As a nation reduces the burden of falciparum malaria, identifying areas of transmission becomes increasingly difficult. Over the past decade, the field of utilizing malaria serological assays to measure exposure has grown rapidly, and a variety of serological methods for data acquisition and analysis of human IgG against falciparum antigens are available. Here, different immunoassays and statistical methods are utilized to analyse samples from a low transmission setting and directly compare the estimates generated.Plasmodium falciparum antigens MSP-1p19, MSP-1p42(D), MSP-1p42(F), and AMA-1. Appropriate parametric distributions and seropositivity cutoff values were determined by statistical measures.A subset of samples (n = 580) from a 2012 Haitian nationwide malaria survey was employed as sample population of low falciparum endemicity. In addition to the Haitian samples, samples from 247 US residents were used as a reference population of ‘true seronegatives’. Data acquisition was performed through standard ELISA and bead-based multiplex assays assaying for IgG antibodies to the Data from both assays showed a strong positive skew, and the lognormal distribution was found to be an appropriate statistical fit to the Haitian and American populations. The American samples served as a good serological true negative population for the multiplex assay, but not for ELISA-based data. Mixture model approaches to determine seronegative and seropositive populations from the Haitian data showed a high degree of distribution overlap—likely due to the historical low falciparum transmission in this nation. Different fittings to the reversible catalytic model resulted depending upon the immunoassay utilized and seropositivity cutoff method employed. Data were also analysed through fitting to penalized B-splines, presenting another possible analytical tool for the analysis of malaria serological data.Standardization of serological techniques and analyses may prove difficult as some tools can prove to be more useful depending on the area and parasite in question, making clear interpretation a vital pursuit. The presented analysis in the low-endemic nation of Haiti found malaria-naive US residents to be an appropriate seronegative reference population for the multiplex assay, and this assay providing consistent estimates between MSP-1 and AMA-1 antigens of percent seropositives for this low-endemic population.The online version of this article (doi:10.1186/s12936-015-0955-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum remains a significant global health concern with approximately 200 million cases and 600,000 deaths annually [P. falciparum has placed on humans and their ancestors, it is rightfully stated that “malaria is the strongest known selective pressure in the recent history of the human genome” [P. falciparum antigens as targets for a humoral response. Many of the most immunogenic antigens include membrane bound proteins that are found on the surface of invasive merozoites, which are released from infected erythrocytes following schizont-induced rupture of the host cell.Malaria caused by the apicomplexan parasite annually . With thP. falciparum antigens (and the serum IgG specific for these antigens) is age. If sustained transmission, no matter how low, is present in a geographical area, persons in that area have a greater cumulative risk of lifetime exposure as they age. In regions of moderate or high transmission for P. falciparum, children progressing through their first decade of life show rapid increases in the acquisition of antibodies against the parasite [P. falciparum [One of the important factors that determines an individual’s carriage of memory B cells educated against parasite –5. The rparasite , which pparasite , 8. A pelciparum , even thP. falciparum biomass within an area has been shown to relegate infections much more heavily towards sub-patent, sub-microscopic asymptomatic presentations [P. falciparum transmission intensity in areas with low parasite prevalence [As malaria incidence within an area decreases, the ability to detect active infections becomes increasingly difficult. The reduction of ntations –12. For ntations . Sensitintations . Furtherevalence , 14, 15.P. falciparum endemicity by comparison of immunoassays, variable classifications, and cutoff value determinations. Data is also presented from a large sample of persons never exposed to malaria to show IgG distributions in a malaria-naive population.Many strategies have been utilized for the analysis and interpretation of malaria serological data. Unlike active infection detection which primarily focuses only on the presence or absence of infection, serological datasets can provide a wealth of information, especially when combined with other covariates such as age. Recently, quantitative immunoassays, such as ELISA and bead-based flow assays, have been the tests of choice, allowing relatively short sample preparation time and robust data generation. In addition, multiple methods have been employed with efforts to maintain the continuous nature of the data as well as dichotomize the sample population into classes of ‘seropositive’ or ‘seronegative’. When dichotomizing variables, further various approaches have been used in order to determine an assay value at which the data can be partitioned—generally referred to as the cutoff value. Here is presented analyses of serological data from a region of low The survey protocol was approved by CDC’s internal review board and the Haiti ethical review committee in 2012 at the Ministry of Health. All persons participating in the survey were assigned a six-digit identification number that could not be traced back to the individual.Samples from persons assumed to have never been infected with malaria were gathered from blood donated to a community blood bank in Memphis, TN, USA. All blood units were from persons that had screened negative for HIV and hepatitis B viruses and had no reported history of international travel in the six previous months.P. falciparum endemicity, a tracking results continuously (TRaC) survey was conducted in 2012 as part of Global Fund’s Round 8 activities that builds on the Strategic Plan against Malaria in Haiti. A 2011 nationwide survey reported Haiti to have a low national parasite prevalence of less than 1 % as estimated by PCR [For the area of low d by PCR . The 201P. falciparum merozoite surface protein 1 (MSP-1) fragments were employed, including two allelic forms of the 42kD fragment of MSP-1: MSP-1p42(D) and MSP-1p42(F) from the 3D7 and FVO strains, respectively, and one form of the 19 kD fragment (MSP-1p19) fused to glutathione S-transferase (GST) cloned from P. falciparum isolate 3D7. The sequence encoding the MSP-1p19 fragment previously described