Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals.Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand’s highest burden area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers.Within the studied community, 90.2 % of Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.The online version of this article (doi:10.1186/s12936-016-1393-4) contains supplementary material, which is available to authorized users. Plasmodium species that cause human malaria  are characteristics of malaria epidemiology in the region.In Thailand, control efforts have been highly effective in curbing malaria nationwide , but malThe Government of Thailand aims to achieve malaria elimination by 2030 , thus idOur study site in the Myanmar-border province of Tak, once Thailand’s highest malaria burden region, experienced a drastic reduction in transmission recently , 16, andPlasmodium species found in Thailand was investigated by qPCR. The population’s antibody responses against P. falciparum and P. vivax proteins were profiled to examine the development of antibody acquisition relative to age and season, and the targets of antibody responses in persons with asymptomatic and febrile malaria in an age-related manner.In the present study, the molecular screening and antibody profiling by microarray were expanded to include samples from all ages and three seasonal time points to obtain a more accurate assessment of the current epidemiology of falciparum and vivax malaria in Tak. For blood samples collected during community mass blood surveys and passive case detection activities at the hospital and clinic, the presence of all five P. knowlesi, are known to infect humans in the region [P. vivax and P. falciparum are vastly predominant [The study was conducted in the village Mae Salid Noi  and the town of Mae Tan  in Tak Province, northwestern Thailand along the Thai–Myanmar border [e region , 19, butdominant , 20, 21.n = 485) and August (n = 398) of 2013, and January 2014 (n = 464) from individuals ranging from 0.7 to 92 years of age . The health history, including absence or presence of malaria symptoms as described below, was recorded weekly for each study participant from October 2012 to June 2014. The sample size (n = 1347) enabled us to determine parasite prevalence by qPCR with 2.6 % margin of error, using alpha 0.05.Whole blood samples were collected during three cross-sectional mass blood surveys (MBS) in the study village Mae Salid Noi, in March (n = 67) and August (n = 53) 2013, and January 2014 (n = 177). Age of PCD participants ranged from 0.8 to 84 years old .Additionally, 297 whole blood samples were collected from individuals with suspected malaria during passive case detection (PCD) at the Mae Tan malaria clinic and hospital in March . Samples were centrifuged to separate cellular and plasma fractions, then immediately frozen at −80 °C for shipment to University of California Irvine for analysis. Upon thawing, plasma was removed and stored at −80 °C until use. Total genomic DNA was isolated from 100 μL of pelleted cellular fraction using DNeasy Blood and Tissue kit in the QIAcube automated system , using the Blood and Body Fluid Spin Protocol with Manual Lysis V1. Purified genomic DNA samples were eluted with 200 μL of Buffer AE and kept at −20 °C until use.Plasmodium parasites was performed at University of California Irvine using a two-tier strategy for qPCR consisting of an initial screening of all 1644 samples for the presence of Plasmodium genus-specific products using SYBR Green, followed by TaqMan assays to determine the Plasmodium species in samples positive in the genus-specific assay.Field microscopy was performed by local trained staff who provided the first result, and positive cases were treated per national malaria treatment guidelines. Individuals found positive in subsequent expert microscopy and molecular screenings were not treated. Expert microscopic examination was conducted at Mahidol University by an expert microscopist with over three decades of experience. Molecular detection of Plasmodium-genus screening, primers were designed to hybridize with a region of 18S rRNA gene conserved amongst P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), P. ovale (Po) and P. knowlesi (Pk). The forward primer sequence was 5′-GTATTCAGATGTCAGAGGTG-3′, and the reverse primer was 5′-CCTACTCTTGTCTTAAACTAGT-3′. Amplification was performed in 20 μL reactions containing 2 μL of genomic DNA, 10 μL FastStart SYBR Green qPCR Master Mix , 0.2 μM of each primer and 3 mM MgCl2, in a CFX96 Touch Real-Time PCR Detection System . After initial denaturation at 95 °C for 10 min, 45 cycles of 94 °C for 30 s, 60 °C for 30 s, 68 °C for 1 min were followed by a final step of 95 °C for 10 s and a melting curve from 65 to 95 °C with 0.5 °C increments for 5 s. Samples were considered positive if Cq was smaller than 41 and there was a product with melt peak in the temperature ranging between 74 and 75.5 °C. All assays included positive and negative controls. The detection limit of this method was determined to be 0.05 parasites/μL using serially diluted P. falciparum cultures.For the P. falciparum, P. vivax, P. malariae and P. ovale as described in Rougemont et al. [P. knowlesi as described in Divis et al. [Samples positive in the genus screening were examined in uniplex TaqMan assays using Plasmo 1 and Plasmo 2 primers and species-specific probes for t et al.  and for s et al. , with thPlasmodium DNA by qPCR. Samples collected at Mae Salid Noi village during community MBSs were classified as (1) community malaria (if sample was qPCR-positive for Plasmodium), or (2) community healthy (if sample was qPCR-negative). Samples collected at the hospital/malaria clinic from suspected malaria patients were classified as (3) febrile malaria (if qPCR-positive), or (4) non-malaria illness (if qPCR-negative). Malaria symptomatology is defined as fever (>37.5 °C), fatigue, myalgia, headache and nausea, occurring alone or in combination.Samples were classified into four major categories, according to collection site and presence or absence of P. falciparum and 515 P. vivax polypeptides printed as in vitro transcription translation (IVTT) reactions, that were down-selected from larger microarray studies based on seroreactivity and antigenicity to humans. Gene accession numbers follow annotation published on PlasmoDB [The protein microarray used in this study, named Pf/Pv500 , was described in detail in Baum et al. , and on PlasmoDB . For larFrom the community MBS, 298 plasma samples were probed: 41 community malaria, and 257 community healthy samples, including the corresponding longitudinal samples of community malaria samples, and samples from individuals age-, gender-, and season-matched to the community malaria samples. Of these, 150 samples collected in March 2013 were randomly selected for the seroconversion study, 15 samples for each of 10 age groups. Additionally, 83 samples collected during PCD at the malaria clinic and hospital were probed, including 32 malaria fever and 51 age- and gender-matched non-malaria illness patients. As unexposed controls, 16 samples from healthy blood donors from the United States, with no travel history to malaria endemic regions, were used for serology comparisons. All microarray probing was performed on the same day, and as previously described in Baum et al. .P. falciparum and P. vivax polypeptides on the microarray the following steps were taken: (1) the median background signal of antibody binding to 24 spots of IVTT reaction without DNA template  was calculated for each individual sample; (2) the raw values of antibody binding to P. falciparum and P. vivax polypeptides were divided by their corresponding median NTC value, generating fold-over-control (FOC) values; (3) FOC values were log2-transformed for data normalization. Normalized data was used for statistical analyses and for figure representations of the data. (4) To determine which polypeptides were seroreactive to plasma from the Thai sample cohort, Significance Analysis for Microarrays (SAM) [n = 211) and 16 USA unexposed controls. The test was performed using MeV 4.8.1, with the following parameters: median and 90th percentile of false discovery rate (FDR), 1.2 and 9.3 %, respectively; median and 90th percentile of number of false significant genes, 3.9 and 30.5, respectively. This resulted in 326 polypeptides being considered significantly seroreactive in exposed Thai samples, and all further analyses considered only this set. Individual plasma samples were considered seropositive for a polypeptide if the sample’s signal intensity value was above the upper 99 % confidence interval value of the unexposed control group. Breadth of response was determined by the number of antigens an individual or group of samples were seropositive to, based on the above criteria for seropositivity. For analysis of antibody responses, the non-parametric multiple comparison Steel–Dwass and the Wilcoxon test were used for pairwise comparisons of means, using JMP11.2. Significance tests were 2-sided and set at 0.05 level for type I error. Z-scores were calculated as the number of standard deviations above or below the mean of the unexposed group. Annual rates of seroconversion and seroreversion for the immunogenic polypeptides were calculated using Systat 11  by fitting age-specific seroprevalence data to a reversible catalytic model using the maximum-likelihood method that assumes binomial error distribution: Pt is the proportion of seropositive individuals in each age group t, λ is the annual rate of seroconversion and ρ is the annual rate of reversion to seronegativity. The model was fitted to seroprevalence curves to each seroreactive antigens using ten age groups, shown in Additional file T test for comparison of slopes of linear regression lines was performed as per Zar [For analysis of antibody binding to ys (SAM)  was perfe taken:  the medi per Zar . A two-pPlasmodium parasites was detected by qPCR in 41 (3.0 %) of 1347 blood samples tested. The highest infection rate was observed in March 2013, with 4.3 % positive samples (21/485), and decreased significantly in August to 1.3 % (5/398), and then increased to 3.2 % in January 2014 (15/464). Four individuals had confirmed malaria in repeat samples, 5  and 10  months apart. Of the 41 community malaria cases, three (two children and one adult) (7.3 %) presented fever at sample collection, while 33 (80.5 %) were asymptomatic and had no malaria symptoms or illness complaints during the 5-month period preceding and following sample collection. The remainder five (12.2 %) developed fever 1–4 months after presenting a positive qPCR for Plasmodium parasites.From the community of Mae Salid Noi, DNA of Plasmodium parasites for the samples collected. Amongst samples from the community surveys, P. vivax and P. falciparum were the most commonly detected species, while P. ovale and P. malariae were detected only in mixed-species infections  of 297 samples by qPCR, with the infection rate declining from 19.4 % (13/67) to 11.3 % (6/53) and 7.3 % (13/177) along the March–August–January time line. Most frequently found in these patients were mixed-species infections of 0.8 % of Plasmodium infections in the Mae Salid Noi community was 14 years, with a mean of 22.8 (17.1–28.5); while the median age of febrile malaria patients in the hospital/clinic was 26.5, with a mean of 27.6 (22.6–32.6). The proportion of qPCR-positive samples in the age groups of 11–20, 21–30 and 31–40 years was significantly higher in the hospital/clinic samples than in the community samples, resulting in the higher median age for malaria patients , apical membrane antigen 1 (AMA1), liver stage antigen 1 (LSA1), serine repeat antigens (SERA) 3, 4 and 5; erythrocyte