive response from RHDL-MoH to a severe malaria outbreak from May to August 2012. These interpretations are further supported by a recently published entomological study of our Amazonian ICEMR that found that the highest monthly entomological inoculation rates (EIRs) are associated with seasonal transmission in both sites, with an important factor being seasonally high river levels that enable mosquito breeding [An. darlingi  and higher human biting rates (HBRs) in LUP between February and June (maximum EIR in April) in successive years 2011 and 2012; while for CAH, the highest monthly EIR (2.52) with also confirmed P. vivax- and P. falciparum-infected An. darlingi occurred during the 2012 malaria outbreak. Interestingly, despite decreased HBR levels leading to a much lower estimated EIR in CAH in October 2012 (period of baseline survey), the entomological study found a high infection rate of collected mosquitoes (1.47%) which indicated onward malaria transmission in the area [The richness of these datasets is important to consider with regard to how future analyses will guide anti-malaria interventions. In riverine, rural LUP, malaria prevalence by PCR may indicate the seasonal-time dependent parasite carriage level in an endemic area  with no major recent changes in the population socio-demographics and in local control measures. In contrast, in road/deforestation and riverine rural CAH, prevalence of parasite carriers (with confirmed gametocytaemia by LM in some of them) reflects the parasite reservoir that enables persistence of malaria transmission by both breeding . Indeed,the area , and thethe area  followinthe area .P. vivax and P. falciparum—were asymptomatic (64–85%) and sub-microscopic (75–77%), despite differences in malaria prevalence and variations in malaria transmission over time. A limitation of such cross-sectional surveys is that they might not correctly estimate the proportion of infections that later might become symptomatic or have higher parasite density levels (as detectable by LM) [As previously described elsewhere in the Peruvian Amazon –21, the e by LM) , 34. Fewe by LM) , 22. A 6e by LM) , 34. Anoe by LM) , 27, offP. vivax and P. falciparum. Household overcrowding, the only factor found to be significantly associated with P. falciparum infection in LUP, has also been reported in several studies conducted in areas where transmission is exclusively due to P. falciparum, or co-endemic with P. vivax [P. falciparum infection and other proxy variables for socio-economic status in LUP, it is important to note that overcrowding can also reflect poor economic household conditions. The complex association between poverty and malaria is well known, which likely operates in both directions [Considering the site-specific socio-demographics, household characteristics and ecological conditions in LUP and CAH, risk factor and spatial analyses of malaria infections were performed separately for each site. The low number of infections in LUP detected during the survey in low transmission season would primarily explain the difficulty in identifying malaria risk factors and spatial clusters of high transmission in the site for both P. vivax , 36. TheP. vivax , leadingrections , 39: pooP. vivax and P. falciparum malaria. This outbreak likely included both: parasite carriers that remained despite the response to a first severe outbreak occurred between May and August 2012, and new infections by either similar or different parasite populations than those of the first outbreak. The increased risk for P. vivax in males and individuals aged >15 years together with the confirmed infection of mosquitoes in the site [An. darlingi mosquitoes bite mainly outdoors [Regardless of how the relationship of species-specific malaria with potential risk factors in CAH might be explained, it must be considered that our parasitological baseline survey probably captured the incipient onset of a second local outbreak of the site  suggestsoutdoors , and by outdoors , 40.P. vivax infections and consequently suggest that transmission is not entirely outdoors; the electricity availability as risk factor for P. falciparum may be related with an increase of evening outdoor activities  of household members [P. falciparum species in CAH  may explain the significant relationship of P. falciparum infections with younger age groups [P. falciparum infections had low parasite densities non-detectable by LM [P. vivax decreased significantly faster with age than that of P. falciparum, suggesting that acquiring clinical immunity is different species-specific, as has been seen in other co-endemic countries such as Papua New Guinea [The differences in malaria risk between species with respect to the availability of electricity at household can be explained by the different connotations that this public service can have. While the lack of electricity may reflect poor socio-economic status associated with increased  members . On the e groups –44, as wle by LM . The resw Guinea , Thailanw Guinea , Vanuatuw Guinea , Ethiopiw Guinea , and Braw Guinea , 49. TheP. vivax and P. falciparum only in the CAH site. Since the cluster located on the south of CAH for P. vivax was found to be associated with an increased presence of male individuals, age groups older than 15 years and the lack of electricity at households, an adjusted spatial analysis for these covariates was performed resulting in the disappearance of the cluster. The observation indicating that the aggregation of P. vivax infections was mainly determined by the aggregation of groups with high-risk than by specific ecological conditions within the site has also been reported in other villages of the Peruvian Amazon [An. darlingi, and the high mobility of infected individuals through the unique and highly accessible road could further increase the dispersal of parasites. Conversely, the cluster for P. falciparum remained in the closest area to the river even after being adjusted for age and electricity availability . Its highest prevalence may be explained by the unprecedented floods from Itaya River in 2012 resulting in large and multiple breeding sites in the area, but also by the presence of a fluvial port to which individuals (resident and non-residents) arrive after being exposed to malaria due to travel-related economical activities in other endemic riverine villages.The high heterogeneity in malaria transmission in the Peruvian Amazon creates opportunities for targeted interventions . Howevern Amazon . Local mParasitological measures (by LM and PCR), and individual and household characteristics collected during the baseline survey of a 3-year population-based malaria cohort study in the Amazonian Lupuna and Cahuide villages, together with contextual data obtained from malaria surveillance systems and health facilities, allowed for a good characterization of their population and malaria transmission patterns. On a population basis in both sites, low parasite rates were observed, with findings that asymptomatic and sub-patent infections predominated, compared to symptomatic and patent infections. However, the site-specific analysis of malaria prevalence and reported incidence showed substantial heterogeneity in malaria transmission at the micro-geographical level and over time, likely influenced by the site-specific socio-demographics, household characteristics, and ecological conditions in the villages. Riverine and road construction/deforestation contexts must be taken into account in order to carry out effective anti-malaria control and elimination efforts.Additional file 1: Table S1. Baseline socio-demographic characteristics of study participants by villages. Table S2. Baseline household characteristics of study participants by villages.Additional file 2: Figure S1. Weekly reported malaria cases in Cahuide (B) in 2012, and main control interventions implemented. (1) Mass screening and treatment (MS&T) and indoor spraying with residual insecticide with >90% of household coverage;  MS&T; (4) distribution of LLINs.