by Burghaus and Holder [GGA TCC AAC ATT TCA CAA CAC CAA TGC GTA-3′ and 5′-GCG GAA TTCTTA GTT AGA GGA ACT GCA GAA AAT ACC-3′, respectively. The BamHI and EcoRI restriction endonuclease sites used for directional cloning into pGEX 4T-2  are underlined in the sequences, and an in-frame stop codon  was included in the reverse primer. Standard protocols for PCR amplification, cloning into BL21 strain Escherichia coli , expression, and purification of a GST fusion protein product have previously been reported [2HPO4 at pH 7.2 (PBS) , and concentrated using a Centricon-10 centrifugal filter device . The final concentration of the GST-MSP-1p19 fusion protein was >0.5 mg/mL  with a yield of approximately 0.25 mg/L of E. coli culture. The external domain of Apical Membrane Antigen-1 (AMA-1) and MSP-1p42 antigens were produced at Walter Reed Army Institute of Research (WRAIR) under previously published conditions [A 6 mm circular punch was taken from the centre of each blood spot, corresponding to 14 μL whole blood, for elution. Samples were shaken overnight at room temperature in 140 μL protein elution buffer containing: PBS (pH 7.2), 0.05 % Tween-20, 0.05 % sodium azide, and stored at 4 °C until analysis. Elution from blood spots provided an initial 1:10 dilution, and samples were further diluted 1:40 in ELISA or Luminex sample diluent for a final whole blood dilution of 1:400, corresponding to a serum dilution of approximately 1:800 with the assumption of 50 % haematocrit in whole blood. Three d Holder  was PCR reported , 19. Fus2SO4 and plates read on a spectrophotometer  at 450 nm. Positive and negative control wells were included on each plate to ensure consistency between readings. At the end of the study, any particular plate was re-run if its controls showed 25 % or more variation among all plates performed for the study.Before analysis, ELISA assays were optimized for antigen coating concentration and dilution of horseradish peroxidase (HRP) conjugate to reduce background signal while still providing strong detection of the specified IgG. After optimization, all samples were run under the same conditions using a standard in-house protocol. Briefly, Immulon 2 HB plates  were coated with 100 μL of 0.25 μg/mL antigen in PBS overnight at 4 °C and then blocked for 2 h the next day with 5 % non-fat milk in PBS, 0.05 % Tween-20 . Samples were diluted in blocking buffer, added in duplicate at 100 μL/well, and incubated for 2 h with gentle shaking. An HRP-conjugated secondary antibody (goat anti-human IgG) was diluted 1:12,000 in PBS-T, and 100 μL added for 1 h. For each well, 100 μL peroxidase substrate  was added and color allowed to develop for 10 min. The reaction was stopped by adding 100 μL 0.2 M H® COOH beads  according to manufacturer’s protocol in the presence of 50 mM 2-(4-morpholino)-ethane sulfonic acid, 0.85 % NaCl at pH 5.0 and an antigen concentration of 20 μg/mL for all antigens. Sulfo-NHS was purchased from ThermoFisher and EDC from Sigma-Aldrich. As a control to test for any serum IgG against GST for the GST-MSP-1p19 fusion protein, a bead was included in the panel which was coupled to GST. Samples were diluted in blocking buffer containing 0.5 % Polyvinyl alcohol (Sigma), 0.8 % Polyvinylpyrrolidine (Sigma), 0.1 % casein (ThermoFisher), 0.5 % BSA (Millipore), 0.3 % Tween-20, 0.1 % sodium azide, and 0.01 % E. coli extract to prevent non-specific binding. Reagent diluent (Buffer C) consisted of PBS-T plus 0.5 % BSA, 0.02 % sodium azide. Filter bottom plates  were pre-wetted with PBS-T and 1500 beads/analyte incubated with sample in duplicate for 1.5 h under gentle shaking. Secondary antibodies tagged with biotin  were incubated for 45 min, and subsequent incubation with streptavidin–phycoerythrin  for 30 min. Plates had a final wash incubation with reagent diluent for 30 min and were read on a Bio-Plex 200 machine by generating the median fluorescence signal for 50 beads/analyte and then the mean fluorescence intensity (MFI) between the duplicate wells. Final MFI was reported for a sample after subtracting MFI values from blank background beads that were included on each plate.The same DBS elutions were also assayed using bead-based multiplex technology. Antigens were coupled to BioPlexP value of >0.05 would indicate a statistically-acceptable fit. The analysis relaxed the stringency to a P value of >0.01 in order to be inclusive for more tests for the purpose of comparisons between immunoassays and among antigens. When considering malaria-naive US residents as a reference population, extreme outliers could significantly affect the statistical nature of distributions, so any optical density value (OD) >0.3 or MFI >250 was eliminated from the ‘true negative’ analysis. The number of removals for each antigen and each immunoassay is listed in Additional file Data were fitted to parametric distributions by statistical software programs SAS 9.3, StataSE 13, and R 3.1.2 and means and standard deviations estimated. Statistical appropriateness of fitted distributions was performed in the SAS software package with the PROC UNIVARIATE procedure and HISTOGRAM statement. The null hypothesis of the Anderson–Darling, Cramér–von Mises, and Kolmogorov–Smirnov tests is that the data of interest are appropriate for the distribution of interest, so a fmm and R’s flexmix command, as well as SAS’s PROC FMM. All use maximum likelihood estimation to fit an unweighted two-component model of ‘seronegatives’ and ‘seropositives’. The mean and standard deviation were collected from the seronegative component and used for further statistical analysis. Seroprevalence curves and estimates for seroconversion and seroreversion rates were based on fitting data to a reversible catalytic model as described previously [Finite mixture regression models were fitted to the dataset by StataSE’s eviously .Continuous data were fitted to penalized B-splines for both ELISA and Luminex assays. A B-spline allows a defined number of connected polynomial functions to be fitted to a continuous variable. A cubic function (degree of 3) with 50 knots was used to generate each spline and a very high penalty function  was imposed to prevent high variation between knots and allow curve smoothing.P. falciparum malaria, spotted onto filter paper, eluted, and titrated using each immunoassay. Through the ELISA assay, high IgG concentrations in Kenyan blood allowed samples to be diluted to 1:105-fold before reaching the lower asymptote of the sigmoidal curve , which aims to identify an indicated mixture of densities using maximum likelihood estimation (MLE). Normal and lognormal distributions were specified for each antigen’s data and attempted to fit to a two-component mixture  or loss of (seroreversion) IgG antibodies against a pathogen by fitting data