membrane protein 1 (PfEMP1); and P. vivax circumsporozoite protein (CSP), early transcribed membrane proteins (ETRAMP), multiple MSP and SERA proteins, amongst many others were recognized, and detailed seroprevalence rates for these antigens are presented in Additional file P. falciparum and P. vivax for the entire Thai cohort are presented in Table Many well-known antigens, such as several Plasmodium proteins on the array, with the number of antigens recognized by individual samples ranging from 11 to 324, median 124.Compared to the background reactivity of unexposed controls, Thai samples had significantly higher responses to the proteins on the microarray  , children in the youngest age group had the lowest magnitude and breadth of antibody responses, while adults over 50 had the highest; the averaged antibody responses to the 326 Plasmodium proteins increased significantly  in intensity and breadth (with one exception) with the increase of age, as shown in Additional file P. falciparum and P. vivax were acquired differed between the two species, as shown in Fig. For the samples from the community MBS n = 98, childP. vivax antigens (Wilcoxon p < 0.0001); but by the age of 21–30 a switch in the dominant target species is observed, with antibodies against P. falciparum exceeding those against P. vivax, a trend that increased with age. As breadth is partially defined by intensity of the response to individual antigens, a similar trend was observed for the number of antigens recognized by the different age groups , but this difference began to disappear in samples from donors in their second decade of life; from age 21 to over 60, samples showed a broadening of their antibody repertoires that recognized significantly more P. falciparum antigens than P. vivax.Up to 20 years of age, the intensity of response , indicating that the rate of accumulation of antibodies against P. falciparum is faster than against P. vivax in this population.Although the higher intercept values in regression equations of P. falciparum and P. vivax antigens. SCR values for P. vivax antigens  were significantly higher than for P. falciparum antigens  (Wilcoxon p = 0.03), indicating that seropositive rates to P. vivax tended to start higher at the youngest age and continued relatively high throughout life. Lower SCR values for P. falciparum antigens indicate a low seroprevalence in the youngest groups, but steady acquisition of seropositive states with aging. Loss of antibodies, as determined by the SRR, was higher for vivax antigens  than for falciparum  (p < 0.01), indicating that antibodies generated against P. falciparum were significantly more stable, while antibodies to P. vivax were less stable and were lost at a faster rate than those against P. falciparum.Similarly, calculated annual seroconversion (SCR) and seroreversion (SRR) rates  and adults (>15 years old) from the village using longitudinal samples collected from the same individuals at three time-points, 5 months apart. For confirmed P. falciparum gametocyte-specific protein Pf111 (index 432 and 435), SERA 3 (index 450) and MSP7 (index 466). Plasmodium falciparum erythrocyte membrane protein 1 and SERA 4  showed a significant decay between the time points before and after the rainy season (March 2013 and January 2014). Adults had more stable responses and fewer antigens varied significantly along the seasons , p < 0.0001); while single P. vivax infections were significantly more frequent amongst the asymptomatic cases than in febrile malaria cases (OR 7.35 (2.18–24.73), p = 0.001). Single infections with P. falciparum were found in equal proportions between the two groups (OR 1.74 (0.53–5.73), p = 0.36).Mixed Plasmodium species, i.e., in P. falciparum, in P. vivax and in mixed Pf + Pv infections, regardless of age.The intensity and breadth of their antibody responses are shown in Fig. P. falciparum and 7 P. vivax antigens and their Wilcoxon p values are presented in Additional file In the oldest age group, plasma from febrile malaria patients aged 51 and older showed a drop in intensity of binding significantly below the level of antibody responses by asymptomatic cases. It is only in this group that individual antigens were found to elicit significantly higher antibody responses in asymptomatic carriers than febrile patients. These 21 P. falciparum and P. vivax antigens, and antibodies profiles associated with seasonal changes and with clinical and subclinical malaria.In a pilot study in the village of Mae Salid Noi and town of Mae Tan, a high level of submicroscopic infections was found amongst asymptomatic and symptomatic adults . Now, frPlasmodium infection rates estimated by qPCR were nearly 14-times higher than those estimated by expert microscopy, and despite the overall low parasite prevalence in the population (3.0 %), over 90 % of infections were both asymptomatic and submicroscopic at the time of the survey. This is consistent with the mounting evidence from other low transmission areas in the Amazon, Africa and Southeast Asia, where a previously unrecognized reservoir of Plasmodium infection is rising in the form of asymptomatic submicroscopic parasitaemia [While the detection limit of expert microscopy is approximately 100 parasites per μL , the qPCsitaemia , 28–31. sitaemia , the Malsitaemia .P. falciparum and P. vivax. All mixed-species infections detected by qPCR were misdiagnosed as single-species infections by light microscopy, primarily by not detecting cryptic P. falciparum in the blood smears. The inability to detect and treat malaria infections is a serious threat to the health care of individual patients and to malaria control and elimination. Molecular diagnosis is not practical in the field and hospital or clinic settings, therefore training of hospital technicians and improvement of malaria rapid diagnostic tests are urgently needed, particularly in light of the presence of multidrug-resistant parasites in Thailand [Mixed-species infections were common in both the community survey and passive case surveillance in the hospital and clinic, where 25 % of asymptomatic infections and 68 % of febrile malaria cases were caused by co-infections of primarily Thailand , 34.Notwithstanding the small number of parasitaemic samples found in the three MBSs of the community by qPCR, serological measures indicated exposure to malaria parasites in all individuals tested. However, the youngest children showed limited responses, reacting to a small number of antigens, a serological indication of the success of malaria control efforts in the area because very low antibody titers are expected to correlate with reduced parasite exposure events. These children are an attractive target group for serosurveillance because detection of antibodies in this group, after drastic reduction or elimination of local transmission, indicates resurgence of malaria in a population , 36. SerPlasmodium in the youngest group would render it especially susceptible to malaria infection and disease, and indeed, in this study half of community malaria cases were of persons below 15 years old. However, despite the lack of high titers or broad antibody repertoires against the parasite, the vast majority of the infections were submicroscopic and asymptomatic, and remained so for at least 5 months. This challenges the notion that individuals with little exposure to malaria have limited ability to suppress parasitaemia and would become symptomatic, but is consistent with epidemiological findings from other low transmission areas [P. falciparum in children from Papua New Guinea were not associated with protection from malaria, rather with higher exposure to parasites [It could be expected that the low levels of antibodies against on areas , 39, 40.arasites . The higP. falciparum and P. vivax . AbundaP. vivax . In thise region , a trendPlasmodium has been used to show the success of elimination programmes in several countries [Plasmodium varies between antigens [As transmission levels drop further, serology is being recognized as a valuable tool to monitor malaria epidemiology . Examinaountries –46. As tantigens , 48 and antigens –51 and lantigens –54 compoThe kinetics of antibody responses to hundreds of proteins were explored in the longitudinal cohort, but there was little change at the level of individual markers in the 10 months studied. Children showed the most changes across time, indicative of reinforcement of memory responses or acquisition of new antibodies with time, while there were minimal changes in the adults’ responses. This is similar to what was observed in Kenya, where significant increase in breadth and intensity of antibody response after the malaria season was observed in children below 8 years old, but not in adults . NonethePlasmodium are indicative of higher exposure rather than of protection to malaria [P. falciparum [P. vivax [Another aim of this study was to identify antigenic targets that elicited higher antibody levels in asymptomatic carriers when compared to febrile patients, and that could be suggested as to play a role in protective responses. However, antibody titers were significantly higher in those who experienced clinical symptoms, especially in the younger age groups. Here, it may be once again the case that high levels of antibodies to  malaria . Not onllciparum  and P. vP. vivax . The exhP. vivax . NonetheP. vivax , 60, 61.P. falciparum and mixed species infections, and 90 % of P. vivax infections went unrecognized and untreated. Amongst the febrile cases, microscopy failed to detect 75 % of P. vivax infections; and of the mixed-species infections, all went unrecognized, being misdiagnosed as a single-species infection or completely undetected. Because current surveillance methods focus on case management, malaria transmission cannot be interrupted if asymptomatic infections are infectious to mosquitoes and contribute to transmission [In summary, the findings of the present study have implications for surveillance, control and elimination of malaria in Thailand. In particular, the data suggests children aged 3–15 years old are a sensitive group for serosurveillance monitoring changes in transmission due to malaria interventions. Additionally, in the community 100 % of smission . Currentsmission . The mousmission , 62, 63.Plasmodium infections in Tak is a challenge to Thailand’s efforts to eliminate malaria by 2030. The shortcomings of current diagnostic and treatment methods argues for a urgent change in malaria control approaches, with mass drug administration, serological and high-throughput detection methods needing to be explored as potential next-generation tools for malaria control and elimination in areas of low-transmission.The high prevalence of undetected and untreated