Plasmodium vivax, merozoite surface protein 1 (MSP1), MSP1 paralogue, MSP4, MSP5, MSP8, and MSP10 have been reported from homologs of Plasmodium falciparum by gene annotation with bioinformatics tools. These GPI-anchored proteins contain two epidermal growth factor (EGF)-like domains at its C-terminus. Here, P. vivax merozoite surface protein 8 (PvMSP8) are considered as potential targets of protective immunity.Thirty-one glycosylphosphatidylinositol (GPI)-anchored proteins of P. vivax-infected patients and immune mice. Moreover, the target epitope of ant-PvMSP8 antibodies and subcellular localization of PvMSP8 was also determined.Recombinant PvMSP8 (rPvMSP8) was expressed, purified, and used for the assessment of humoral and cellular immune responses in P. vivax. IgG2 antibody subtype was the predominantly responses to this antigen. Antibody response to PvMSP8 increased up to day 7 and after that slightly decreased within a month. The longevity of anti-PvMSP8 antibody was stably sustained up to 12-year recovery patient samples. Most anti-PvMSP8 antibodies recognized two epitopes that were located outside the C-terminal EGF-like domain. The cellular immune response in P. vivax-exposed individuals produced high levels of IFN-γ and IL-10 upon PvMSP8 antigen stimulation in vitro.The rPvMSP8 was successfully expressed and purified as soluble form as ~55 kDa. PvMSP8 was localized to the outer circle of pigments associated with the food vacuole. The rPvMSP8 protein had a high antigenicity (73.2% in sensitivity and 96.2% in specificity) in patients infected with P. vivax infection. The subcellular localization of PvMSP8 confirmed that it was associated with the parasite food vacuole in blood-stage parasites. A further characterization of this protein will be useful for blood stage P. vivax vaccine development.All data in this study suggest that PvMSP8 antigen has a potential to induce both humoral and cellular immune responses in patients with The online version of this article (doi:10.1186/s12936-017-1837-5) contains supplementary material, which is available to authorized users. Plasmodium species [Plasmodium yoelii MSP8 (PyMSP8) fused with P. yoelii MSP1-19 induced MSP8-restricted T cell response and high and sustained levels of protective PyMSP1-19- and PyMSP8-specific antibody responses [Plasmodium falciparum MSP8 (PfMSP8) (ΔAsn/Asp) is useful for protective efficacy, together with the fusion partner of poor immunogens such as PfMSP1-19/MSP8 (ΔAsn/Asp) [P. falciparum MSP8 knock-out study showed that it is not required for asexual stage parasite growth and replication [Plasmodium species.The merozoite surface protein 8 (MSP8) is one of the glycosylphosphatidylinositol (GPI)-anchored proteins of blood-stage malaria parasites. It contains a signal sequence at N-terminus and two epidermal growth factor (EGF)-like domains at C-terminus with significant homology to those of  species –4. In presponses . As a maAsn/Asp) . The domAsn/Asp) . Howeverlication . Thus thPlasmodium parasites internalize host cell haemoglobin, which is degraded in a specialized compartment, the FV [Plasmodium blood stages; in other words, it is absent from the mosquito and liver stages and is not found in other apicomplexan parasites. In addition to PfMSP8, the C terminus of another well-known GPI-anchored protein, PfMSP1, also constitutes the FV [PfMSP8 has been confirmed to localize to the parasitophorous vacuole (PV) of infected erythrocytes. Intriguingly, its C terminus is found in the food vacuole (FV) . Plasmod, the FV . Some ch, the FV . This his the FV , 13. ThePlasmodium vivax-infected patients’ sera [P. vivax parasite, in this study, a high-level and long-lived immune response was observed against PvMSP8 in vivax malaria patients and a high immunogenicity was detected in rPvMSP8-immunized mice. Dominant epitopes were also mapped in the C terminus of PvMSP8 and its subcellular localization in the blood stage was shown to be at the FV.However, as a homologue of PfMSP8, little is known about PvMSP8 as potential targets of protective immunity. Only one previous study showed the recognition of recombinant PvMSP8 with ts’ sera . To propPvmsp8 was obtained from the PlasmoDB website . Protein domains were further predicted using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) [GGGCGGATATCTCGAGGGAAACGTTAGCCCACCC-3′; and PvMSP8R, 5′-GCGGTACCCGGGATCCTTAGCAGTATATTCCGTCTCCCTCA-3′ were used for DNA amplification. Then, the PvMSP8 DNA was cloned into the pEU-E01-His-TEV-MCS vector . The rPvMSP8 protein was expressed using a WGCF expression system and purified using a Ni–NTA agarose column , as described elsewhere [Gene sequence of erg.de/) , 16. Recerg.de/) . Brieflylsewhere . The proFemale BALB/c mice, at 6–8 weeks of age  were immunized with 20 μg of rPvMSP8 and phosphate buffered saline  with complete Freund’s adjuvant  using intraperitoneal route of administration. Three and 6 weeks after immunization, the equal volume of antigen with incomplete Freund’s adjuvant (Sigma-Aldrich) was boosted. Mouse blood samples were taken after the final booster injection, 2 weeks later. The antisera against PvMSP1 was also produced following the same procedure as PvMSP8 . Animal P. vivax were collected from malaria patients in Thailand, as described previously [The schizont-stage-rich parasites of eviously . Brieflyeviously  and mousn = 56) were collected in 2012 from patients in Shwe Kyin area of Myanmar, and the Thai samples (n = 56) were collected from symptomatic, smear-positive patients from the Mae Sod district of western Thailand. For the 1-month follow-up study, 25 patients from the Shwe Kyin area were assessed at 7, 14, 21, and 28 days after treatment. The serum samples from healthy individuals with a vivax malaria history were collected from Chinese residents who had an episode of vivax malaria infection in malaria endemic areas of Anhui Province, China, in 2012, and who did not have a reinfection episode of vivax malaria in the preceding 5 (n = 30), 12 (n = 30), or 30 (n = 30) years  from endemic countries and 80 samples from healthy individuals, assessed as being negative using microscopy, were collected in the Republic of Korea. The Myanmar samples (P. vivax subjects (n = 15) who had recovered from P. vivax infection after 8–10 weeks were collected at malaria clinics in Tha Sae, Chumphon Province, which is located in the southern peninsular region of Thailand. Moreover, heparinized blood from healthy individuals (n = 15) who had no history of exposure to malaria were collected. The samples were used for peripheral blood mononuclear cell (PBMC) preparation.For the cellular immunity study, 10 mL of heparinized blood from 2-)-coated slides were prepared as previously described [Amine . The phenotypes of T cells that responded to rPvMSP8 were analysed by intracellular cytokine staining after in vitro stimulation with 10 μg/mL of rPvMSP8, 20 ng/mL of PMA/ionomycin, or medium alone. Cells were stained with monoclonal antibodies (mAbs) against surface determinants of CD3, CD4, and CD8 and intracellular cytokine markers of anti-IFN-γ and anti-IL-10 antibodies, according to the manufacturer’s instructions.Lymphocyte stimulation assay was carried out for measuring of cellular immunity in 15 recovered A splenocyte proliferation assay was performed using splenocytes removed from mice that had been immunized with rPvMSP8. The cells were stimulated with rPvMSP8 (2.5 μg/mL), Con A (5 μg/mL), LPS (10 μg/mL), or medium alone. Culture supernatants were collected after 72 h of incubation and assayed using a BD CBA Flex Set kit (BD Biosciences), according to the manufacturer’s instructions.The correlation between the antibody reactivity of different concentrations of the recombinant proteins and duplicate spots of protein arrays was observed using GraphPad Prism software, version 5.0  and PASW Statistics 18.0 . Sensitivity and specificity were measured by the percentage of patients who had a positive test result and the percentage of healthy individuals who had a negative test