to a reversible catalytic conversion model , 9, 33. rated in ) and estrated in –37. A goP. falciparum antigens would be the likely result of active or recent parasite exposure, or evidence of multiple exposures throughout life [Since transforming continuous data to binary results in an inherent loss of information, data were also analysed by maintaining immunoassay signal intensities. High IgG titres against out life . Penalizout life , but MSPPlasmodium and vector antigens have been well characterized, and multivariate approaches to malaria serology may yield robust analyses in the future. As P. falciparum exposure incidence is reduced in a population, levels of IgG against falciparum will reduce as well, and defining what constitutes an individual as seropositive becomes more difficult. Here, direct comparisons are presented of multiple methods for acquiring and analysing falciparum serological data from an area of low P. falciparum endemicity. It was found that the Multiplex bead assay provided a higher titration capacity, and a denser histogram of data when used to generate assay from a low-endemic sample population. Additional analyses found that a malaria-naive sample set was an appropriate reference when using a bead-based assay and that ELISA estimates for seropositives within a population varied greatly depending on the method used to develop a seropositivity cutoff value. The novel method of applying B-splines to serological data provides an additional technique for displaying this type information.Malaria serological data have shown great potential for the elucidation of varying transmission regions within an area. Numerous


Plasmodium falciparum malaria is controlled. Transcriptomic studies in humans have so far been limited to retrospective analysis of blood samples from clinical cases. In this prospective, proof-of-principle study, we compared whole-blood RNA-seq profiles at pre-and post-infection time points from Malian adults who were either asymptomatic (n = 5) or febrile (n = 3) during their first seasonal PCR-positive P. falciparum infection with those from malaria-naïve Dutch adults after a single controlled human malaria infection (n = 5). Our data show a graded activation of pathways downstream of pro-inflammatory cytokines, with the highest activation in malaria-naïve Dutch individuals and significantly reduced activation in malaria-experienced Malians. Newly febrile and asymptomatic infections in Malians were statistically indistinguishable except for genes activated by pro-inflammatory cytokines. The combined data provide a molecular basis for the development of a pyrogenic threshold as individuals acquire immunity to clinical malaria.Identifying molecular predictors and mechanisms of malaria disease is important for understanding how  Plasmodium, afflicting ~200 million people globally per annumPlasmodium falciparum is responsible for the majority of malaria-related mortality. Plasmodium infection in the host liver is an asymptomatic process but quickly progresses to the blood stage, during which the merozoite form of the parasite invades and replicates within erythrocytes. In non-immune individuals, it is the cyclical rupture of infected erythrocytes and release of daughter merozoites during blood-stage infection that coincides with the paroxysms of fever and chills typical of clinical malaria.Malaria is a significant public health burden caused by parasites of the genus P. falciparum-induced inflammation via a regulatory state mediated in part by the anti-inflammatory cytokine IL-10 produced by CD4+CD25+Foxp3- T cells that also produce IFNγ and TNF6in vitro using parasite-stimulated peripheral blood mononuclear cells obtained from individuals with varying degrees of prior malaria exposure. Ex vivo transcriptional profiling of peripheral blood cells has provided broader and more direct insight into cellular immune responses to natural Plasmodium blood-stage infections in malaria-experienced individuals5910111210P. falciparum.Although the role of antibodies in controlling both parasite density and the symptoms of malaria is well established2P. falciparum infection of the malaria season occurredP. falciparum infection of the malaria season and compared these gene-expression profiles to those of previously malaria-naïve adults who were infected with P. falciparum in a CHMI studyP. falciparum-induced interferon and inflammatory responses during acute infection despite having higher parasite densities than naïve individuals.We recently conducted a prospective cohort study of malaria-experienced individuals in Kalifabougou, Mali during which we collected blood samples by finger prick every 2 weeks and determined by PCR when each individual’s first P. falciparum PCR-negative, uninfected baseline and during their incident, P. falciparum PCR-detectable blood-stage infection, were included in the group RNA-seq analyses . Among the 6 Malians for whom urine samples were available, none were positive for Schistosoma haematobium by microscopy. No Malian individuals were infected by the bloodborne filarial nematode Mansonella perstans by PCR of blood gDNA at either the uninfected baseline or the first P. falciparum infection of the malaria season.Thirteen subjects, each with paired samples collected at their analyses ; Table 1The average number of mapped reads per sample was 71 million paired-end reads with a range of 24 to 130 million paired-end reads . Using aP. falciparum infection status, regardless of whether the top 50% or the top 10% most variably expressed genes were used . Hierarchical clustering of the transcriptome correlation matrix of all samples, after removal of site-specific batch effects, segregated individuals into 2 major clusters defined by ere used . InteresP. falciparum infection between malaria-naïve and malaria-experienced individuals with and without disease, we employed RNA-seq analysis of uninfected/infected sample pairs in a multifactorial design to account for intra-individual variation and enhance statistical powerP. falciparum infection more similarly to each other than to malaria-naïve individuals irrespective of febrile symptoms by both principal component analysis (PCA) and hierarchical cluster analysis using the top 50% (8208) most variably expressed genes  and Ingenuity canonical pathways by Fisher’s exact test. Using a cut-off of P < 0.05 (adjusted for multiple comparisons using the Benjamini-Hochberg method), acute febrile malaria significantly affected 196 pathways in the ΔNF class and 198 pathways in the ΔEF class  and interferon-related genes  in the ΔNF class  from each of the 6 contrasts outlined in the Methods  to Ingenn scores . Overalln scores . IL1RN, n scores . AlthougNF class . InteresP. falciparum infection. Consistent with pathways and