P. vivax and P. falciparum merozoite antigens in individuals living in areas of varying P. vivax endemicity in Pará state, Brazilian Amazon region.Measurement of malaria endemicity is typically based on vector or parasite measures. A complementary approach is the detection of parasite specific IgG antibodies. We determined the antibody levels and seroconversion rates to both P. vivax and P. falciparum was determined in 1,330 individuals. Cross sectional surveys were conducted in the north of Brazil in Anajás, Belém, Goianésia do Pará, Jacareacanga, Itaituba, Trairão, all in the Pará state, and Sucuriju, a free-malaria site in the neighboring state Amapá. Seroprevalence to any P. vivax antigens (MSP1 or AMA-1) was 52.5%, whereas 24.7% of the individuals were seropositive to any P. falciparum antigens (MSP1 or AMA-1). For P. vivax antigens, the seroconversion rates (SCR) ranged from 0.005 (Sucuriju) to 0.201 (Goianésia do Pará), and are strongly correlated to the corresponding Annual Parasite Index (API). We detected two sites with distinct characteristics: Goianésia do Pará where seroprevalence curve does not change with age, and Sucuriju where seroprevalence curve is better described by a model with two SCRs compatible with a decrease in force of infection occurred 14 years ago (from 0.069 to 0.005). For P. falciparum antigens, current SCR estimates varied from 0.002 (Belém) to 0.018 (Goianésia do Pará). We also detected a putative decrease in disease transmission occurred ∼29 years ago in Anajás, Goianésia do Pará, Itaituba, Jacareacanga, and Trairão.The prevalence of antibodies to recombinant antigens from We observed heterogeneity of serological indices across study sites with different endemicity levels and temporal changes in the force of infection in some of the sites. Our study provides further evidence that serology can be used to measure and monitor transmission of both major species of malaria parasite. Plasmodium vivax compared to P. falciparumP. vivax infection has been estimated from 106 to 313 million cases per year across the world P. vivax is currently the most predominant malaria species P. vivax representing 75–80% of these Efforts in mapping malaria transmission have demonstrated a wider geographical distribution of the parasite P. falciparum malaria burden in Brazil, which predominated in the past P. vivax infections. The high frequency of asymptomatic P. vivax carriers in the Brazilian endemic area Plasmodium species. In this setting of high proportion of asymptomatic carriers, it is critical to have in hand good epidemiological tools in order to assess not only the status quo of disease dynamics, but also to monitor any change in disease transmission due to malaria control interventions.The control programs have had a significant impact on In Brazil, variations or changes in malaria transmission have been previously associated with intensive use of land and environmental transformations due to farming, deforestation, or gold mining Plasmodium antigens across different South and Central American countries P. falciparum and/or P. vivax have confirmed these findings in Africa A large sero-epidemiological study, conducted at the end of the 1960's showed an association between malaria endemicity and the prevalence of antibodies against P. vivax infected individuals complicates control significantly and makes statistical inferences over parasite prevalence measures more problematic P. falciparum malaria transmission. However, there is a scarce number of studies aiming to test whether seroprevalence for P. vivax antigens can be an equally good epidemiological tool to measure and monitor changes in malaria transmission rates. With this goal, we have conducted a cross sectional study where we determined the antibody prevalence to P. vivax and P. falciparum for individuals living in areas with varying malaria endemicity levels in Pará state, Brazilian Amazon region.Estimation of malaria transmission is routinely based on vector and parasite measures. The possibility of relapses in The present study was conducted in six municipalities in Pará state in the northern region of the country . GeograpThe samples were collected in ten localities within the municipalities considered to be representative of each area. The number varied from one to three localities per municipality. These municipalities and the corresponding localities were: Anajás city ; Jacareacanga (Porto Rico and São José); Goianésia do Pará ; Itaituba (São Luiz do Tapajós); Trairão (Três Boeiras); and Belém city (Cotijuba island). A malaria-free area, Sucuriju village in Amapá state, was also included in the study. The sampling coverage in relation to total residents of each study site was the following: Anajás (12.0%), Belém (15.4%), Jacareacanga (19.3%), Goianésia do Pará (29.3%), Itaituba (48.0%), Sucuriju (61.0%), and Trairão (81.0%). See This study was carried out in full accordance with all International and Brazilian accepted guidelines as written informed consent was obtained from all participants. The Ethics committee reviewed and approved all the consent procedures. A signed consent form was approved for all adults and provided by parents or guardians for children under 15 years old. For individuals between 16 and 18 years old, the consent procedure was similar to adult participants. This study was approved by the Federal University of Pará (3686/2005) and by the Evandro Chagas Institute (0014/2007 and 0031/2010) Ethics committee.http://portalweb04.saude.gov.br/sivep_malaria) One cross-sectional survey was performed in each study site between 2006 and 2010. The total sample size was 1,330 individuals and ranged from 113 (Anajás) to 262 (Goianésia do Pará) with an average sample size of 190 individuals per municipality. According to the malaria surveillance office of the Brazilian Ministry of Health  and merozoite surface protein 1 (MSP1) using quantitative enzyme-linked immunosorbent assay (ELISA) as described elsewhere P.vivax two recombinant proteins of MSP1 (Belém genotype), both contained the 19 KDa C-terminal portion of MSP1 (MSP119) in fusion with histydine tag (His6) or Glutationa S-transferase (GST) P. falciparum malaria antigens AMA1 (3D7) and MSP119 (Wellcome) P. falciparum and P. vivax, respectively) were added in duplicate to each plate. The sera were tested at a final dilution of 1∶1000 for both P. vivax recombinant proteins, and 1∶1000 and 1∶2000 for P. falciparum MSP119 and AMA1, respectively. The plates were washed and horse-radish peroxidase conjugate rabbit anti-human IgG antibody  was added to all wells at a dilution 1∶5000 in PBS/T. Antibody responses were detected after development with o-phenylenediamine dihydrochloride substrate solution (OPD) for 20 minutes. Reactions were stopped with 25 µl per well of 2M H2SO4. Plates were read immediately at 492 nm on a Molecular Devices Versa Max ELISA reader and optical density (OD) values recorded.All sera were tested for IgG antibodies to recombinant blood-stage Duplicate ELISA OD values were averaged and normalized against the positive control sample on each plate. OD data was then converted to antibody titers, expressed in Arbitrary Units (AU/ml), using a standard curve obtained from an appropriate hyperendemic sera control. Antibody titers were calculated using the formula: titer  =  dilution/[maximum OD/(OD test serum – minimum OD) -1] (33). Seropositivity was determined by fitting a mixture model to normalised OD values. The model assumed two Gaussian distributions, one for sero-negative individuals and another for sero-positive individuals Seroprevalence was stratified into yearly age groups and then analyzed using a reverse catalytic modeling approach under a binomial sampling assumption, as described elsewhere Plasmodium species was calculated for each individual and then log-transformed in order to obtain Gaussian distributions approximately. The corresponding analysis was carried out using the Michaelis-Menten model as available in the ‘vgam’ package for the R software P. falciparum antibody titers from Jacareacanga, we fitted two Michaelis-Menten curves using the age cut-off obtained from the best catalytic model for the corresponding seroprevalence curve.To analyze age-adjusted antibody levels, the highest titer level for antigens to the same Spearman's correlation coefficient. We also used logistic regression to correlate previous malaria exposure with age adjusting for municipality and gender.Finally, a correlation analysis between pairs of different site-specific measures  was performed using the non-parametric All statistical analysis was done in the R software  using our own scripts.−15; logistic regression adjusted for gender, age and municipality) and older individuals are more exposed to malaria than younger ones. There was also a strong correlation between the respective Annual Parasite Index (API) as reported by Brazilian Ministry Health and reported malaria exposure . Data on previous exposure to malaria would appear to reflect the official statistics of the study area.The number of individuals recruited at each site ranged from 113 to 262 . The perP. vivax or P. falciparum antigens, or to either Plasmodium species, also showed variation between municipalities (P.vivax antigens ranged from 19.6% (Belém) to 77.5% (Goianésia do Pará), while seroprevalence for any P. falciparum antigens is estimated to be between 0.8% (Sucuriju) and 59.3% (Jacareacanga). As expected, seroprevalence to any P. vivax antigens was higher than that to any P. falciparum ones in every study site. Parasite prevalence was low and ranging from 0 to 5%. Official API was highly correlated to the seroprevalence to any P. vivax antigen, but only moderately correlated with that to any P. falciparum antigen . This likely reflects the difference between the contributions of P. vivax and P. falciparum to malaria burden, with API representing all malaria cases.Seroprevalence to any palities . SeropreP. vivax seroprevalence, the best model for the data assumes a stable transmission throughout time but with different transmission intensities across sites with the exception of Sucuriju in the Amapá state  . All positive individuals were infected with P. vivax, except for one case in Goianésia do Pará which was infected with P. falciparum  were estimated in different study sites . For P. pá state . SCR estficient) . In the lciparum . There aP. vivax antibody titers also increase with age and then reaching a plateau (P. vivax antibody level plateau is achieved at a younger age (∼20 years old) due to higher malaria transmission and exposure. In the second cluster - Itaituba, Sucuriju, and Belém - the peak antibody level is reached at an older age (∼30 years old), reflecting a low malaria exposure. Similar clustering can be observed using age-adjusted seroprevalence curves  to 1 ib/p/yr except in Jacareacanga where the historical SCR (λ1) suggests an EIR of >20ib/p/yr.All onducted . Historionducted . Using SP. falciparum than in P. vivax antibody data . Our surveys were also based on a convenience sampling approach rather than structured community based sample. This could have led to bias in those captured by the survey due to proximity of sampling location and/or health seeking behaviour when the survey was conducted at health facilities. We have previously conducted surveys for serological analysis using communal meeting points Other limitations are related to the timing, type and intensity of the sampling approach. In this study data collection was performed over a 5 year period for the different sites. Whilst the different transmission estimates can be compared and contrasted, temporal comparison between sites reflecting changes in malaria transmission over time or with age is more complicated. However, the profile likelihood approach to estimate changes in transmission identifies a range of years/age over which transmission has changed. In this case, the estimated standard deviations were low and, therefore, broad estimate of 29 years for a change in In summary, we have used serological data from anti-malaria antibody responses to can describe areas of different malaria endemicity in Pará state, Brazil. We have demonstrated that seroprevalence data shows good correlation with government-derived API and is able to detect changes in malaria transmission in the study sites.Figure S1Theoretical predictions for the relationship between age, mean antibody titers, and seroprevalance.A. Expected relationships between age and antibody titers for any P. vivax antigens using a Michaelis-Menten modelling approach. B. Expected relationships between age and seroprevalence for any P. vivax antigens using appropriate reversible catalytic models (see (TIFF)Click here for additional data file.