result, respectively. The significance of differences in mean fluorescence intensity (MFI) values between every two groups were performed with the Mann–Whitney U test.The predicted molecular weight of PvMSP8 (PVX_097625) is 54.7 kDa, with 487 amino acids containing a signal peptide, asparagine-rich region in N-terminal region and two EGF domains and GPI-motif in C-terminal region Fig. a. The reP. vivax, PvMSP8 was localized to the outer circle of pigments associated with the FV . Furthermore, PvMSP8 produced similar IgG titers among isolates of vivax malaria patients from Korea, Myanmar, and Thailand, as well as from malaria-naïve individuals  from China. A significantly higher IgG reactivity was observed in 5- and 12-year recovery sera, although the IgG titer was lower than that observed for acute infection. Moreover, in most cases, IgG antibody levels in individuals who infected malaria 30 years previously had decreased to baseline levels  located in the N terminus were highly reactive  in comparison to that observed in the absence of antigen stimulation  also predominated among the anti-malarial antibodies developed by unprotected subjects, whereas cytophilic subclasses (IgG1 and mainly IgG3) were the most abundant isotypes produced by malaria patients, who are protected from malarial parasites , 22. Conlearance . In addilearance . In thisHere, the authors also observed that long-term maintenance of IgG antibodies against PvMSP8 was detected in individuals from Anhui Province, China , and that CD8lls Fig. a, b. Thinfection , as wellnfection , 29. CD8P. vivax infection and represent an important advance in the understanding of blood-stage immunity to P. vivax, at least in part. Intriguingly, the manner via which a potent immune response, such as the one shown here, can be induced remains to be studied, together with the FV localization of PvMSP8. To confirm the protective ability of PvMSP8 antibodies, functional assays, such as a short-time growth inhibition assay, need to be developed in the future.The study results demonstrated the presence of PvMSP8-antigen-induced humoral and cellular immune responses in


Plasmodium falciparum malaria in Sweden, Charlson comorbidity score ≥1 as well as diabetes and obesity were significantly associated with severe malaria in both nonimmune travelers and immigrants from endemic countries.In this nationwide observational study of 937 adults diagnosed with  Plasmodium falciparum malaria.Noncommunicable diseases and obesity are increasing in prevalence globally, also in populations at risk of malaria. We sought to investigate if comorbidity, in terms of chronic diseases and obesity, is associated with severe We performed a retrospective observational study in adults (≥18 years of age) diagnosed with malaria in Sweden between January 1995 and May 2015. We identified cases through the surveillance database at the Public Health Agency of Sweden and reviewed clinical data from 18 hospitals. Multivariable logistic regression was used to assess associations between comorbidities and severe malaria.P = .004). Charlson comorbidity score ≥1 was associated with severe malaria , as was diabetes among individual diagnoses . Median body mass index was higher among severe (29.3 kg/m2) than nonsevere cases (24.7 kg/m2) (P < .001). Obesity was strongly associated with severe malaria, both independently  and in combination with an additional metabolic risk factor  . The associations were observed among nonimmune travelers as well as immigrants from endemic areas.Among 937 adults , patients with severe malaria had higher prevalence of chronic diseases (28/92 [30.4%]) compared with nonsevere cases (151/845 [17.9%]) (P. falciparum. Noncommunicable diseases should be considered in the acute management and prevention of malaria.Comorbidities, specifically obesity and diabetes, are previously unidentified risk factors for severe malaria in adults diagnosed with  Obesity and noncommunicable diseases (NCDs), such as diabetes, hypertension, and cardiovascular disease, have increased globally, including in malaria-endemic regions . In addiPlasmodium falciparum infections was found among individuals with type 2 diabetes in Ghana ) than nonobese  was associated with severe malaria , as was obesity combined with ≥1 additional metabolic risk factor, corresponding to 2 of 3 criteria for metabolic syndrome  aOR, 6.. Only 5 P = .001), and 9.1% (11/121), 2.3% (9/385), and 3.4% (13/388) (P = .004) for diabetes in patients with residency ≥15 years, <15 years, and non/low-endemic origin, respectively. Stratified analyses resulted in positive associations between both obesity and diabetes and severe malaria in all categories; however, only significantly among patients of endemic origin with residency ≥15 years . Interaction analysis could not verify that patient origin, and time of residency modified the association of either diabetes or obesity with severity .Obesity and diabetes were most prevalent among patients of endemic origin with long residency in Sweden: 16.5% (15/91), 4.8% (12/248), and 3.8% (8/209) for obesity  are well recognized to increase severity of infections , 30. BotObese patients had higher parasitemia compared with nonobese patients , a feature also described in obese mice , and hypNumerous studies have shown that immigrants from high-endemic regions are at lower risk of severe and fatal malaria compared to nonimmune travelers . RecentlICD codes, and medication lists. Moreover, prevalence data on chronic diseases and BMI present before the malaria episode produce an acceptable proxy for longitudinal data. However, a majority of the HIV tests were taken in conjunction with the malaria episode and severe cases were more often tested, which could imply a detection bias for HIV. Nonetheless, 18 of 21 HIV cases were known before, and among the 3 newly detected, none had severe malaria. Data on BMI were lacking from a large proportion of patients. Incorporating the factors associated with missing data (Supplementary Table 2) in a multiple imputation model resulted in similar effect of obesity on severity as the complete case analysis. Systematic health screening including BMI and glucose control, preferably in prospective studies, are needed to confirm our results. Possibly, some patients had yet undiagnosed chronic diseases, such that nondifferential misclassification might have diluted the effects.Our study has several limitations. The retrospective design could lead to misclassification of exposures and outcome variables. The current data were entered as part of a larger epidemiological study without priori hypotheses. Moreover, in Sweden, fever after tropical visits is managed by infectious diseases specialists using standardized protocols. A high sensitivity of comorbidity assessment was achieved by using medical records, registered The strength of our study is that it includes clinical data from a large population of travelers, with a mixed constitution of individuals previously exposed and unexposed to malaria, diagnosed at multiple centers. Case detection was based on the national surveillance system with high detection sensitivity . The resWe believe our findings have implications beyond the management of malaria in travelers. Sub-Saharan Africa is facing a double burden of disease; while continuing to deal with malaria and other infections, there is a rapid upsurge in NCDs . This isP. falciparum malaria. We show that obesity, diabetes, and combinations of metabolic risk factors were associated with severe malaria, both in nonimmune travelers and immigrants from sub-Saharan Africa. The findings are of high clinical relevance for the acute management of malaria in travelers; there is also an urgent need for awareness and further investigations of these risk factors in malaria-endemic areas.This is, to our knowledge, the first study investigating the impact of NCDs on severity of Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Wyss_etal_CID_85276_Supplementary_Material_cleanClick here for additional data file.