upstream regulator analyses, the ΔNF class had greater overexpression of interferon modules  than the ΔEF and ΔEA classes , which are expected to induce more modest gene expression. Other possible reasons include the 2-week resolution for active surveillance for P. falciparum-induced gene expression between the malaria-experienced classes. Yet, direct comparison of ∆EF vs. ∆EA revealed only 70 DEGs using the same significance thresholds. Inspection of the top DEGs within the ∆EA class with an unadjusted P value <0.05 provides some explanation for this discrepancy  analysis between classes using only pre-infection baseline data, as the NF class and Dutch study site were synonymous. Interestingly, removal of study-site batch effects, which also removes biological variation, still resulted in higher within-class correlations than between-class correlations among pre-infection baseline transcriptomes, a finding that could be reflective of either the disparate genetic and/or environmental backgrounds between classes and/or differences in prior malaria-exposure. Although this observation requires evaluation in a larger study, the notion that pre-infection cellular signatures can predict host responses during blood-stage infection is compelling, given recent evidence that pre-perturbation cell populations can predict antibody responses induced by influenza vaccinationP. falciparum transcriptomes using whole blood obtained during acute malariaP. falciparum-specific read counts, but the low-density infections in both the NF and EA classes precluded us from obtaining sufficient P. falciparum read counts in these samples for DGE analysis with the EF class. Future attempts at dual RNA-seq with low-density infections will likely require isolation of host and parasite genetic material prior to RNA collection.Dual host-pathogen RNA-seq has previously been used to investigate interactions between the human and P. falciparum blood-stage infections that would otherwise induce intense inflammation in naïve individuals at much lower parasite densities. Differences in the molecular intensity of febrile malaria are accompanied by qualitative differences, with prominent activation of B-cell receptor signalling that is unique to malaria-experienced individuals. Taken together, these data provide a molecular basis for the development of a pyrogenic threshold as individuals acquire natural immunity to malaria disease. Larger studies are needed to validate these findings and to gain further insight into the cellular immune response during asymptomatic Plasmodium infections.In summary, we provide transcriptomic evidence of disease tolerance in human malaria, demonstrating regulated interferon-mediated inflammatory responses in malaria-experienced individuals during http://www.clinicaltrials.gov (NCT01322581). The CHMI study was approved by the Central Committee for Research Involving Human Subjects of The Netherlands (NL33904.091.10) and complied with the Declaration of Helsinki and Good Clinical Practice.Written informed consent was obtained from all subjects. The Malian cohort study was approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The study in Mali is registered on P. falciparum malaria transmission is intense and seasonal, occurring from June through DecemberPlasmodium infection and weekly active and passive surveillance for clinical malaria. Exclusion criteria included a haemoglobin level <7 g/dL, axillary temperature ≥37.5 °C, acute systemic illness, underlying chronic disease, use of antimalarial or immunosuppressive medications in the past 30 days, or pregnancy. Haemoglobin typing for HbS and HbC was performed with a D-10 instrument (Bio-Rad). Malaria episodes were pre-defined as parasitaemia of ≥2500 parasites/μL, an axillary temperature of ≥37.5 °C within 24 hours, and no other cause of fever discernible by physical exam. By finger prick we collected whole-blood for RNA (200 μl whole blood in 400 μl of Tempus solution [Applied Biosystems]), dried blood spots (DBS) on filter paper , cell pellets, and plasma from healthy, uninfected children and adults at enrolment , during bi-weekly scheduled visits, and at their first malaria episode of the ensuing transmission season. After transport from the field site, RNA, blood pellets, and plasma were stored at −80C until analysis. Point-of-care blood smears were performed for individuals symptomatic at any clinic visit, with anti-malarials given for any Plasmodium positive smears per the Malian national guidelines. For each subject, the first P. falciparum infection of the malaria season was detected retrospectively by PCR analysis of the longitudinally collected DBSThe malaria-experienced individuals for this study are part of a larger on-going prospective cohort study of naturally acquired malaria immunity15P. falciparum-specific antibodies approach adult-like levels in this cohortP. falciparum infection  and 2) individuals who were asymptomatic at the time of their first PCR-detected P. falciparum infection  for whom samples were available for RNA-seq.Here, we focus on individuals >13 years old, which is the age when levels of Schistosoma haematobium eggs by microscopy after urine filtration. Presence of Schistosoma mansoni eggs was assessed by microscopy of stool using the Kato-Katz techniqueNecator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis were detected following DNA extraction from stoolMansonella perstans was also performed on gDNA extracted at both the uninfected baseline and the first P. falciparum infection of the malaria season with two 3-mm DBS punches using an established protocolScreening for helminths at uninfected baseline (enrolment) was performed as previously describedP. falciparum infection  included five individuals who were recruited as infectivity controls for a previously described CHMI study (NCT01218893)P. falciparum-specific antibodies by ELISA at enrolment, and none had travelled to a malaria-endemic area within 6 months prior to the study. In October 2011, individuals were challenged simultaneously by exposure to bites of five Anopheles stephensi mosquitoes infected with the NF54 P. falciparum strain and monitored by daily thick blood smear starting on days 5–6 post-challenge. Whole blood was collected for RNA in PAXgene blood RNA tubes  on the day prior to challenge; days 5, 6 and 9 after challenge; the day of treatment; and day 35 after challenge. In addition to blood-smear diagnosis, parasite density was also retrospectively quantified by qPCR of blood DNA samples collected up to twice daily from day 5 until day 21 after challenge to determine the time to PCR-positivity as described previouslyThe malaria-naïve individuals in this study, all of whom were febrile during Plasmodium falciparum 3D7  genomes in parallel using