Plasmodium vivax is very rarely seen in West Africa, although specific detection methods are not widely applied in the region, and it is now considered to be absent from North Africa. However, this parasite species has recently been reported to account for most malaria cases in Nouakchott, the capital of Mauritania, which is a large country at the interface of sub-Saharan West Africa and the Maghreb region in northwest Africa.To determine the distribution of malaria parasite species throughout Mauritania, malaria cases were sampled in 2012 and 2013 from health facilities in 12 different areas. These sampling sites were located in eight major administrative regions of the country, within different parts of the Sahara and Sahel zones. Blood spots from finger-prick samples of malaria cases were processed to identify parasite DNA by species-specific PCR.P. vivax alone, 296 (62.7 %) Plasmodium falciparum alone, and 13 (2.8 %) had mixed P. falciparum and P. vivax infection. All cases were negative for Plasmodium malariae and Plasmodium ovale. The parasite species distribution showed a broad spectrum, P. vivax being detected at six of the different sites, in five of the country’s major administrative regions . Most cases in Nouakchott were due to P. vivax, although proportions vary significantly among different health facilities in the city. In the northern town of Zouérat, all cases were due to P. vivax, whereas almost all cases in the south of the country were due to P. falciparum. All P. vivax cases tested were Duffy blood group positive.Out of 472 malaria cases examined, 163 (34.5 %) had P. vivax is recognized to be a widespread cause of malaria in Mauritania, occurring in diverse regions. This should be noted by the World Health Organization, as it has significant implications for diagnosis, treatment and control of malaria in the northwestern part of Africa.It is important that  Plasmodium falciparum [Plasmodium malariae or Plasmodium ovale, while Plasmodium vivax is known to occur in only some areas of the continent [Most malaria cases in Africa are caused by lciparum , with a ontinent , 3. Follontinent , 5. Althontinent , 6, 7. M2, lying between 15 and 27°N latitude, and 5 and 17°W longitude. Malaria is considered to be the third most common cause of clinical presentation to health facilities in the country. Although a large part of the territory is Saharan desert, malaria is most common in areas of Sahel in the south of the country, and has been increasingly seen in Nouakchott, the capital city that contains approximately 25 % of the country’s inhabitants [P. vivax is more commonly seen than P. falciparum in patients with malaria in Nouakchott [P. vivax has been very rarely or never seen at most other sites in West Africa [P. vivax, which is rare among other West African ethnic groups living mostly in the south of the country [Anopheles arabiensis has been facilitated by surface water environments, created by loosely regulated urbanization over recent decades in an area that was originally desert [The country of Mauritania is at the extreme northwestern limit of malaria distribution in Africa. It has a population of approximately 3.5 million people, and covers a large area of 1,030,700 sq kmabitants –10. Receuakchott –12. Thist Africa –15. One  country . A seconP. falciparum, apart from occasional cases reportedly being due to P. malariae and P. ovale [P. vivax has not been clearly described at other sites, except for a few infections detected in 2009 and 2010 in the Hodh El Gharbi region in the south of the country [Outside of the capital Nouackchott, the limited slide microscopy surveys conducted historically have indicated that almost all malaria cases are due to P. ovale . P. viva country , althougPatients with malaria were sampled from among those attending 14 health facilities at 12 different geographical sites in eight administrative regions of Mauritania in 2012 and 2013 . Tests that are negative for P. falciparum but positive for Plasmodium are routinely considered to be likely due to either P. malariae, P. ovale, or P. vivax, although resolution of these alternative species is not done at diagnosis in most health facilities. All cases analysed were local residents who did not report having travelled within the previous 2 weeks, and who were invited to provide finger-prick blood samples. These were collected on filter paper and air-dried prior to storage with desiccant in sealed polythene bags. All samples were obtained with informed consent from patients, and guardians of patients who were under 18 years of age. Regardless of inclusion in the study, patients were treated at the health facilities with artemisinin combination therapy. Current recommendations of the National Malaria Control Programme are for first-line treatment combination of artesunate and amodiaquine, and second-line treatment option of artemether and lumefantrine, with primaquine being added in the case of confirmed P. vivax infection. The study was approved by ethics committees of the Ministry of Health of Mauritania and the London School of Hygiene and Tropical Medicine.Patients were diagnosed with uncomplicated clinical malaria by local health facility staff on the basis of clinical signs including the presence of fever, supported by positive rapid diagnostic tests detecting circulating malaria parasite lactate dehydrogenase . Species-specific identification of malaria parasites by nested PCR was performed using a published method , with a A proportion of individuals sampled were genotyped to test for Duffy blood group negativity, based on the GATA-1 gene promoter allele that leads to null expression in West African populations, using a previously published protocol .Plasmodium genus-specific as well as species-specific PCR analyses. Of these, 296 (62.7 %) had P. falciparum alone, 163 (34.5 %) P. vivax alone, and 13 (2.8 %) had mixed P. falciparum and P. vivax infections. None of the samples was positive for P. ovale or P. malariae. Apart from Nouakchott, in which infections were already known to occur, P. vivax is described here for the first time from five widely separated sites in four other administrative regions of Mauritania . The proportions of malaria cases with P. vivax and P. falciparum showed extreme variation among the different sites  of cases sampled from Teyarett Health Centre had P. vivax alone, in contrast with 37 % (16/43) at the National Hospital Centre (Fisher’s Exact P < 0.0001). In 2013, 85 % (62/73) of cases sampled from Teyarett Health Centre had P. vivax alone, in contrast with 43 % (3/7) at the Cheikh Zayed Hospital Centre (Fisher’s Exact P = 0.013).In the capital Nouakchott, P. vivax positive cases did not show any individual to be homozygous Duffy negative.Duffy promoter genotyping of 82 P. vivax is a widely distributed cause of clinical malaria in Mauritania. Cases occur over a large area, and are not restricted to the capital Nouakchott from where they were previously described. Remarkably, there is no information on the occurrence of this parasite species in country malaria reports for Mauritania given by the World Health Organization [P. falciparum in the central and southwest parts of the country , and in Nouakchott where its relative frequency as a cause of malaria varies between different health facilities. Notably, P. vivax was the only parasite species seen in malaria patients from Zouérat in the north of the country (Tiris Zemmour region), whereas in the southeast of the country it is clear that P. falciparum dominates and P. vivax is rarely seen.In contrast to the situation in other West African countries, nization . This stP. vivax might be maintained in areas with minimal transmission in the centre and north of the country due to long persistence of infection in individuals experiencing occasional relapse from dormant hypnozoite stages in the liver. As samples were from individuals who had not reported travelling within the previous 2 weeks, most of the cases were probably acquired locally, although some might have been due to relapses of P. vivax infections acquired previously. A study of malaria in northern Senegal near the border with Mauritania surveyed households of index cases [P. falciparum infections are associated with travel to more highly endemic parts of Senegal. Surveys based around index cases may also be useful to understand the epidemiology of P. vivax as well as P. falciparum in different parts of Mauritania. Although there is a previous report of a P. vivax-infected individual in Nouakchott who was Duffy negative [P. vivax tested were shown to be Duffy positive. Indeed, where P. vivax was seen to be clearly the predominant cause of malaria, for example in the Teyarett area of Nouakchott and in the northern town of Zouérat, most of the local population are white Maures, an ethnicity highly associated with Duffy positivity. Therefore, this parasite species will probably remain rare in the extreme south of Mauritania where most of the population are Duffy negative, but its widespread distribution in the central and northern parts of the country needs to be monitored and addressed.The epidemiology of malaria in these communities needs to be better understood. It is possible that ex cases , revealinegative , in the P. vivax and P. falciparum differ globally [P. vivax as a widespread cause of malaria in communities living in desert and Sahel areas of West Africa has not been previously appreciated. The situation described here in Mauritania, and recent data on a limited number of positive samples in Senegal [P. vivax is likely to be endemic in many communities living in other parts of northwestern Africa which have yet to be surveyed with species-specific methods [P. vivax emerged prehistorically as a zoonosis from African apes prior to becoming endemic in humans and then spreading to other continents [P. vivax distribution in Mauritania is part of a relict African parasite population, or whether the species was re-introduced in historical times.Although the distributions of globally , it is r Senegal  and nort Senegal , 15, sug methods . There intinents . It remaPlasmodium vivax causes a substantial proportion of malaria cases in diverse parts of Mauritania. Far from being restricted to the capital Nouakchott, P. vivax is detected from five of the major administrative regions of this large country, including one northern town in which all cases sampled were caused by this species. This has significant implications for diagnosis, treatment and control of malaria in this part of northwest Africa, so reporting and advice by the World Health Organization and other authorities need to reflect the epidemiological situation.