Plasmodium falciparum and Plasmodium vivax in the coastal and Amazonian regions. This success in malaria control in recent years has led Ecuador to transition its malaria policy from control to elimination.The recent scale-up in malaria control measures in Latin America has resulted in a significant decrease in the number of reported cases in several countries including Ecuador, where it presented a low malaria incidence in recent years (558 reported cases in 2015) with occasional outbreaks of both P. vivax and 0.6% P. falciparum), much higher than expected and comparable to that reported in endemic areas of neighbouring countries with higher malaria transmission. Serology using ELISA and immunofluorescence indicated 27% respondents for P. vivax and 22% respondents for P. falciparum.This study evaluated the general knowledge, attitude and practices (KAP) about malaria, as well as its prevalence in four communities of an endemic area in northwest Ecuador. A total of 258 interviews to assess KAP in the community indicated that most people in the study area have a basic knowledge about the disease but did not use to contribute to its control. Six hundred and forty-eight blood samples were collected and analysed by thick blood smear and real-time PCR. In addition, the distribution of the infections was mapped in the study communities. Although, no parasites were found by microscopy, by PCR the total malaria prevalence was 7.5% (6.9% Results suggest that despite a great malaria reduction in Ecuador, transition from control to elimination would demand further improvement in malaria diagnostics, including active case detection to identify and treat parasite asymptomatic carriers, as well as community participation in its elimination. Plasmodium falciparum) and chloroquine + primaquine (Plasmodium vivax). Malaria surveillance activities are focused in the Northwest coast and in the Amazon regions, the current endemic areas [Malaria is endemic in the coastal and Amazon regions of Ecuador in areas below 1500 m of altitude, however, the number of malaria cases has decreased >99% since 2001 and only 558 cases were reported in 2015  reported only ~20 cases [Presently transmission is low and unstable with periodic outbreaks of both he Andes , 5. The das city . In cont20 cases . In 2014Anopheles albimanus is considered the main vector species in northern Ecuador and southwest Colombia [Despite the low transmission, several challenges remain for Ecuador to progress towards the elimination goal. Major challenges are the accurate estimation of malaria prevalence, particularly in view of the number of asymptomatic cases potentially harbouring low levels of parasitaemia not detectable by microscopic examination and the instability of malaria control activities. As in many other regions, microscopic examination of thick blood smears is the gold standard for malaria diagnosis due to its good performance to confirm symptomatic infections. However, many infections have been shown to be submicroscopic and asymptomatic , leadingColombia , the knoColombia , 16. TheThe aim of this study was to better understand the epidemiological situation in the northern region of Ecuador, in view of the current malaria elimination plans. Four communities of Esmeraldas province were investigated by using sociodemographic and epidemiological surveys.Plasmodium vivax and P. falciparum are both transmitted in different proportions in these regions, which display an unstable transmission pattern. Pedregal is a semi-urban neighbourhood of the city of San Lorenzo, has a population of 270 inhabitants, and is located at sea level. Most inhabitants are described as African–Ecuadorians. The predominant malaria parasite species in this area is P. falciparum (six cases in 2013). Ricaurte is a semi-urban community in the Tululbí River 15 km southeast of El Pedregal; it has a population of 1300 inhabitants. La Boca is a rural community located 10 km southeast of Ricaurte at sea level, in the intersection of the Bogotá and Tululbí rivers; it has a population of 580 inhabitants predominantly African-Ecuadorian; El Guadual is a rural community located 40 km southeast of La Boca in the common border of Esmeraldas, Carchi, and Imbabura provinces; it is located at a higher altitude (600 m.a.s.l) and it has a population of 250 inhabitants predominantly mestizo; sporadic cases of P. vivax and P. falciparum have been previously reported in this community. A summary of the geographic and demographic characteristics of the study sites is shown in Table Four endemic settings in Ecuador’s San Lorenzo canton in Esmeraldas province were selected because of the relatively higher malaria prevalence, as compared to other parts of the country: El Pedregal, Ricaurte, La Boca, and El Guadual. All are located close to the road between Ibarra and San Lorenzo city Fig. . PlasmodA total of 649 individuals living in 258 houses of the study communities were studied. Blood samples were randomly chosen from residents of the four communities using the software Epidat to obtain a 95% confidence. Samples were collected in El Pedregal (177), Ricaurte (231), La Boca (165), and El Guadual (73). The blood was collected by finger prick and spotted on filter paper (Whatman 3MM) from inhabitants aged 2–70 years old. In addition, thin and thick blood smears were taken from each individual. All houses where georeferenced and geo-localized in a map.A simple KAP questionnaire was designed, approved by the Ethical Review Committees and used to survey a sample of the population. The questionnaire had a total of 13 questions: four questions of general knowledge about malaria transmission, three about prevention of malaria and six about malaria history.The study protocol was approved by the Ethical Review Committees of Pontificia Universidad Catolica del Ecuador (# CBE-016-2013). Written informed consent (IC) was provided by study participants and/or their legal guardians (informed assent) for both the KAP survey and sample collection.Plasmodium species confirmation.The thin and thick blood smears taken from each individual were stained by the standard Giemsa method and used to confirm malaria infection . ParasitP. falciparum and P. vivax DNA positive and negative controls were used in each batch of tests including the extraction of both negative and inhibition control. A sample was considered negative if there was no increase in the fluorescent signal after a minimum of 40 cycles. Parasitaemia quantification was performed using a parasite specific standard curve made with serial blood dilutions of a reference field isolate. Each reaction plate included a standard curve for parasite quantification and positive results were independently confirmed. All positive results were reported to the local health authorities.DNA was extracted using the PureLink Genomic DNA Mini Kit , and real time PCR was performed as previously described , 18, 19,The serum was extracted from blood samples on filter paper as previously described . The driP. falciparum or P. vivax infected red blood cells were used to produce antigen slides, which were processed as previously described [P. vivax and P. falciparum antigens were used as follows: for P. vivax a long synthetic peptide corresponding to the amino terminus of the circumsporozoite (CS) protein (CS-N) and an amino terminal recombinant fragment (r200L) of the merozoite surface protein 1 (MSP-1); for P. falciparum a synthetic fragment corresponding to the amino flank (CS-N fragment) was used. These antigens have been previously shown to be highly immunogenic [P. vivax and asexual blood stages antigens of both P. vivax and P. falciparum were determined by ELISA as described elsewhere [For IFAT, escribed . Serum sunogenic –25. IgG lsewhere . BrieflyStudy data were collected and managed using REDCap electronic data capture tools hosted at the Caucaseco Research Center, Cali, Colombia . REDCap 1; X2, …, Xn) drawn from some unknown distribution f(y), where y is a geographical coordinate. Xi1 is the latitude of the sample and Xi2 is the longitude of the sample. To estimate the shape of this distribution f, we used a Gaussian kernel with bandwidth h1 of 0.0005 longitude and h2 of 0.0005 latitude. Where the density of the function f(y) is estimated using the following expression:To generate a smooth representation of the discrete malaria case data collected during the study, a Gaussian kernel estimator was used to produce a population density map, a disease incidence map and a disease risk map per each village in the study. The population data of each village was smoothed using a Gaussian kernel estimator where the surveyed population data per household were treated as samples . Regarding malaria prevention practices, >90% of the surveyed families identified the use of bed nets and closing doors and windows as the main activities to prevent malaria, while other activities such as use of mosquito repellent and insecticide-treated bed nets were not as commonly recognized except by families in El Pedregal (P = 0.04). A small proportion of the families (10–30%) indicated to take actions against malaria outside of the house like cleaning grass and bushes.The general malaria KAP survey was performed on a sample of the population (n = 258 houses), and one person was surveyed in each house. In terms of knowledge, it was found that only a small percentage of the population (5–10%) see malaria as the main health problem that affects their communities when