TopHat2 For all Dutch samples, total RNA was extracted from whole blood using the PAXgene Blood miRNA Kit (Qiagen). RNA was converted to cDNA and amplified using Ovation RNA-Seq System V2 (Nugen), which uses single-primer isothermal amplification equivalent to 12 cycles of PCR amplification, and library preparation was performed with the TruSeq Nano Kit (Illumina) per the manufacturers’ instructions. For the Malian samples, total RNA was extracted from whole blood using the Tempus Spin RNA isolation kit (Thermo Fisher) and depleted of rRNA and globin RNA before amplification using the ScriptSeq Complete Gold Kit (Illumina). Directional RNA-seq libraries were prepared using the ScriptSeq v2 kit with barcode adapters (Epicentre), which includes a 15-cycles of PCR amplification. For both Malian and Dutch samples, sequencing of 2 × 100 bp paired-end reads was performed on a HiSeq 2000 using V3 reagents (Illumina). The Illumina sequences were trimmed of bases with a Phred quality score of less than 15 and any contaminating adapters used in the preparation of cDNA and sequencing libraries by the Trimmomatic programGene expression analysis of RNA-seq read counts mapping to the human genome was performed using edgeR (version 3.10.2)infected − logCPMuninfected (delta logCPM) values were used for each subject. We compared the effect of infection within subjects as well as the differences between classes while taking into account the pairing of samples by subject. We used the following model formula which has class as the main effect with nested interactions for subject and infection status: ~Class + Class:Subject + Class:Infection. Thus, differences in batch, sex, and age were accounted for by the subject effect, as each subject’s pre-infection baseline served as his or her own control. DGE analysis was performed by fitting counts for each gene to a negative binomial generalized log-linear model using the glmLRT function for the following within class comparisons, set up as contrasts:Expression data for the top 50% most variable genes by median absolute deviationBetween-class comparisons were also performed:P values, and false discovery rates from results of the model were imported into Ingenuity Pathway Analysis  to determine pathway enrichment scores and perform upstream regulator analyses and into a custom R script for modular transcriptional repertoire analysis19Gene symbols, log-fold change ratios, unadjusted P. falciparum reference genomes sought to determine relationships between the host and parasite transcriptomes in minimally processed finger prick whole blood samples, with the goal of comparing parasite transcriptomes in both asymptomatic and febrile infections. Such a strategy yields sufficient human but not P. falciparum mapped counts, presumably due to the overwhelming abundance of human transcripts in whole blood un-enriched for parasite material, with adequate library sizes for P. falciparum only observed at high parasite densities  as indicated in the results or figure legends. Statistical significance was set at FDR <0.05 unless noted otherwise.Statistical analyses were performed in R 3.2.3 .


Plasmodium vivax predominance.Most commonly used malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections which are frequent in low transmission settings. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too laborious for field deployment. In this study, the applicability of a malaria diagnosis kit based on loop-mediated isothermal amplification (mLAMP) was assessed in malaria endemic areas of Colombia with P. falciparum accounted for 23.9% and P. vivax for 76.1% of the infections and no cases of mixed-infections were identified. Microscopy sensitivity for P. falciparum and P. vivax were 100% and 86.1%, respectively. mLAMP sensitivity for P. falciparum and P. vivax was 100% and 91.4%, respectively. In the ACD study, mLAMP detected 65 times more cases than microscopy. A high proportion (98.0%) of the infections detected by mLAMP was from volunteers without symptoms.First, a passive case detection (PCD) study on 278 febrile patients recruited in Tierralta (department of Cordoba) was conducted to assess the diagnostic performance of the mLAMP method. Second, an active case detection (ACD) study on 980 volunteers was conducted in 10 sentinel sites with different epidemiological profiles. Whole blood samples were processed for microscopic and mLAMP diagnosis. Additionally RT-PCR and nested RT-PCR were used as reference tests. In the PCD study, P. vivax low-endemicity settings and under minimum infrastructure conditions, it displayed sensitivity and specificity similar to that of single-well RT-PCR for detection of both P. falciparum and P. vivax infections. Here, the dramatically increased detection of asymptomatic malaria infections by mLAMP demonstrates the usefulness of this new tool for diagnosis, surveillance, and screening in elimination strategies.mLAMP sensitivity and specificity were comparable to RT-PCR. LAMP was significantly superior to microscopy and in  Plasmodium vivax predominance, we conducted field studies using the LAMP kit implemented in remote settings. We found that LAMP sensitivity and specificity were comparable to RT-PCR for detection of both P. falciparum and P. vivax infections and dramatically increased detection of asymptomatic malaria infections. This simple detection method for very low parasitemia raises opportunities and new strategies for malaria elimination.The ability to detect and treat asymptomatic infections will be fundamental to eliminate malaria. This requires highly sensitive screening tests close enough to cases to enable rapid treatment. Very low parasitemias can be detected by molecular methods such as PCR; however, these techniques require considerable training and are restricted to reference laboratories. A new field-stable diagnostic kit for malaria based on loop-mediated isothermal DNA amplification (LAMP) is now commercially available. This LAMP kit is able to detect down to 1 parasite/µl of blood in less than 1 hour. In order to evaluate the feasibility of this LAMP kit as a tool for the detection of asymptomatic malaria in malaria endemic areas of Colombia with  Malaria causes over 207 million clinical cases and ∼627,000 deaths worldwide every year representing an enormous global social and economic burden Plasmodium species on thick blood smear (TBS) has been the standard method for malaria diagnosis for decades and currently represents the gold standard diagnostic method for malaria control programs, its field accuracy, errors in species identification and its operator-dependence significantly limits its usefulness particularly in regions of low malaria transmission with high prevalence of asymptomatic low-density infections. Microscopy has usually