Malaria epidemiology in Madagascar is classified into four different areas, ranging from unstable seasonal transmission in the highlands to hyperendemic perennial transmission areas in the costal level. Most malaria studies in Madagascar are focused on symptomatic children. However, because of the low transmission in some areas with correspondingly low level of semi-immunity, adults are also at risk, in particular pregnant women. The objective of this study was to gain information on the genetic epidemiology of malarial infections in pregnant women in order to provide information for malaria control and elimination programmes in Madagascar.Plasmodium falciparum antibodies by immunofluorescence test and for malarial infection by realtime-PCR. The prevalence of chloroquine and sulphadoxine-pyrimethamine resistance markers was also determined in all Plasmodium falciparum samples by PCR-RFLP as well as the multiplicity of infection through genotyping six neutral microsatellites.Between May and August 2010, we carried out cross-sectional surveys targeting healthy pregnant women in six locations, three in the coastal area and three in the highlands at 850–1300 m. 1244 blood samples were screened for anti-Plasmodium falciparum and 9.2% were carrying parasites, at the coast 95.6% and 14.8%, respectively. In the mean, 1.2 clones were detected in infected pregnant woman in the highlands and 1.5 at the coast. A higher level of monoclonal infections was found in the highlands (85.4%) compared to the coast (61.8%). Resistance markers for sulphadoxine-pyrimethamine were present only in two sites.In the highlands, 67.4% of the women presented antibodies against Immunity is triggered in Malagasy highland populations when they are infected with malaria parasites, but these populations could also serve as a reservoir for epidemics.The online version of this article (doi:10.1186/s12936-015-0704-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum, Plasmodium vivax and to a minor degree Plasmodium malariae and Plasmodium ovale [Anopheles funestus that once had disappeared after extensive insecticide spraying in the 1950s, but reappeared in the 1980s [Asymptomatic carriage of malaria parasites is common in adults in endemic regions and provides a reservoir for gametocytes, the forms of the parasite infectious to mosquitoes and thus responsible for cross-species transmission . These aum ovale . The maihe 1980s .The country has been classified in four distinct malaria epidemiologic zones based on the geography and the length and intensity of malaria transmission. Malaria transmission is stable and perennial at the East Coast, stable and long seasonal at the West Coast and unstable and seasonal in the Central Highlands and the Semi-desert zones of the South . On the P. falciparum infections in the country [Since 2008, The National Malaria Control Programme (NMCP) of Madagascar received international support through funds from the Global Funds, the World Health Organization and the US President’s Malaria Initiative as help in their fight to eradicate malaria from the island . Neverth country . Most st country .P. falciparum antibodies, (iii) the multiplicity of infection and (iv) the presence of drug resistance markers for sulphadoxine-pyrimethamine (SP) and chloroquine (CQ).This study explored the prevalence of asymptomatic malaria carriers in two of the four epidemiological areas in Madagascar. Blood samples collected in 2010 from 1,244 asymptomatic pregnant women from three coastal and three highland cities were investigated for: (i) the presence of malaria parasites, (ii) the seroprevalence of anti-A cross-sectional survey was carried out between May and July 2010 in antenatal clinics in six different locations  blood sample was taken and the plasma was extracted by centrifugation. The supernatant was stored at −20°C for serological analysis. Five hundred microlitre of the pellet was taken and diluted with the same amount of 8 M Urea solution (v/v) that is appropriate for conserving the sample at +4°C for later DNA extraction.Plasmodium falciparum was performed by an in-house indirect immunofluorescence antibody test (IFAT). Plasma samples were diluted to 1:40 with PBS plus milk powder 3%. Twenty μL of the diluted plasma were applied to an antigen field, and were incubated for 30 min at 37°C. One positive control and one negative control were included in each series. The slides were washed three times for 5 min in PBS and dried before adding fluorescein-labelled total human anti-Ig conjugate  diluted at 1/200 in PBS containing Evans blue (0.1 g/L). After incubation for 30 min at 37°C in a humid chamber, the slides were again washed three times for 5 min in PBS. The slides were read under a fluorescence microscope (Zeiss) with UV-illumination at a magnification of 250. Samples fluorescing at 1/40 dilution were considered positive. An exact titer of the positive samples was not determined.Serology for the detection of antibodies (IgG + IgA + IgM) directed against Plasmodium strain was cultured in red blood cells obtained from German Red Cross Blood Bank as described elsewhere [The in-house immunofluorescence test itself was manufactured using the laboratory strain 3D7 as antigen. The lsewhere . ErythroDNA was extracted from 400 μL of peripheral venous blood-EDTA/8 M urea (v/v) using the QIAamp® DNA Blood mini Kit according to the manufacturer’s protocol (Qiagen).Plasmodium species with human as host were assessed by real-time PCR methodology based on Fluorescence Resonance Energy Transfer technology. P. falciparum was detected with primers and probes targeting the cox1 gene and P. ovale, P. malariae and P. vivax with primers and probes targeting the 18S rDNA  and 0.5 U Taq polymerase . The multiplex PCR to detect P. malariae, P. ovale and P. vivax used three forward primers; each primer is specific of one species and one reverse primer for the three species. The mixture differed from the first one by primer concentration: 0.7 μM of forward primer for P. malariae, 0.5 μM of each forward primer for P. ovale and P. vivax, 0.4 μM of reverse primer.On a LightCycler 480 (Roche), the PCR was carried out in a total volume of 20 μL containing 5 μL of extracted genomic DNA or plasmids including the gene targeted for each species as positive control. The mixture to detect For both PCRs, the following PCR programme was used: 2 min at 95°C; 10 touchdown repeated cycles of 5 sec at 95°C, 10 sec at 63°C to 58°C, 7 sec at 72°C; and 45 repeated cycle of 5 sec at 95°C, 10 sec at 58°C, 7 sec at 72°C. Melting analysis was performed by denaturing for 30 sec at 95°C and cooling for 2 min to 45°C followed by heating at the rate of 0.1°C/s from 45°C to 75°C.ARA2 , PfPK2 , TA60 , TA81 , TA87 , Poly alpha . The multiplicity of infection (MOI) was quantified as the “highest number of fragments” for one of the microsatellites genotyped.To determine the minimal number of distinguishable clones per isolate, we genotyped 6 different neutral highly polymorphic, microsatellites 2, 200 μM dNTPs, 50 nM for each primer . Cycling conditions were as follows: 2 min at 94°C; 25 repeated cycles of 20 sec at 94°C, 20 sec at 45°C and 30 sec at 65°C; 2 min at 65°C. Amplified fragments were analysed on an ABI 3130 Genetic Analyzer and by GeneMapper® software version 4.0 (Applied Biosystems) to measure the variable length in the samples.The six microsatellite loci were amplified by semi-nested PCR according to a previous publication  with fewpfdhfr at codons N51I, C59R, S108N and I164L, pfdhps at codons A437G and K540E, pfcrt at codon K76T and pfmdr1 at codon N86Y were genotyped as described previously [P. falciparum strains  was used as controls for the PCR and the digestion.For this study, the polymorphism of eviously . BecauseFor this study, the aim was to detect at least one pregnant woman with malaria parasites if the real prevalence was 2.5% with a 90% probability. Using binomial distribution as a proxy for a hypergeometrical distribution, the required sample size of 152 was calculated. Therefore aimed to include at least 160 women per study site. With 200 pregnant women per study site, the 90% probability to detect at least one parasitized women holds even if the real prevalence is only 2%.For the statistical analysis of the study univariate tests were performed. All categorical variables were analysed conducting Fisher’s exact tests. As a normal distribution of the MOI could not be assumed Mann Whitney’s U test and Kruskall Wallis test as the non-prarametric equivalent to an ANOVA were conducted for this variable. The two-sided significance level for all tests was set to 0.05 and not adjusted for multiple testing as the study was planned as a descriptive study.Overall, 1,244 pregnant women were included into the study. In all study locations, the age distribution was right skewed with a median age of 25 years (range 12–50). Subsequently, different variables were analysed according to two age groups defined as up to 19 years and, 20 years and over. Nine out of 601 women questioned from the highlands claimed to travel within the last half of a year and only one of these travels was to a coastal location.The proportion of primiparae was similar in the highlands and at the coast . The only noteworthy mosquito protective measures that were used frequently were bed nets (70.3%). The use of bed nets was more common in the coastal cities of Mananjary (90.5%), Manakara 91.2%) and Ifanadiana (94.4%) compared to the highland cities of Tsironoamandidy (56.7%), Moramanga (67.0%) and Ambositra (21.5%). Intermittent presumptive treatment in pregnancy (IPTp) was reported by 41.3% overall. The proportion of women who had received IPTp was higher at the coast than in the highlands (p > 0.001)  were PCR-positive for P. falciparum were included.At the coast, the prevalence of infection was lower in pregnant women using prophylaxis than in women without prophylaxis. With IPTp, the prevalence was 14.0% vs. 18.9% and with bed net use; it was 14.2% vs. 21.6%, respectively Table . For theP. falciparum monoclonal infection was 61.8% at the coast against 85.4% in the highlands (p = 0.005)  with PfPk2. The average number of different clones found in infected pregnant woman was 1.4 and it was higher at the coast level  than in the highlands   and CQ (pfcrt K76T and pfmdr1 N86Y); 118 (77.6%) samples were successful genotyped for at least one of the eight markers. Figure dhfr triple mutant 51I, 59R, 108N were polymorphic only in Manakara and Tsiroanomandidy with a prevalence of 20% (6/30) and 25% (2/8), respectively. The allele dhfr 164L was genotyped in three samples from the coast. The allelic frequency of dhps 437G was variable among the different locations (p = 0.02). With 71.6%, the prevalence of the mutant was higher at the coast compared to the highlands with 27.3% (p < 0.001). Overall positive samples for dhfr and dhps genotyping (N = 93), 6 (6.5%) samples were carrying the quadrupule mutant dhfr 51I, 59R, 108N + dhps 437G. Very few samples carried mutations on pfmdr1 genes and the wild type allele of pfcrt K76 and dhps K540 were fixed in all location.Overall 152 isolates were screened for eight drug resistance markers against SP . However, the weak clinical immunity together with a relevant parasite reservoir could initiate malaria epidemics like in Ambositra (1200 m above sea level) where the seroprevalence in pregnant women were around 30% and seems to be contracted in older category of age. Indeed, the malaria epidemics in the highlands between 1986 and 1988 were devastating with around 70,000 to 100,000 deaths every year [Even in the highest study location, 1,200 m above sea level, 7% of healthy participants were ery year . Indeed,ery year .Anopheles gambiae, at altitudes above 1,200 m [In this study, the use of bed nets or chemoprophylaxis was associated with a reduction of infections in participants from the coast. Currently, malaria control in the Malagasy highlands is mainly based on indoor insecticide spraying . It cann 1,200 m .dhfr gene involved in pyrimethamine resistance was previously found only in Madagascar [dhfr 51I, 59R, 108N that is associated with a high resistance of P. falciparum against antifolate drugs [dhps 437G + dhfr 51I, 59R, 108N in Manakara (11.8%) and two samples in Tsiroanomandidy (15.4%) were found. Accumulation of mutations in these two genes is associated with in vivo SP resistance [An unusual haplotype carrying the single nucleotide polymorphism (SNP) I164L on the dagascar . This SNte drugs . On the sistance . Similarsistance . Even ifpfcrt 76T was low in Madagascar compared to continental Africa [in vivo resistance to CQ in Madagascar that is not associated with the mutation pfcrt 76T but with the mutation pfmdr1 86Y was found and perhaps yet unknown mechanism were involved [pfmdr1 86Y was low compared to previous data [pfmdr1 N86 SNP is associated with recrudescence of malaria infection after artemether-lumefantrine treatment in Benin. Studies on emerging ACT resistance can provide more information for the guideline of malaria treatment in Madagascar.Prevalence of the principal resistance marker for CQ l Africa . Howeverinvolved ,25. In oous data ,23-25 maous data  has showP. falciparum strains in the two coastal areas are in accordance with what was described in areas of moderate to high endemicity [Lower numbers of different parasite strains were found in infected women from the highlands compared to the coast. This was expected, as multiplicity is a function of transmission likelihood. The average of two to four demicity . Low pardemicity . The finfalciparum parasite carriage and also with the reduction of the number of clones of parasites in one infection, especially in the coastal area where the population are targeted by the mass distribution of long-lasting insecticidal nets and IPTp.In this study, the use of chemoprophylaxis (IPTp/SP) and vector control (bed net) was assessed among pregnant women from costal and highland locations of Madagascar. The use of those preventive measures against malaria was associated with the reduction of around 50% The findings of this study are in accordance with the conclusion of the nationwide evaluation of malaria infection and coverage of malaria control interventions in Madagascar, which took place in 2012–2013 . MalariaThis study demonstrated that asymptomatic malaria parasite carriers occur in Madagascar up to altitudes of 1,200 m with seroprevalences under 50%. The constellation of an existing reservoir and low immunity in the population is prone for malaria epidemics. The prevalence of drug resistance markers is still not alarming compared to some areas in the African mainland but monitoring might be valuable in Madagascar where malarial parasites seem to have different drug resistance mechanism than in other areas. As the Malagasy National Malaria Control Programme is moving toward the elimination of malaria in the highlands, monitoring of the existence of parasites in asymptomatic individuals becomes more important. More sensitive technology like PCR based diagnostic for the detection of submicroscopic parasitemia provides useful information for malaria control.