compared to other diseases such as respiratory diseases. In addition, 50–75% of the surveyed people in the communities declared to know how malaria is transmitted and 90–100% knew that a mosquito is responsible for malaria transmission. Likewise, most surveyed people (60–75%) correctly identified the main symptoms of malaria such as fever and headache. Only in El Guadual, most families identified chills as an important symptom. This was significantly different from the other three communities .The most prevalent parasite was P. vivax or P. falciparum. Specifically, 23.08% of volunteers presented antibodies against PvCS-N, 27.23% against PvMSP1 (Pvr200L) and 21.69% against PfCSN. The community with a higher percentage of responders for all three antigens was El Pedregal , followed by La Boca , and Ricaurte . El Guadual had the lowest percentage of responders against two of the three antigens  . In La Boca there was a high concentration of malaria in places where the population density was low, thus clearly delineating an area of high risk Fig. ; this coEcuador has experienced ~99% reduction in malaria clinical cases in the last 15 years and is now pointing towards an elimination phase. Despite periodic focal outbreaks, the country has shown a malaria transmission decreasing trend. In 2014, Ecuador reported the lowest malaria incidence of the last decade but in 2016 the reported incidence (917 cases), almost doubled that of the previous year. The study indicated the presence of a significant number of asymptomatic malaria parasite carriers in the study sites, with parasitaemia detectable only by qPCR which is probably an indication of an official under reporting of cases.The periodic outbreaks, particularly the most recent one, appear to depend on several features. First, as most countries do, the National Malaria Control Programme (NMCP) of Ecuador uses microscopy and more recently RDT for malaria diagnosis. Although these two methods have great value for passive case detection of symptomatic cases, they show very limited sensitivity to detect asymptomatic cases which frequently display submicroscopic parasitaemia levels. Second, the intense population mobility from and to Colombia, may contribute to frequent reintroductions of malaria parasites. These and other issues may slow down the elimination process.P. vivax (6.9%). The study also indicated a good level of malaria knowledge in the study communities, however, the surveyed population declared not to be participating significantly in control activities, even though the contribution of the endemic communities as well as that of several other malaria stakeholders to malaria elimination is considered essential.In the last several years there has been an increasing number of studies indicating the presence of asymptomatic submicroscopic malaria cases in all endemic areas of the world; unfortunately, in Ecuador there is a lack of studies addressing the malaria prevalence with more sensitive methods. Indeed, in contrast with the official records, this study confirmed a prevalence of asymptomatic infections between 2 and 13% in the different study sites, greater than expected and with a higher rate of Despite the fact that moving from malaria control to elimination, may appear as simple as intensifying control activities, elimination requires a significant sustained effort with critical strategic programme changes to ensure the goal of reaching zero cases.Anopheles as suggested by previous studies [Because the NMCP does not usually seek identification of asymptomatic infections, they frequently remain undiagnosed, and even if they are searched, thick smears have limited sensitivity. Furthermore, there is no policy about treatment of these infections, and therefore asymptomatic carriers may be contributing to maintaining the parasite transmission to  studies , 9, 26.  studies , but com studies  and the  studies , suggest studies . These r studies . In thisOn the other hand, the presence of asymptomatic cases indirectly indicates that despite the low transmission intensity the community has developed substantial levels of clinical immunity, even though overall, less than one-third of the population displayed positive titers to both parasite species. Even in El Pedregal where the highest reactivity was recorded, less than half of the study population was positive. The seroreactivity found was in general low, but the frequency of seropositive individuals appeared to correlate with the malaria prevalence, it was higher in El Pedregal which displayed greater number of submicroscopic infections. While this study used a basic serological approach, more information could have been obtained from an analysis of age dependent antibody response and IFAT in all the studied population.P. falciparum cases, in this study, P. vivax was by far the most prevalent parasite . In this province only four cases of P. vivax were reported in 2013 [P. vivax infections only detectable by q PCR, and is in contrast with a greater prevalence of P. falciparum reported in southern Colombia during the same period [P. vivax infections at low parasitaemia and develop immunity against this parasite [P. vivax in El Pedregal and La Boca.It is surprising that, although Esmeraldas province is the only area of Ecuador still regularly reporting e period , 28. A pparasite , consistP. vivax in an area where it is rarely reported brings new challenges from the treatment point of view. Ecuador uses a radical cure treatment that includes a 3-day chloroquine course and a 7 day primaquine treatment. The need for primaquine treatment for P. vivax, in an area not previously reported, will have important consequences in a possible elimination setting, considering that the status of glucose-6-phosphate dehydrogenase (G6PD) deficiencies is uncertain. Even though P. vivax treatment has been performed without G6PD deficiency testing in Ecuador, models predict that the north of the country, an area where P. vivax is rarely reported has a higher prevalence of G6PD deficiency than other areas where vivax infections are more common [P. vivax patients will need to go hand in hand with the determination of the G6PD status for each patient.The high prevalence of e common . As elimThe communities of El Pedregal, Ricaurte and La Boca with a greater historical exposure to malaria presented better knowledge about malaria but comparatively lower than in the adjacent coast of Colombia . In contAlthough most people had a basic knowledge about malaria, practices to prevent malaria transmission were not very common. However, geo-referencing of malaria cases allowed mapping specific disease foci which facilitate targeting by control and elimination activities including education and community participation.P. vivax asymptomatic and submicroscopic infections. Although this may be due to significant clinical immunity, it evidences diagnosis challenges for the NMCP and in addition may be contributing to the maintenance of transmission. The elimination goal in Ecuador would imply important changes including more sensitive diagnostic methods and active case detection, and probably community participation to eliminate mosquito breeding places among others.Taken together, the results of this study demonstrate higher malaria prevalence than expected in the north coast of Ecuador that may be related to environmental factors, with most cases corresponding to


Plasmodium falciparum prevalence (PfPR) is a widely used metric for assessing malaria transmission intensity. This study was carried out concurrently with the RTS,S/AS01 candidate malaria vaccine Phase III trial and estimated PfPR over ≤ 4 standardized cross-sectional surveys.PfPR was estimated by site and age category.This epidemiology study (NCT01190202) was conducted in 8 sites from 6 countries , between March 2011 and December 2013. Participants were enrolled in a 2:1:1 ratio according to age category: 6 months–4 years, 5–19 years, and ≥ 20 years, respectively, per year and per centre. All sites carried out surveys 1–3 while survey 4 was conducted only in 3 sites. Surveys were usually performed during the peak malaria parasite transmission season, in one home visit, when medical history and malaria risk factors/prevention measures were collected, and a blood sample taken for rapid diagnostic test, microscopy, and haemoglobin measurement. PfPR was recorded for Nanoro, Burkina Faso (52.5%). PfPR significantly decreased over the 3 years in Agogo (Ghana), Kombewa (Kenya), Lilongwe , and Bagamoyo (Tanzania), and a trend for increased PfPR was observed over the 4 surveys for Kintampo, Ghana. Over the 4 surveys, for all sites, PfPR was predominantly higher in the 5–19 years group than in the other age categories. Occurrence of fever and anaemia was associated with high P. falciparum parasitaemia. Univariate analyses showed a significant association of anti-malarial treatment in 4 surveys  and bed net use in 2 surveys  with lower risk of malaria infection.Overall, 6401 (survey 1), 6411 (survey 2), 6400 (survey 3), and 2399 (survey 4) individuals were included in the analyses. In the 6 months–4 years age group, the lowest prevalence (assessed using microscopy) was observed in 2 Tanzanian centres (4.6% for Korogwe and 9.95% for Bagamoyo) and Lambaréné, Gabon (6.0%), while the highest PfPR differed substantially between sites and age groups. In children 6 months–4 years old, a significant decrease in prevalence over the 3 years was observed in 4 out of the 8 