been considered less expensive and easier to handle than other malaria diagnostic methods developed more recently such as nucleic acid amplification test (NAAT); however, it is laborious, time consuming, and requires well trained personnel which are frequently not available in remote malaria-endemic areas. This creates uncertainty about the cost-effectiveness of microscopy in low transmission settings 10 times amplification within 15 to 60 minutes), it is faster than PCR, and it can be performed in basic laboratories without the need for specialized infrastructure.Although microscopic detection of Plasmodium mitochondrial DNA (mLAMP) displayed sensitivities ranging between 89.5–93.3% and a specificity between 95.9–100% Using nested PCR as reference standard Plasmodium species and allows for P. falciparum speciation. New versions of this kit are in development P. vivax speciation P. vivax and P. falciparum are present at different proportions.Diagnostic tools like mLAMP which are capable of detecting very low parasite densities (1 parasite/µL) and reliable under field-conditions are urgently needed for active case detection to accelerate malaria elimination P. vivax and P. falciparum malaria parasites:A total of 10 sentinel sites were selected from three well characterized malaria endemic setting of Colombia with different malaria transmission intensities and different prevalence of P. vivax (82.4%) followed by P. falciparum (17.4%) and mixed malaria infections (0.2%) Tierralta is a municipality located in the Department of Cordoba in the northwestern part of Colombia with ∼90,000 inhabitants composed of ∼2% indigenous-groups and 44.4% rural population. The predominant malaria parasite species in this region is P. vivax (85%) followed by P. falciparum (15%). The sentinel sites selected in this region were Punta Soldado, Zacarias and La Delfina.Buenaventura is located on the Pacific coast of Valle Department in the west of Colombia, with ∼350,000 inhabitants which are predominantly Afro-Descendant (72.4%). The predominant malaria parasite species in the region is P. falciparum (79.2%) followed by P. vivax (20.8%). The sentinel sites selected in this region were Robles, Candelillas and Bucheli.Tumaco is located in the Department of Nariño in the southwest of the country near the Ecuador border. It has a population of ∼187,084 inhabitants composed mainly of Afro-Descendants (88%). The predominant malaria parasite species in the region is 278 febrile adults and children attending San Jose Hospital in Tierralta between April and December, 2013 were invited to participate in the study and requested to sign an informed consent (IC) previously approved by the Caucaseco Scientific Research Center Institutional Review Board (IRB). For mLAMP processing, whole blood were collected from each patient by arm venipuncture using heparinized tubes. TBS was done from finger prick and PCR from EDTA tubes.A total of 980 adult and children volunteers were enrolled under a cross-sectional survey carried out in the ten sentinel sites between October and December 2013 during the rainy season despite the transmission in Colombia is perennial. The cross- sectional study was performed in two steps: a) a door-to-door census of the entire population of each site and b) blood-sample collection in volunteers from a subset of randomly selected houses. After written IC/assents were obtained from adult and minors (1–17 years of age) and their parents/guardians, respectively, a trained member of the study staff collected clinical, demographic and epidemiological information from each participant using a standardized questionnaire. Additionally, a trained team of physicians collected the blood for lab tests (1 mL/each) which were processed as described below. Microscopy and mLAMP were performed in the field whereas RT-PCR was performed in Caucaseco Scientific Research Center (SRC) in Cali, Colombia. For mLAMP processing, blood samples (60 µL) were collected from finger pricks using disposable Pasteur pipets and delivered into vials containing 60 µL of lysis buffer  that were maintained at room temperature (25–30°C) for ∼24 hour until reach the processing site.For training and initial validation of mLAMP, a subset of samples with confirmed malaria diagnosis was used. A total of 150 blood samples were extracted by the boil and spin method and were evaluated using the mLAMP kit. The results were compared to those of RT-PCR performed using purified DNA . Discordant samples were tested in triplicates using nested RT-PCR (3-well RT-PCR).Using approximately 100 µL of blood previously collected by finger prick, both thick and thin duplicate smears were prepared, stained with Giemsa stain Two laboratory technicians in each sentinel site and with no previous experience on molecular methods were trained during 3 days on LAMP procedures for samples collection, processing, amplification and detection. Demonstration of procedures were done during day 1, supervised performance of the test during day 2, and blind testing of reference samples during day 3.In Tierralta and Buenaventura, DNA extraction and mLAMP were performed in small local laboratories whereas samples in lysis buffer from Tumaco were sent by plane for processing in Cali. Stability of blood in lysis buffer at 37°C for 24 h was assessed to ensure quality of DNA . DNA extPlasmodium species (Pan-mLAMP) as well as specifically detecting P. falciparum (Pf-mLAMP). All reaction tubes were placed into a LAMP incubator for DNA amplification at 65°C for 40 minutes and reaction inactivation at 80°C for 5 minutes. Results were read by visual fluorescence by two independent readers and a third reader was called if discordances occurred. Positive and negative controls were included for each batch of reactions. For mLAMP implementation in the field, laboratory personnel and field technicians were trained on all study procedures in a three-day workshop. Complete standard operating procedures for the mLAMP assay and boil and spin method are available online mLAMP kits  were transported and stored at ambient temperature and used before the expiration date as recommended by the manufacturer. For each sample, 25 µL of diluted DNA were transferred into reaction tubes with dried reagents designed to detect all P. falciparum and P. vivax DNA positive and negative controls were used in each batch of tests including the extraction of both negative and inhibition control. A sample was considered negative if there was no increase in the fluorescent signal after a minimum of 40 cycles. Parasitemia quantification was performed using a parasite specific standard curve made with serial blood dilutions of a reference field isolate. Each reaction plate included a standard curve for parasite quantification.Assays were