In addition to evidence for a protective role of antibodies to the malaria blood stage antigen merozoite surface protein 1 (MSP1), MSP1 antibodies are also considered as a marker of past malaria exposure in sero-epidemiological studies.Plasmodium falciparum and Plasmodium vivax MSP142 in healthy Cambodian adults was conducted at two sites as part of an active, observational cohort evaluating the efficacy of dihydroartemisinin-piperaquine (DP) for uncomplicated malaria .In order to better assess the potential use of MSP1 serology in malaria chemoprophylaxis trials in endemic areas, an analysis for the prevalence of antibodies to both 42 and PvMSP142, respectively), and titers higher in those who lived in a higher transmission area, although there was little correlation in titers between the two species. Those volunteers who subsequently went on to develop malaria had higher baseline MSP142 titers than those who did not for both species. Titers to both antigens remained largely stable over the course of the 4–6 month study, except in those infected with P. falciparum who had multiple recurrences.Rates of baseline sero-positivity were high (59 and 73 % for PfMSP142 serology as either a screening criterion and/or biomarker of exposure in chemoprophylaxis studies. Further work remains to identify useful markers of malarial infection and/or immunity.These findings illuminate the difficulties in using MSP1 As US armed forces continue to deploy to malaria endemic areas for both military and humanitarian missions, development of efficacious anti-malarial chemoprophylaxis with convenient regimens and minimal side effects remains a top priority for the Department of Defense . Weekly Plasmodium falciparum malaria in either treatment arm, true efficacy could not be determined due to lack of a surrogate endpoint for malaria exposure [Historically, chemoprophylaxis agents have been licensed based on the results of placebo-controlled studies in semi-immune volunteers. However, this approach raises two ethical dilemmas—justification for the use of placebo and post-trial access for the study population . Non-infexposure , and reqexposure . AnotherP. falciparum controlled human malaria infection (CHMI) study by Moon et al. [42 were not induced in malaria-naive, healthy volunteers taking mefloquine chemoprophylaxis with strict clinical and parasitologic monitoring; however, it may be possible that assessment MSP142 as a surrogate biomarker in endemic populations may be of more utility.A surrogate biomarker for malaria exposure would ensure future active comparator trials are interpretable. Antibodies to blood stage malaria antigens, such a merozoite surface protein 1 (MSP1), have been evaluated in sero-epidemiological surveys as estimates of malaria exposure , 9. The n et al. , antibodP. falciparum and Plasmodium vivax infections in roughly equal proportion, focal transmission by forest-dwelling Anopheles mosquitoes with sporadic infections, all occurring in the epicenter of anti-malarial resistance [42 titers in a cohort of healthy asymptomatic Cambodian soldiers in a malaria endemic area [The US and Cambodian militaries have recently been working to develop new anti-malarial chemoprophylaxis agents . Malariasistance –15. Cambmic area  in orderClinicalTrials.gov identifier NCT01280162) [P. falciparum or P. vivax malaria and were treated with 2 or 3 days of DP. Blood was drawn for antibody titers at screening/enrollment, time of malaria infection, 42 days after completion of DP therapy, and any time of malaria recurrence. Recurrent cases were distinguished as recrudescence or reinfection by genotyping for MSP-1, MSP-2, and GLURP (glutamate-rich protein) allelic variants as previously described [P. vivax recurrences was not performed.Serum samples were isolated from 5 ml of peripheral blood drawn from volunteers enrolled in an IRB-approved study conducted in Anlong Veng District, Oddor Meanchey Province, Cambodia. From September 2010 until February 2011, a two-arm, randomized, open-label trial of 2- versus 3-day treatment regimen of dihydroartemisin-piperaquine (DP) nested within an active observational cohort study was conducted as previously reported , was provided by Dr. Evelina Angov, manufactured at WRAIR as described in [E. coli expressed, recombinant protein with 391 amino acids, of which 17 are non-MSP-142 amino acids fused to the N-terminus .The 3D7 allele of the 42-kDa recombinant protein, ribed in . This isPlasmodium vivax merozoite surface protein-1 (MSP-142), was provided by Dr. Sheetij Dutta, WRAIR, and is also an E. coli expressed, recombinant protein based on the P. vivax Sal I allele, 380 amino acids long, consisting of 18 non-MSP-142 amino acids fused to the N-terminus part of MSP-142 (amino acids 1350–1789) [P. falciparum MSP-142, kindly provided by Dr. Evelina Angov.50–1789) . Also as42 and PvMSP142 at 1 µg/ml and allowed to incubate overnight at 4 °C. Plates were washed four times and blocked with 3 % bovine serum albumin-phosphate buffered saline with Tween 20 (BSA-PBST) for 1 h; the samples were loaded at serial twofold dilution. After 2 h incubation at 37 °C, plates were washed four times and incubated for 2 h at 37 °C with peroxidase-conjugated anti-human IgG(γ)  at 1:8000. The plates were washed four times and incubated with ABTS substrate  for 30 min at room temperature. The reaction was stopped with stop solution  and plates read on an automatic plate reader Spectromax340PC. The absorbance at 405 nm was determined for each well with the resulting data applied to a four parameter logistic curve using Softmax Pro Version 5.2 software . The serum dilution required to yield an optical density of 1.0 was defined as the titer. Positive controls consisted of plasma samples from highly reactive sera. Negative controls consisted of pooled plasma from individuals from Thailand obtained through the Thai Red Cross. Before running all study samples, 10 random serum samples were selected to compare ELISA titers among the three main haplotypes of PfMSP142 . The titers were similar among the three alleles (data not shown), suggesting strong serologic cross-reactivity in this small sample subset from Cambodia. Thus the Pf3D7 allele was selected for all study ELISAs.Plates were coated with 100 µl PfMSP119 then sequenced to determine allelic haplotype. Primer sequences  were as described previously [19 which yielded 435 and 426 bp of PCR product, respectively. Genomic DNA was extracted by using MagNA Pure compact instrument  according to manufacturer’s instruction. The primary and secondary PCR reactions were carried out in 25 µl reaction volume on Peltier Thermal Cycler . Master Mix of both PCRs was comprised of 1× PCR buffer with MgCl2 , 0.1 µM of primers, 0.2 mM of dNTP  0.1 U/µl of Taq polymerase , and extracted DNA (adjusted concentration to 3 ng/µl). PCR conditions were as follows: pre-denaturation at 94 °C for 1 min followed by 40 cycles (35 cycles for secondary PCR) of denaturation at 94 °C for 45 s, annealing at 53 °C for 45 s (55 °C for secondary PCR), extension at 60 °C for 5 min. The PCR products were loaded on 1 % agarose gel, stained with ethidium bromide, and visualized under UV illumination . For the secondary PCR, PfMSP119 variants; E/TSR/L, E/TSG/L, E/KNG/F, and Q/KNG/L with GenBank accession number HM569746, HM569747, HM569748, and HM569750, respectively, were used as reference strains in sequence alignment. PCR products were purified by using a PCR purification kit  prior to sequencing. Sequencing was performed at AIT Biotech, Pte, Ltd., (Singapore) and was analysed and aligned by using BioEdit Sequence Alignment Editor Software (version 7.2.5).Genotyping of PfMSP1 42-kDa region was determined by using semi-nested PCR, and PfMSP1eviously . The PCRP. falciparum MSP1 was 225 units and for P.vivax MSP1, 379 units. Cut-off for seropositivity was defined as >3 standard deviations (SDs) above the geomean, or 360 and 494 units for P. falciparum and P. vivax, respectively. Comparison of numbers of volunteers seropositive or seronegative was performed by using Fishers exact tests with two-tailed p values. To compare level of titers, the data was log transformed to obtain normal distribution and the geomean titers reported. Titers were compared either using unpaired t tests or one-way ANOVA with Tukeys post-test comparisons and comparisons between vivax and falciparum titers done using Pearson’s correlation coefficient. The multiple regression analysis was conducted separately using SAS v 9.2.Prism v 6.0 was used for statistical analyses. The geometric mean antibody titer at each time point was determined with 95 % confidence intervals. Negative controls were determined by ELISA analysis of serum samples from ten non-malaria infected healthy Thai adults living in Bangkok. The geomean titer of negative controls for 42 were 904 units  and for PvMSP142 were 2207 units . Malaria transmission in Cambodia is seasonal, with transmission increasing during the rainy season from May to October, with peaks in September and November [P. falciparum or mixed P. falciparum/P. vivax malaria were seropositive for PfMSP142 with a geomean titer of 12,300 units, nearly 15× higher than those who did not have P. falciparum . There were 32 of 41 patients with P. vivax malaria, and 88 % were seropositive for PvMSP142, with geomean titer 4242 units, roughly 2× higher than those without P. vivax . Among the 214 volunteers negative for malaria at enrollment, preexisting exposure based on serology was uneven at the two study sites—Site A, neighboring a small village and Site B, in a forested area  and mean PvMSP142 titers of 3637 versus 1076 units, respectively .A total of 256 adults were screened for the study with serum samples obtained from all but one. Geometric mean titers and (range) for PfMSP1November . The majP. falciparum and P. vivax MSP1 titers in site A were significantly higher in those with malaria within 12 months than for those not having malaria within the past year or never, p = 0.0117 and p = 0.0057, respectively (one-way ANOVA with Tukeys post-test). At site B, titers to both antigens were not different among the three groupings of malaria timings: PfMSP1 (p = 0.7734) and PvMSP1 .When timing of malaria episodes was grouped into <12 months ago, more than 12 months ago and never . The same was true for PvMSP142 with 88/101 (87 %) seropositive at site B versus 69/113 (61 %) at site A . Seropositivity rates per last reported malaria episode (three groupings) are shown in Fig. 42 and PvMSP142, respectively, with even higher rates for Site B: 61 and 87 %. Titers for both MSP142 antigens were similar if the volunteer reported having malaria 1, 2, 3, or >3 times in the past year . Although nearly half (49 %) of volunteers were seropositive for both P. falciparum and P. vivax MSP1 at enrollment and only 17 % seronegative for both, there was only a weak correlation of titer levels between falciparum and vivax MSP142 at site A , and no correlation at site B .For malaria negative volunteers at enrollment, Site B participants were more often seropositive to PfMSP1P. vivax, 10 P. falciparum, two mixed P. falciparum/P. vivax, and one Plasmodium malariae cases. P. falciparum and P. vivax MSP142 titers of 49 were assessed to evaluate the hypothesis that those with higher titers at enrollment would be protected against subsequent infection with malaria, possibly confounding a