study sites.Local Trial registration Clinical Trials.gov identifier: NCT01190202:NCT. GSK Study ID numbers: 114001The online version of this article (doi:10.1186/s12936-017-2078-3) contains supplementary material, which is available to authorized users. Plasmodium falciparum prevalence (PfPR), defined as the prevalence of asexual blood stage infection in human hosts. PfPR is a widely accepted and long-standing measurement of the level of malaria risk in a community, with proven biological, epidemiological and statistical properties and consistent with the historical hypo-, meso- and hyper-endemic malaria categorizations [PfPR may vary seasonally, due to variable ecological conditions, and is influenced by the population immunity against malaria [The burden of malaria remains the highest in sub-Saharan Africa, with 90% of cases and 92% of malaria deaths occurring in this region alone in 2015 . An effeizations , 5. PfPR malaria .PfPR, has decreased to almost half and this was correlated with a 40% decrease in the incidence of clinical disease [Between 2000 and 2015, malaria transmission in Africa, as assessed by  disease . This wa disease .PfPR using microscopy, over a period of 3 years in sites from several sub-Saharan African countries, concurrently with and in the same environs as (but independently from) a Phase III clinical trial of the GSK’s candidate malaria vaccine RTS,S/AS01 [This study was designed to evaluate estimates of S,S/AS01 –11. AssoS,S/AS01 . Study mS,S/AS01 .This multicentre, epidemiology study was conducted between March 2011 and December 2013, and comprised up to 4 cross-sectional surveys. Among the 11 sites (distributed in 7 countries) which implemented the phase III trial of the candidate malaria vaccine RTS,S/AS01, 8 centres in Burkina Faso, Gabon, Ghana, Kenya, Malawi, and Tanzania agreed to participate and were included in this study. Five centres conducted 3 surveys and 3 centres  conducted 4 surveys, with the first 2 surveys carried out between March 2011 and January 2012  or households (for the latter) were randomly selected according to a computer-generated list. For each selected child ≥ 6 months to < 5 years old (6 months–4 years), another participant from the same household was considered for enrolment: either an individual 5–19 years old (5–19 years) or aged 20 years or more (≥ 20 years). To allow for a 10% non-response rate, 440 children 6 months–4 years were selected from the demographic surveillance system or, when unavailable, 660 houses were selected from enumeration lists, assuming 65–70% of houses will have children 6 months–4 years of age. To minimize sampling bias, 3 attempts were made to establish contact with a household before replacing it. If more than 1 child aged 6 months–4 years lived in the house, 1 was randomly selected.http://www.gsk-clinicalstudyregister.com (ID: 114001).The trial protocol was approved by the ethical review board and national regulatory authorities at each study site and partner institutions. The survey was undertaken in accordance with the Good Clinical Practice Guidelines . DependiDemographic information, medical history, socio-economic factors, and environmental and ecological factors were collected at each survey, during home visits. A blood sample was collected by finger prick for rapid diagnostic test, thin and thick blood film, haemoglobin measurement, and filter paper blood spot. Blood smears were examined by two independent microscopists, and discrepancies were settled by a third reader. Parasite density was estimated by examination of 100 high powered fields, either against measured blood volume  or againParasite density (parasites/μl) was defined as the geometric mean of 2 blood smear-reading values if the participant status was defined as positive. The parasite densities were grouped into the following predefined classes: low (< 2500 parasites/μl); medium (2500–9999 parasites/µl), high , and very high . Fever was defined as an axillary temperature ≥ 37.5 °C, or a history of fever reported in the 24 h prior to each survey. Anaemia was considered as non-severe for haemoglobin when between 7 g/dl and < 11–13 g/dl, depending on the age group , gender and pregnancy status, and severe for a haemoglobin cut-off of < 7 g/dl, regardless of age and gender , 19. SerThe sample size was computed for each centre separately and used to estimate point prevalence of infection among children in the 6 months–4 years group during one cross-sectional survey. The effective sample size was computed to obtain an estimation of the prevalence of infection with a relative standard error ≤ 0.25. To meet this criterion of precision, the effective sample size was estimated at 400 eligible participants per study centre. For participants in the 5–19 years or ≥ 20 years groups, a relative standard error ≤ 0.35 was considered acceptable, as the sample size of 200 eligible participants in both age categories per centre was assumed to be sufficient.PfPR and serology data.Analyses were carried out on data from all evaluable participants, i.e. those meeting all eligibility criteria, complying with protocol procedures and with available laboratory blood sample for P. falciparum infection.Descriptive analyses were conducted for demographic and medical history characteristics, socio-economic factors and environmental and ecological factors. For each centre, unadjusted odds ratios (OR) with 95% confidence intervals (CIs) were computed for the association of malaria control measure or risk factor with the risk of PfPR was conducted by year, site, age group and year of age for children younger than 5 years. Pooled prevalence across centres was done by age class using generalized estimating equations in order to take into account the correlation between participants from each site/country [The analysis of /country , 22, andUnivariate analysis using logistic regression was conducted to determine the association between infection and anaemia, malaria control measures, or malaria risk factors. ORs  were estimated in the entire population for each centre separately and also adjusted with the centre as a random effect.Statistical analyses were performed using SAS version 9.2.The number of participants enrolled and reasons for exclusion from analyses are presented in Fig. Additional file PfPR varied substantially between centres. No consistent patterns of change over the 3 years could be identified across age groups .In the 6 months–4 years age group, the estimated pooled PfPR was recorded in Nanoro (52.5%). In this age group and at the time of survey 3, the infection prevalence significantly decreased in Agogo (Ghana), Kombewa (Kenya), Lilongwe , and Bagamoyo (Tanzania), as shown by non-overlapping 95% CI. A trend for decreased PfPR was observed over the last 3 surveys for Nanoro, while in Kintampo, PfPR tended to increase over the last 3 surveys  and Lambaréné, Gabon (6.0%). The highest PfPRs were higher in the 5–19 years age group compared to the 6 months–4 years group. The lowest PfPRs in individuals aged 5–19 years were recorded at the 2 Tanzanian sites, for which a significant decrease was documented from survey 1 to survey 2. The highest values were observed for Nanoro, with a PfPR of 59.1% at survey 1 and 70.7% at survey 4, but showing a decrease if only the last 3 surveys were considered, with a peak at survey 2 (PfPR = 81.8%). PfPR was also high in Kombewa, varying from 42.0% (survey 2) to 54.2% (survey 3) over the 3 surveys conducted. A trend for increasing PfPR was observed for Kintampo, while PfPR decreased in Lilongwe over the study period  to surveys 2 (32.0%) and 3 (42.7%), but PfPR decreased again to 25.5% at the time the fourth survey was conducted. High prevalence in adults was also observed in Kintampo, and was relatively stable over the years (between 11.1 and 17.7%), while a trend for decreasing PfPR was observed for Bagamoyo  versus 13.1% (95% CI 12.2–14.0) for survey 1, 17.7% (95% CI 15.6–20.0) versus 12.3% (95% CI 11.4–13.2) for survey 2, 23.8% (95% CI 21.5–26.2) versus 12.1% (95% CI 11.2–13.0) for survey 3, and 21.5% (95% CI 18.6–24.7) versus 9.6% (95% CI 8.3–11.1) for survey 4. The same trend was observed in each age group  tended to be higher in children with higher parasite density, although this differed by site , with estimated centre adjusted ORs of 1.89 (95% CI 1.47–2.35) for survey 1, 1.64 (95% CI 1.40–1.90) for survey 2, 1.61 (95% CI 1.19–2.19) for survey 3, and 1.76 (95% CI 1.40–2.19) for survey 4. ORs varied by site and study survey. For survey 3, infection was not associated with anaemia in Bagamoyo, Tanzania . For all other sites and surveys, OR values ranged between 1.21 (95% CI 0.89–1.65) and 4.08 (95% CI 0.88–18.98) , Lilongwe  and Bagamayo (Tanzania). The use of anti-malarial treatment was associated with reduced odds of P. falciparum infection for all sites.This study showed that, among regions where the RTS,S/AS01 Phase III clinical trial was implemented, observed , a signiPfPR observed in Burkina Faso is not surprising and is in line with previous reports [PfPR in the younger 6 months–5 years age group compared with children over 5 years of age reported in Nouna [PfPR over time was observed when the surveys at the same 3 time points were considered, in line with reports of a consistently decreased prevalence between 2009 and 2011, compared to 2000 [PfPR values in Agogo than in Kintampo were consistently documented in our study. This difference is not surprising, as marked heterogeneity of P. falciparum