performed as described previously Plasmodium species. The second PCR reaction was developed as previously described For positive samples, DNA was extracted using the PureLink genomic DNA kit and 2 µL of DNA were used to carry out the species-specific nested RT-PCR Pf assays were analyzed independently. All mLAMP and RT-PCR discordant samples and an equal number of randomly selected concordant samples (both for Pan and Pf LAMP assays) were re-tested in a blinded fashion by mLAMP and nested RT- PCR in triplicates. The sample was declared as positive if at least 1/3 reaction well was positive.LAMP results with Pan and Information was captured in the field in paper-based case report forms (CRF). Data was digitized using REDCap (version 4.1) and imported into MATLAB (version 2011b) for analysis.The data quality assurance (QA) procedure consisted of setting a quality control (QC) sample of size q less than the total number p of all CRFs for each location. The maximum permissible error for this study was set to 1%.Data analysis was performed using MATLAB (version 2011b). Using descriptive statistics, the general characteristics of individuals admitted to the study were established. Measures of central tendency and dispersion were calculated for qualitative characteristics, whereas absolute frequencies as well as confidence intervals were used for quantitative characteristics. The primary analysis was conducted using RT-PCR as gold standard to determine diagnostic performance characteristics  and positive (PPV) predictive values, and overall diagnostic accuracy) of mLAMP.This study was carried out in accordance with institutional guidelines. The protocol was previously reviewed and approved by the IRB of Caucaseco Scientific Research Center . Written informed consent (IC) was obtained from each volunteer at enrollment. Parents were asked for consent for their child to take part in the study. Information obtained from the participants was managed on principles of confidentiality. Immediately after blood donation, malaria positive volunteers were provided with the antimalarial treatment recommended by the Colombian Ministry of Health.P. vivax, 30 P. falciparum and 50 Plasmodium negative samples) at the Caucaseco SRC. The sensitivity and specify of mLAMP using both Pan and Pf reaction tubes under laboratory conditions were 81% (95%CI: 75–93) and 100% (95%CI: 87–100) respectively for P. falciparum whereas for P. vivax the sensitivity and specificity were 98% (95%CI: 89–100) and 82% (95%CI: 73–89) respectively. Most of the false negative results (3 out of 4) corresponded to samples with low density P. vivax infections as determined by RT-PCR.An initial assessment of the mLAMP kit was done with 150 well characterized blood samples  and 70 P. vivax cases . All the case were non-complicated malaria. 94 out of the total 278 samples prepared by the boil and spin method (33.8%) were positive with Pan-mLAMP malaria specific primers. To determine Pan-mLAMP performance, a corrected data base was obtained after adjusting the discordant results using nested RT-PCR as reference. Pan-mLAMP displayed 100%, sensitivity, 98.9% specificity, 76.9% PPV and 100% NPV for P. falciparum. In contrast, for P. vivax samples, Pan-mLAMP displayed 91.4%, sensitivity, 91.8% specificity, 80.5%, PPV, and 99.6% NPV  P. vivax and 0% mixed infections. Pan-mLAMP detected 98.0% of these asymptomatic infections confirmed by RT-PCR, most of which occurred among participants >15 years old. Malaria prevalence across all sentinel sites was 0.2% by microscopy (n = 2) and 6.6% by Pan-mLAMP (n = 65). Comparison of Pan-mLAMP with corrected single-well RT-PCR for P. falciparum indicated that Pan-mLAMP had 100% sensitivity, 99.9% specificity, 93.8% PPV and 100% NPV. In the case of P. vivax samples, Pan-mLAMP presented 90.9% sensitivity, 99.5% specificity, 90.9% PPV and 99.5% NPV  when compared to microscopy (28.1%). For A significant number of infections with low parasite density were detected when using mLAMP for ACD. The kit achieved a sensitivity equivalent to RT-PCR in the detection of infections of low malaria parasite density. The estimate of malaria prevalence by mLAMP was 6.1% (n = 60) with mostly asymptomatic cases (59/60). The identification and management of asymptomatic carriers has become a new and increasingly important challenge for malaria control programs The processing time for mLAMP in each of the sentinel sites was <1 day, whereas PCR results were available in 7 days. This significant time reduction would directly translate into timely initiation of treatment and reduction of patient withdrawal from treatment. mLAMP requires less training, is faster and requires minimal equipment compared to RT-PCR. The combination of high sensitivity and specificity together with the ease and efficiency of performance and stability at ambient temperature makes mLAMP a promising new tool for malaria surveillance, particularly for the detection and prompt treatment of asymptomatic infections in low transmission settings. In regions of declining malaria transmission, the use of highly sensitive tools such as mLAMP in remote clinics would contribute to the reduction of transmission and to the acceleration of malaria elimination.Plasmodium genus and P. falciparum–specific LAMP kit is able to identify all P. falciparum–infected individuals. Non-falciparum Plasmodium species are also detected but not resolved to the species level, a limitation shared with most RDT tests. Moreover, under the current configuration, the kit does not differentiate mixed infections from P. falciparum mono infections. Despite this limitation, by the combined use of the Pan-malaria and P. falciparum kits, we could identify all P. vivax cases as confirmed by RT-PCR. Reagents able to identify infections with other species and more specifically with P. vivax parasites, as well as mixed infections, are required to guide appropriate treatment of malaria cases in the clinical setting.The current format of the Pan-This malaria mLAMP kit is an easy-to-use and field stable diagnostic tool which brings molecular techniques to areas with low laboratory infrastructure. The availability of this technique opens news perspectives in the implementation of surveillance and response activities in malaria elimination campaigns. The detection and prompt treatment of sub-microscopic asymptomatic infections in less than one hour after sample collections may contribute to the control of transmission and accelerate malaria elimination.S1 TableSTARD checklist.(DOCX)Click here for additional data file.S1 FigSTARD flow chart for PCD S2.(TIF)Click here for additional data file.S2 FigSTARD flow chart for ACD S3.(TIF)Click here for additional data file.S1 TextSample stability.(DOCX)Click here for additional data file.