chemoprophylaxis study conducted in an endemic area. For both antigens, titers at enrollment were highest in those who developed subsequent homologous infection , and higher than those who developed P. vivax infection, 993 units (n = 37) . Similarly, the titers to PvMSP142 at enrollment were higher for those who subsequently contracted P. vivax malaria (6105 units) than those who never developed malaria (1158 units), or P. falciparum malaria (3235 units) .Among181 malaria-negative volunteers enrolled, 50 subsequently developed malaria within 8–114 days after enrollment: 37 42 at enrollment were still more likely to develop subsequent P. falciparum malaria than those who remained malaria-free over the course of the study (p = 0.0065). Likewise, volunteers subsequently developing P. vivax malaria had higher baseline PvMSP142 titers than those remaining malaria-free (p = 0.0006). This positive association also held for seropositivity. Of the 74 PfMSP142 volunteers seropositive at enrollment, 12 % went on to develop P. falciparum malaria compared to 2 % of 62 seronegative volunteers  while 39 % percent of those PvMSP142 seropositive subsequently developed P. vivax malaria versus 1 % of those who were PvMSP142 seronegative .When analysed using multiple regression model, adjusting for time to infection and site of enrollment, those with higher titers to PfMSP142, the relationship between 3D7 antibody titers at enrollment and the infecting P. falciparum 42-kDa haplotype was examined. Protection by 3D7 MSP142 antibodies against homologous P. falciparum 3D7 infection could not be determined since sequencing of the 33 P. falciparum isolates revealed no 3D7/MAD20 alleles. The majority of isolates (26/33 or 79 %) were the 42-kDa CAMP allele, comprised of the MAD20 33-kDa plus the EKNGL 19-kDa haplotypes, while a minority (21 %) bore the FVO allele, comprised of the K1 33-kDa plus the QKNGL 19-kDa haplotype [P. falciparum infections, only one volunteer (178) had a new second P. falciparum infection (versus recrudescence for others). This volunteer had an initial CAMP infection in October followed by FVO infection in January at the time of discharge. The P. falciparum MSP1 3D7 titers rose from 6120 to 293,896 units, the third highest measured of any volunteer, suggesting recent exposure and induction of immune response toward the new parasite, albeit non-protective.Since the ELISA assay plate antigen was the 3D7 allele of PfMSP1aplotype . Of thosP. falciparum infection with P. falciparum recurrence or initial P. vivax infection with P. vivax recurrence.  Group 2 had initial P. falciparum infection followed by P. vivax, and Group 3 had initial P. vivax infection followed by P. falciparum. Titers to PfMSP142 remained relatively stable over the course of the study in those with recurrent mono-infections with the exception of volunteers with initial P. vivax infection then P. falciparum infection, in which titers increased substantially with the P. falciparum infection. The single volunteer (178) who developed a new second P. falciparum infection (vs recrudescence) also had increasing titers at the new P. falciparum infection. Mixed infections induced varying titers although the number of volunteers was small. PvMSP142 titers remained quite stable over time in all groups.To look more closely at how multiple malaria infections may affect titers, the titers of volunteers with one or more recurrences are plotted and organized by groups in Fig. 42 and PvMSP142, there was no statistically significant difference in titers between enrollment and discharge for any of the groups except for the four volunteers who developed multiple P.falciparum infections over the course of the study in whom the titers were much higher at discharge than enrollment . Of the 11 total P. falciparum infections in this group of four patients, 10 had a MSP1 19-kDa haplotype of EKNG. At discharge, titers for these subjects with multiple P. falciparum infections were higher than those who had never had malaria (457 units) as well as those who had one P. vivax infection (497 units)  but not those who had one P. falciparum infection (3784 units). Titers to PvMSP142 did not differ significantly among the groups .In Fig. P. falciparum to healthy volunteers under mefloquine prophylaxis to assess seroconversion rates of P. falciparum MSP142, defined as a fourfold rise in titers. None of the volunteers seroconverted, and only four of six controls, those who did not receiving mefloquine who did develop malaria, seroconverted.The US military continues to develop new anti-malarial drugs and vaccines as part of its mission to protect the war fighter. Historically, anti-malarial chemoprophylaxis studies for licensure in Asia and elsewhere have used placebo-controlled study designs in semi-immune populations, with recent examples including tafenoquine in soldiers of the Royal Thai Army  and azitP. falciparum and P. vivax MSP142 responses during a single malaria season in order to gain insight to malaria exposure and immunity in a low transmission setting with multidrug resistant malaria in northern Cambodia and lay the groundwork for malaria prophylaxis studies. The predominantly resident military population was a mix of self-reported malaria-naïve and semi-immune individuals; some were newly arrived from non-transmission areas while other long-term veterans had likely had multiple exposures over several years while stationed in malarious areas. Transmission varied substantially between the two study locations, with significantly higher attack rates during the cohort study at the forested site B compared to semi-urban site A [P. falciparum and P. vivax MSP142 proteins in asymptomatic, aparasitemic adults varied greatly by individual, with a 1–5 log difference not directly attributable to geographic site nor reported clinical history of prior malaria infection. Geomean titers were higher in those reporting a malaria infection in the past year compared to those with no history of disease, but significant overlap existed, underscoring the lack of utility of a single serological measurement as marker of immunity. Based on sero-positivity rates, prior exposure seemed quite evident, particularly for P. vivax, even in those reporting no clinical history of malaria.This study aimed to characterize background and changes in humoral n site A . BaselinP. falciparum and P. vivax AMA1 could fluctuate widely in pregnant women living in Thailand even when measured biweekly. Another study in northern Thailand found that in individuals with documented falciparum or vivax malaria infection in the past 6 years, only 48 % were sero-positive to PfMSP19 and 11 % to P. vivax MSP119 [42 [The wide variation in baseline serology is similar to prior sero-epidemiology studies in both high and low transmission settings. Fowkes et al.  found thx MSP119 . Even inP119 [42 . This laVolunteers who did not get malaria during the course of the cohort study had essentially no change in titer. Mean PfMSP1 titers hovered just above the upper limit of seropositivity at both time points suggesting this is the natural ‘background’ titer of adults in rural Cambodia, although this would need to be validated during more than one transmission season. Ideally, sero-epidemiological studies conducted in areas with seasonal transmission would have humoral responses measured just before the onset of the transmission season. Since this study was nested in an active drug efficacy study, conducted several months after the rains had begun, baseline titer levels and rates of seropositivity may be overestimated. It is unclear if the high rate of sero-positivity signifies recent or remote infections, and it gives little indication of the number and species of prior infections, particularly for those with low titers. Regardless, the high rates did not appear to confer immunity.42 for the four volunteers with three malaria infections during the study period. The PfMSP1 19-kDa haplotype for 10 of 11 infections for these four patients was EKNG; thus the higher titers do not represent a broadening of the immune responses by infection with different circulating haplotypes. With P. vivax infections, titers appeared either to wane or remain remarkably stable over time. As reported elsewhere, it is likely that the majority of P. vivax infections represented relapses [P. vivax antigens by microarray in Peruvian patients with P. vivax mono-infection and found that both height and breadth of responses were not different in those who were determined to have relapse versus P. vivax re-infection. Moreover, boosting may not be seen due to longer half-lives of MSP1 titers in low to medium transmission areas. Wipasa et al. [Perhaps more importantly, there was little apparent ‘booster effect’ of baseline titers in response to infection, except for PfMSP1relapses , and thirelapses  analyseda et al.  used a mP. falciparum infections were protected from subsequent P. falciparum challenge and seroconverted to CSP but not AMA1 or GLURP [P. vivax malaria, although one study of deployed Thai soldiers found almost three-quarters contracted vivax malaria despite 62 % being PvCSP seropositive at deployment; those who were seronegative did seroconvert with acute illness though the rise in titers was modest and fell quickly [P. vivax malaria did not mount any CSP antibody response. There were not successive increases in CSP titers with relapses, a finding also demonstrated in Cambodian adults in this study, suggesting that a biomarker for P. vivax infection may be even more elusive. Whether pre-erythrocytic or erythrocytic, a single antigen may not suffice as a biomarker; thus, adding immunogenic antigens, such as MSP2 and schizont extract (SE) [Alternative biomarkers to other antigens, such as antibodies to the circumsporozoite protein (CSP), which is located on the surface of the sporozoite, may be less affected by chemoprophylaxis. In an intervention-treatment-vaccination (ITV) study in healthy Dutch volunteers, those taking chloroquine prophylaxis during three successive mosquito-borne or GLURP . A studyor GLURP . Less is quickly . Thirty-act (SE) , or asseact (SE)  plus newact (SE) , 35 may P. falciparum and PvMSP1 in endemic areas has been mixed [42 induces a better homologous and heterologous antibody response than the 3D7 allele of MSP142 [In design of chemoprophylaxis studies in endemic areas, it is possible that innate immunity in semi-immune subjects may attenuate infection, thereby inflating the apparent protective efficacy of prophylactic drugs and overestimate their potential benefit to malaria-naïve individuals. The previous evidence for the protective role of en mixed –39. In ten mixed , 36. Crof MSP142 . In addif MSP142 .Evidence for the establishment and duration of both serologic markers and functional malaria immunity in subjects living in Southeast Asia remain limited. Compounding the complexities of assessment, unlike homologous laboratory strains used in challenge studies, malaria infections in endemic areas are frequently with heterologous strains or mixed species and responses reflecting the more complex transmission patterns such as observed in Cambodia. These factors combine to limit the current utility of serologic biomarkers of malaria immunity in malaria prevention studies. Studies to assess the development of a broader array of serologic biomarkers in controlled settings are needed.