infection was previously reported [The higher  reports . In Noun reports , similarin Nouna . A decli to 2000 . A high  to 2000 , althougreported .P. falciparum prevalence noted in the Tanzanian centres is in agreement with recent estimations of PfPR by rapid diagnostic test in a community survey [PfPRs compared fairly well with the P. falciparum infection prevalence detected microscopically from a study conducted in southern Malawi from 2012 to 2014, although larger values were reported in the same study when real-time polymerase chain reaction (PCR) was used for parasite detection [PfPR was lower in the 6 months–5 years age group than in the 5–19 years one, which is consistent with previous observation of an increase in age of the malaria high-risk population in Gabon between 2008 and 2011 [The lower y survey , and repy survey . Lilongwetection . In Lamband 2011 .PfPRs’ heterogeneity across the 8 study sites represents well the variability of transmission intensity in sub-Saharan Africa [Ultimately, the n Africa , 33, andn Africa .P. falciparum endemic areas, including Africa [Differences across centres were also observed in relation to the use of anti-malarials and control measures, but they are also likely to be associated with other extrinsic factors such as social, behavioural, economic and local policies. Previous anti-malarial treatment  and use of bed nets (in ≥ 2 surveys) was associated with a lower risk of infection for the majority of the sites. During the study, the proportion of participants having taken malaria treatment varied widely in centres with high malaria prevalence; the lowest recorded use of malaria treatment was in Nanoro, Burkina Faso (0.5–2.5% of participants) and the highest in Kintampo, Ghana (up to 21.9% of participants). Of note, to our knowledge, Seasonal Malaria Chemoprevention measures as recommended by the World Health Organization had not been implemented at the time the study ended in any of the study sites . This fig Africa .P. falciparum prevalence remained high, especially in school-aged children [Bed net use (on the night before the survey) was high in all sites throughout the 4 years and was associated with reduced risk of infection. Although recent data confirm the effectiveness of ITNs , reportschildren .Increased use of ITNs and IRS has already been associated with a reduction in malaria burden through a population effect, and the fact that the results of these measures have varied across regions –47 and bP. falciparum-infection in this study. OR analyses confirmed P. falciparum infection to be a significant risk factor for anaemia, with ORs ranging from 1.21 to 4.08 across all sites and surveys (notwithstanding the negative association found in Bagamoyo at survey 3), consistent with results from other studies [As expected, both anaemia and fever were associated with  studies –51.PfPR for all surveys and age groups, seems to be in line with previous findings in Burkina Faso, with 85–90% of parasite-positive individuals showing no fever [PfPR for the 6 months–4 years age group , fever prevalence was also substantial for low to medium parasite densities . These results show that a lower parasite density was associated with fever at sites where PfPR (and therefore MTI) was lower, as children in regions with moderate-to-high levels of MTI may develop partial immunity to malaria more easily and at an early age, manifested through a decrease in symptoms [Overall, fever was associated with high parasite densities . There was a trend towards a higher association between fever and parasite density in the youngest age group, but results varied widely across centres. The lower fever prevalence observed in Nanoro, the site with the highest overall no fever . Howeversymptoms , 52. Of symptoms .A number of key factors contributed to the quality and reliability of results presented in this paper. Standard methodologies were used across centres to ensure comparability of results and to make certain that sampling was representative across catchment areas which participated in the Phase III trial. This allowed for a surplus in the number of randomly selected households in order to take into account non-response and to minimize sampling bias. However, the sampling followed a non-probability method, as the targeted population represented a convenience sample, and therefore, generalization of the results should be made with caution.P. falciparum prevalence was assessed microscopically, a method which underestimates the true level of infection when compared with molecular (PCR) detection [The study has several limitations. Four surveys were conducted only in 3 study sites, as the study ended in December 2013, along with the Phase III trial, and at that time, 5 centres had not conducted the fourth survey. For some centres, the first survey was carried out outside the peak transmission season. etection , 54. ThePfPR for the 4 study years provided a similar ranking as when using incidence of clinical malaria, measured in control infants 6–12 weeks of age at enrollment in the Phase III study during 12 months of follow-up [PfPR was Nanoro, for which the highest malaria incidence was observed in the Phase III trial, while the lowest values were documented for Korogwe in both studies. The order of the other sites also compared fairly between PfPR and clinical incidence of malaria, suggesting that the control group could serve as a surrogate for relative malaria parasite transmission intensity. The data in our study contribute to an improved interpretation of site variations in the Phase III vaccine efficacy study of RTS,S/AS01.Malaria control intervention may have a different effect and/or impact on malaria morbidity at different levels of MTI. Therefore, the assessment of MTI in parallel to efficacy trials for new malaria control interventions is essential for the generalization of trial results to other settings and to guide their implementation –58. Whenollow-up , 11, espAdditional file 1. Data for 5–19 and ≥ 20 years age categories.Additional file 2. Malaria rapid diagnostic test results and prevalence of infections with other parasites.Additional file 3. Odds ratios analysis of P. falciparum infection in relation to reported usage of anti-malarial treatment and prevention.


Treatment decisions by CHWs were validated by microscopy on a reference blood slide collected at the time of consultation, to compare the proportion of children <5 years receiving appropriately targeted ACT treatment, defined as patients with microscopically‐confirmed presence of parasites in a peripheral blood smear receiving artemether‐lumefantrine or rectal artesunate, and patients with no malaria parasites not given ACT.Cluster‐randomized trials were conducted in two contrasting areas of moderate‐to‐high and low malaria transmission in rural Uganda. Each trial examined the effectiveness of ACT treatment was appropriately targeted in 79.3% (520/656) of children seen by CHWs using mRDTs to diagnose malaria, vs. 30.8% (215/699) of children seen by CHWs using presumptive diagnosis (P < 0.001). In the low transmission area, 90.1% (363/403) children seen by CHWs using mRDTs received appropriately targeted ACT treatment vs. 7.8% (64/817) seen by CHWs using presumptive diagnosis (P < 0.001). Low mRDT sensitivity in children with low‐density parasitaemia (<200 parasites/μl) was identified as a potential concern.In the moderate‐to‐high transmission area, mRDTs, ACT treatments delivered by CHWs are more accurately targeted to children with malaria parasites. mRDT use could play an important role in reducing overdiagnosis of malaria and improving fever case management within iCCM, in both moderate‐to‐high and low transmission areas. Nonetheless, missed treatments due to the low sensitivity of current mRDTs in patients with low parasite density are a concern. For community‐based treatment in areas of low transmission and/or non‐immune populations, presumptive treatment of all fevers as malaria may be advisable, until more sensitive diagnostic assays, suitable for routine use by CHWs in remote settings, become available.When equipped with  The study took place in two different malaria transmission settings in rural Uganda to provide evidence to optimise use of ACTs and mRDTs within programmes of integrated community case management (iCCM) that are currently being scaled up in Uganda and other countries.Early diagnosis and effective case management is a central component of the current malaria control strategy The study was conducted in Rukungiri District, south‐western Uganda, inhabited by the Bahororo and Bakiga ethnic groups, whose main occupation is subsistence farming. Anaemia among under‐fives in south‐western Uganda is estimated at 40%, and in other regions of the country, it ranges from 57% to 74% All villages in the two subcounties were invited to participate in the trial: 63 villages in Bwambara, ranging between 981–1203 m, and 64 villages in Nyakishenyi, ranging between 1064–2157 m. A public meeting was held in each eligible community to explain the purpose of the research, and community members were asked to identify 2–3 CHWs per village for training. Written informed consent to take part in the trial was sought from community leaders and all CHWs selected for training, of whom more than 50% had previously worked in the home‐based management of fever (HBMF) programme that was defunct at the time of the study.A cluster‐randomized two‐arm trial design was used to compare