In 2010, the World Health Organization shifted its malaria guidelines from recommending the empiric treatment of all febrile children to treating only those with laboratory-confirmed malaria. This study evaluated the frequency and predictors of malaria over-treatment among febrile malaria-negative children in Kenya.Between 2012 and 2013, 1,362 children presenting consecutively with temperature ≥37.5°C to Kisii and Homa Bay hospitals were enrolled in a cross-sectional study evaluating causes of fever. Children were screened for malaria using smear microscopy and rapid diagnostic tests and managed according to standard of care at the hospitals. The frequency of anti-malarial prescriptions among children with laboratory-confirmed malaria negative children  was determined; and clinical and demographic correlates of overtreatment evaluated using logistic regression. Because of differences in malaria endemicity, analyses were stratified and compared by site.Among 1,362 children enrolled, 46 (7%) of 685 children in Kisii, and 310 (45.8%) of 677 in Homa Bay had laboratory-confirmed malaria; p < 0.001. Among malaria-negative children; 210 (57.2%) in Homa Bay and 45 (7.0%) in Kisii received anti-malarials; p < 0.001. Predictors of over-treatment in Homa Bay included ≥ one integrated management of childhood illness (IMCI) danger sign , fever lasting ≥ seven days , and fever ≥39°C . In Kisii, only fever ≥39°C predicted over-treatment .Malaria over-treatment was common, particularly in Homa Bay, where the prevalence of malaria was extremely high. Severe illness and high or prolonged fever were associated with overtreatment. Overtreatment may result in failure to treat other serious causes of fever, drug resistance, and unnecessarily treatment costs. Malaria is a leading cause of morbidity and mortality among children in sub-Saharan Africa. The most well recognized sign of malaria infection is fever, which accounts for over 30-50% of all paediatric hospital visits in sub-Saharan Africa . HistoriMalaria overtreatment may result in the failure to treat other serious causes of fever, particularly blood stream infections ,8. MortaThis was a cross-sectional study nested within an ongoing surveillance study of febrile illnesses among children at Kisii Provincial Hospital and Homa Bay District Hospital, both in Western Kenya. These hospitals are situated in areas with historically different malaria endemicity: Kisii is hypo-endemic with annual entomologic inoculation rate (EIR) of <1.5 infectious bites per person per year. Homa Bay historically has been holo-endemic with EIR of ≥300 infectious bites per person per year ,17, althClinical staff at the study sites consists of clinical officers and registered nurses with three years of medical training and licensed to independently diagnose and treat diseases, including ordering and interpreting laboratory tests. There are very few medical doctors available for direct clinical care. The new malaria “test and treat” policy was adopted by the Kenyan Ministry of Health in 2011, and subsequently health workers received in-service malaria case-management training [Study staff was trained on enrolment criteria, consent, clinical and laboratory procedures, use of integrated management of childhood illness (IMCI) syndromic management algorithms, and assessment of danger signs. Because the parent study was a cross-sectional study aimed at understanding bacterial etiologies of fever, the study protocol did not stipulate standardized treatment protocols for malaria and clinicians managed sick children according to the standard of care practices at the hospitals.During screening and enrolment, study clinicians and registered nurses systematically captured comprehensive information regarding sociodemographics, anthropometric characteristics, past medical history and care-seeking behaviour, and current clinical and laboratory data using standardized case report forms. Clinicians also recorded diagnoses and prescribed treatments. Children with any of the IMCI danger signs  were classified as having severe febrile illness . All scrLaboratory-confirmed malaria was defined as a positive smear microscopy and/or RDT test. Malaria over-treatment was defined as a negative smear and RDT result that was treated with anti-malarials.Demographic, clinical and laboratory characteristics were compared between study sites using Chi-square or Fisher’s exact tests for categorical variables and two sample t-tests or Wilcoxon rank sum tests for continuous variables.a priori following a thorough literature review was first examined in a bivariate logistic regression. These included malnutrition, mother, and child’s HIV status, presence of IMCI danger signs, fever of ≥ seven days, having previously sought care, pre-treatment with anti-malarial prior to hospitalization. To identify independent correlates of malaria over-treatment, all of the following a priori selected confounders were included simultaneously in a multivariate logistic model: child’s age, sex, whether the child was enrolled from out- or in-patient departments, primary caregiver’s level of education and income. The latter two variables were used as surrogates for family socioeconomic status. Because the sites had marked differences in malaria endemicities that might impact clinician suspicion, analyses were stratified and compared by site. All analyses were done using Stata statistical software . Strengthening the reporting of observational studies in epidemiology (STROBE) guidelines were followed in reporting this study [The proportion of children laboratory-confirmed negative malaria but were treated with anti-malarials was estimated and compared between sites using Chi-square test. The association between malaria over-treatment (primary outcome) and patients’ clinical and demographic characteristics that had been selected is study .vs 16.1%; p < 0.001). Children in Homa Bay were more likely to have HIV-infected mothers  or to be HIV infected themselves  than those from Kisii. However, children from Homa Bay were less likely to be wasted, and to have previously sought health care elsewhere for their current illness than children from Kisii .Between April 2012 and November 2013, 1,362 febrile children were enrolled into the parent study; 685 in Kisii and 677 in Homa Bay. Figure Among febrile children, 26% had laboratory-confirmed malaria infection; 45.8% in Homa Bay and 6.7% in Kisii; prevalence ratio = 6.82; 95% CI: 5.22-8.90. Among laboratory-confirmed malaria cases (n = 356), 96.1% were positive by both RDT and smear, 10 (2.8%) were positive by RDT alone and 4 (1.1%) were positive by smear alone. In Kisii, the prevalence of bacteraemia among children with and without malaria was 4.1% (26/639) and 2.2% (1/46), respectively, p = 0.524. The prevalence of bacteremia among children with and without malaria was 4.5% (45/1006) and 2.0% (7/35), respectively; p = 0.034.All children with laboratory-confirmed malaria (n = 356) were treated with anti-malarials regardless of their initial provisional presumptive diagnosis. Most (82.3% received artemisinin combination therapy (ACT), while 17.7% received quinine. Children treated with quinine were more likely than those treated with ACT to have danger sign(s) present 76.2% (48/63) vs. 47.1% (138/293); p < 0.001. Among children with laboratory-confirmed negative malaria (639 in Kisii and 367 in Homa Bay), the proportion of those treated with anti-malarials was significantly higher in Homa Bay as compared to Kisii (57.2% vs. 7.0% p < 0.001). Overall, among those over-treated, children treated with quinine were more likely than those treated with ACT to have danger sign(s) present 76.2% (48/63) vs. 47.1% (138/293); p < 0.001.Because the site characteristics and patients’ demographic and clinical characteristics were so distinct, analyses were stratified and compared by site. In Kisii, odds of malaria over-treatment were significantly higher for children with high-grade fever (odds ratio (OR) = 2.14; 95% CI: 1.21-4.37), those who had taken anti-malarials in the week preceding hospitalization  and those whose mothers were HIV-infected  in bivariate analysis , a history of fever lasting a week or more , and presence of at least one IMCI danger sign . Notably, there was no significant association of malaria over-treatment with HIV-infection.In Homa Bay, signs and symptoms of severe sickness were associated with over-treatment. The unadjusted odds of malaria over-treatment for children with both a negative blood smear and RTD test result were significantly higher for children with high-grade fever , a history of fever lasting a week or more , and at least one IMCI danger signs present  . In Homa Bay, children more likely to be treated with both anti-malarials and antibiotics included those who presented with a history of fever lasting a week or more  or manifested an IMCI danger sign(s) . In Kisii, although children presenting with high-grade fever of ≥39°C but without malaria were more likely to receive both anti-malarials and antibiotics , the presence of IMCI danger signs was not predictive of dual treatment .More children with negative laboratory malaria diagnostic tests were treated with both anti-malarials and antibiotics in Homa Bay than in Kisii  of febrile children with laboratory-confirmed negative malaria in a high malaria transmission area were prescribed anti-malarials compared to only 7.0% in a low malaria transmission area. In Kisii, the only strong predictor of malaria over-treatment was a fever of >39°C. In Homa Bay, signs of more severe illness  were the strongest predictors of over-treatment with anti-malarials. In high malaria prevalence areas, clinicians may have over-treated sick children because they did not trust negative test results, and/or feared missing possible malaria cases, with potentially fatal consequences . The finThe higher proportion of children with danger signs in Homa Bay may, in part, reflect limited healthcare exposure, delayed access to, or poor engagement with, the health care system and inappropriate home treatment of children with fever. Additionally, significantly fewer children in Homa Bay  had received all age-appropriate vaccines and children in Homa Bay were also significantly more likely to be HIV-infected or exposed. Because study procedures, inclusion criteria, and training of study clinicians were uniform across study sites, it is unlikely that the higher proportion of very sick children in Homa Bay was due to systematic selection bias or differential misclassification.This study found that children exhibiting signs of severe disease were more likely to be treated for both malaria and for presumptive blood stream infection. More than 80% of the children at both sites were presumptively prescribed antibiotics. This suggests that in the absence of point-of-care diagnostics for other causes of fever, clinicians are reluctant to miss the opportunity to avert potential morbidity and mortality and will presumptively treat children, even when guidelines suggest otherwise. Malaria-negative children were more likely to be prescribed antibiotics compared to children with positive results , suggesting that negative malaria test may have led clinicians to consider and treat alternative causes of fever.The finding of significant malaria over-treatment, particularly in very sick children in endemic areas, is consistent with a recent Cochrane review of the effect of RDTs on the prescription of anti-malarials in Africa . This reWhile this study had several strengths, notably the inclusion of data from two sites in Kenya with widely differing underlying malaria endemicity, there were also some weaknesses. Malaria over-treatment was only evaluated in relation to patients’ demographic and their clinical symptoms. Other factors such as clinicians’ level of training, number of clinical staff and patient load, patients’ expectations, diagnostic laboratory capacity, and regular supervision and public health promotional activities also likely affect clinicians’ malaria diagnosis and treatment decisions. Lastly, although the inclusion of only children with fever ≥37.5°C was intended to enhance the clinical yield of microbiologic investigations, this may limit the generalizability.Febrile patients, particularly those with signs of more severe illness in areas of high malaria transmission, continue to be treated with anti-malarials despite negative laboratory tests, against current WHO “test-treat” policy. This may result in missed opportunities to accurately diagnose and treat alternative causes of fever. There is need for the strengthened training in fever case management, supervision, monitoring and evaluation of malaria treatment practices by clinicians in order to ensure adherence to malaria test results, particularly in areas of high malaria endemicity.