the effectiveness of mRDTs used by CHWs to diagnose and treat malaria with ACT [intervention] to presumptive diagnosis and treatment of malaria with ACT [control]. The unit of randomisation was the village, with all CHWs in the same village receiving the same type of training. Within each transmission area (subcounty), villages were randomly allocated, using a random number table in Epi Info, to either the intervention (mRDT) or control (presumptive treatment) arm.Community health workers in both arms of the trial were trained in recognising signs and symptoms of malaria, how to administer antimalarial treatment and when to refer Figure , Table 1www.actconsortium.org/RDThomemanagement). Training workshops lasted 3 days for CHWs in the control arm and 4 days for CHWs in the mRDT intervention arm. The latter received an additional 1‐day training to cover the rationale for diagnostic testing in febrile patients, performing an mRDT and interpretation of the test result based on training materials from WHO Participatory training workshops in malaria case management were held for CHWs in each arm, based on a trainer's manual and accompanying set of pictorial job aids . The key messages were that not all fevers are malaria and hence a diagnostic test was advisable before treatment with ACT; and a quick test (mRDT) could test for malaria, and these tests were available from government health facilities and trained CHWs in the intervention villages.For the first 6 months of implementation (July–December 2010), CHWs in both arms received additional close support supervision by project staff through parish‐level CHW meetings to assist adoption of the new procedures into everyday practice, to promote accurate and complete record keeping and to give advice on how to handle difficult situations that could affect adherence, for example caretakers insisting on treatment even after a negative RDT result. At the end of the 6 months, supervision was scaled back to periodic contacts when CHWs came to collect their supplies and a monthly allowance for paraffin and soap intended to support good hygiene and work in the evening/night . CHWs were also provided with pictorial job aids, training certificates, bicycles and T‐shirts at the start of the programme. Although CHWs in Uganda are volunteers, expected to work for free, programmes often provide small ‘refunds’ or tokens, which are generally expected as compensation.Data on diagnosis and treatment were recorded prospectively on all children seeking treatment for fever in carbon‐copy duplicated CHWs' registers, provided for this purpose. The treatment register for CHWs in the control arm was similar to the intervention arm except for data on mRDT result. CHWs in both arms were trained on how to prepare a thick blood film for every child with fever who consulted them, for later microscopy, subject to verbal assent given by the caretaker accompanying the child. Treatment decisions made by CHWs were validated by light microscopy on the reference blood slide collected at the time of consultation, to compare appropriate targeting of ACTs between arms over a 12‐month period (January–December 2011). The primary endpoint was the proportion of febrile children with malaria receiving appropriately targeted treatment with a first‐line antimalarial, a composite outcome defined as: children with microscopically‐confirmed presence of parasites in a peripheral blood smear (slide positive) who received either artemether–lumefantrine or rectal artesunate, and febrile children with no malaria parasites (slide negative) who did not receive either of these two artemisin‐based treatments . The other co‐primary outcome was prompt effective treatment defined as the proportion of febrile children who received appropriately targeted ACT treatment from a CHW within 24 h of onset of malaria symptoms, while the secondary outcome was over‐prescription defined as the proportion of children not parasite‐positive (slide negative) who received inappropriate ACT treatment from CHW .k = 0.35 in the moderate‐to‐high transmission area during the period of close support supervision, a minimum sample size of 6 patients per cluster (180 patients per arm) was required to demonstrate a 50% increase in appropriately targeted treatment with use of mRDTs from 50% to 75%, with 90% power at the 5% significance level, in a cluster‐randomised trial with 30 clusters per arm. The equivalent sample size in the low transmission area, with 30% malaria prevalence, 32 clusters per arm and inter‐cluster variation k = 0.45, to demonstrate an increase from 30% to 65% was 2 patients per cluster (128 patients per arm). Taking into account the lower proportion of cases seeking treatment within 24 h of onset of symptoms (89% and 83% in each site), sample size requirements to detect a comparable increase in prompt appropriately targeted treatment were 9 patients and 3 patients per cluster (270 and 192 per arm), in the high‐to‐moderate and low transmission areas respectively.Based on an observed prevalence of malaria of 50% and inter‐cluster variation ® Ag. P. falciparum (HRP2) Card test, www.PremierMedCorp.com; intervention arm only) performed. The mRDTs passed independent quality‐assurance batch testing by WHO‐FIND, Institut Pasteur, Cambodia. Thick blood smears were haemolysed and stained with 10% Giemsa for 10 min and examined under light microscopy with oil immersion objective lens (×100). Asexual parasites were counted against 200 white blood cells and parasite densities calculated assuming a standard white blood cell count of 8000/μl blood. A blood smear was declared negative if no parasites were detected in 100 high power fields. Reference slides included in the analysis of the primary endpoint were subjected to two independent readings by light microscopy. The second reading was performed by experienced technicians at Vector Control Division, Ministry of Health in Kampala, blinded to initial results. Any discrepant results between the first and second readings were resolved by a third technician blinded to all previous results. As the number of consultations in Bwambara (moderate‐to‐high transmission area) exceeded the sample size requirements, a subsample of blood slides was randomly selected for analysis of the primary endpoint. To reflect seasonal variation in the prevalence of malaria, two slides were randomly sampled per month from each village during the main evaluation period from January to December 2011 for double reading . Malaria was less common in Nyakashenyi, with fewer consultations, and thus, all available slides were re‐examined by expert microscopy.A finger‐prick blood sample was collected by the CHW at the time of consultation, a thick blood film for microscopy prepared, and mRDT , after the training and 6‐month period of close support supervision had ended. During 2011, a total of 13 035 children were seen by CHWs in 63 villages in Bwambara subcounty , with 5715 children seen by CHWs in the mRDT intervention arm and 7320 in the control arm  had parasite densities ≤200 parasites per μl . Despite greater delay in seeking treatment in the mRDT arm, the proportion of children who received appropriately targeted treatment within 24 h was also significantly higher in the intervention arm .Among febrile children seen by CHWs in the mRDT intervention arm, 37.5% were treated with either artemether–lumefantrine or rectal artesunate (hereafter referred to collectively as ACT) compared with almost 100% of children seen by CHWs using presumptive diagnosis . The proportion of children who received appropriately targeted treatment within 24 h was also significantly higher in the intervention arm .When compared with malaria status by microscopy, 5.8% of slide‐negative children in the mRDT intervention arm received an ACT, a significant reduction in overprescription compared with control arm, 02 Table . HoweverP < 0.001 in both sites). The effect was more marked in the low transmission area where more than 90% of the patients received appropriately targeted treatment in the mRDT arm. This could be attributable to the combination of high specificity of mRDT in a setting of low and unstable malaria transmission, where nearly 100% of mRDT negatives were negative by light microscopy, and high adherence to mRDT results by CHWs. In contrast, where CHWs followed presumptive diagnosis, more than 90% of patients subsequently found to be slide negative had received an unnecessary ACT treatment. This concurs with previous reports that overuse of ACTs is likely to be higher in areas with low malaria prevalence This study confirms that use of mRDTs by CHWs can reduce the number of ACTs used, as documented in previous studies More than 80% of patients with fever in the intervention and control arms in both transmission settings consulted CHWs for treatment within 24 h after onset of symptoms, which is much higher than has previously been observed at health facilities in Uganda The sensitivity of mRDTs in this study was lower than has been previously observed among CHWs The benefits of using mRDTs to improve targeting of ACTs and limit their overuse need to be weighed against the risks of not treating an infected child The findings presented here underscore the value of mRDT‐based diagnosis of malaria in reducing overuse of ACTs in community case management and should be of particular interest to malaria programme managers and policymakers, nationally, regionally and internationally as countries scale up iCCM