Plasmodium falciparum malaria.Naturally acquired immunity to clinical malaria is thought to be mainly antibody-mediated, but reports on antigen targets are contradictory. Recognition of multiple antigens may be crucial for protection. In this study, the magnitude of antibody responses and their temporal stability was assessed for a panel of malaria antigens in relation to protection against clinical P. falciparum-antigens apical membrane protein (AMA)-1, merozoite surface protein (MSP)-119, MSP-3, glutamine-rich protein (GLURP-R0) and circumsporozoite antigen (CSP) were assessed by enzyme-linked immunosorbent assay (ELISA) for 99 children with plasma available at all time points. Parasite carriage was determined by microscopy and nested PCR.Malian children aged two to 14 years were enrolled in a longitudinal study and followed up by passive and active case detection for seven months. Plasma was collected at enrolment and at the beginning, in the middle and after the end of the transmission season. Antibody titres to the 19, and the number of antigens recognized increased with age. After malaria exposure, antibody titres increased in children that had low titres at baseline, but decreased in those with high baseline responses. No significant differences were found between antibody titers for individual antigens between children remaining symptomatic or asymptomatic after exposure, after adjustment for age. Instead, children remaining asymptomatic following parasite exposure had a broader repertoire of antigen recognition.Antibody titres to all antigens, except MSP-1The present study provides immune-epidemiological evidence from a limited cohort of Malian children that strong recognition of multiple antigens, rather than antibody titres for individual antigens, is associated with protection from clinical malaria.The online version of this article (doi:10.1186/s12936-015-0567-9) contains supplementary material, which is available to authorized users. Despite the success of major public health control efforts in recent years , malariaPlasmodium parasites [The most commonly studied antigens in this respect are merozoite antigens, which are expressed both by mature liver schizonts as well as during the late schizont and merozoite-stage of blood-stage arasites . Studiesarasites . One hyparasites . Other iarasites . Additioarasites -10, whicarasites .19 and glutamine-rich protein (GLURP-R0), alone or in combination, may be associated with protection from clinical malaria in a hyper-endemic area. It was further assessed whether this association was temporally stable or dependent on the time point of sampling.Using a set of samples collected at four time points from a cohort of children followed up longitudinally for seven months, it was investigated whether a high concentration of antibodies against different pre-erythrocytic, blood-stage and cross-stage antigens, namely circumsporozoite antigen (CSP), merozoite surface protein (MSP)-3, apical membrane protein (AMA)-1, MSP-1Plasmodium falciparum is the predominant Plasmodium species in this region and accounts for more than 95% of malaria cases [et al., unpublished data).This study was conducted in Malian children from Samako, a village of about 3,000 people located in the Sudanese savannah zone of the Upper Niger valley (district of Kati) about 70 km southwest of Bamako, the capital of Mali. Samako is 5 km from Bancoumana where the Malaria Research and Training Centre has established a malaria vaccine site since 2000. ia cases . Transmiia cases . The stuia cases . From JuWritten informed consent was obtained from parents or legal guardians who consented on behalf of their children. All laboratory procedures were carried out within the guidelines of good laboratory practice. Ethical clearance to conduct the study was sought from the ethical committee of the Faculty of Medicine, Pharmacy and Odonto-Stomatology at the University of Science, Techniques and Technologies of Bamako .One physician and one biologist were based in the village to follow up a cohort of children aged two to 14 years during one malaria transmission season. In December 2011, 171 children were enrolled in the study and subsequently attended three additional cross-sectional visits during the transmission season in July 2012 (n = 134) and September 2012 (n = 137) and after the end of the transmission season in February 2013 (n = 111). Ninety-nine children attended all three cross-sectional visits and provided samples during and after the 2012 transmission season and were selected for longitudinal immunological analysis in this study.3, taking into account that low parasite densities are not always the causative factor of clinical symptoms [From July until December clinical malaria infection was monitored by active and passive case detection. The active case detection was carried out by house visits and cross-sectional survey. On a 14-day basis, field workers conducted active house visits to all the children to assess malaria infection. Participants were instructed to register any possible malaria symptoms in a diary. Passive case detection of clinical malaria episodes was carried out at the village health clinic. Children who visited the health clinic were identified using an individual identification card and were screened by a doctor. When children reported any of the malaria symptoms , a rapid diagnostic test  and thick smear were performed and axillary temperature was checked. A clinical malaria episode was defined by presence of any symptom of malaria including fever of 38°C or more, associated with a parasitaemia of at least 5,000 trophozoites/mmsymptoms . ChildreP. falciparum during follow-up  and stored at room temperature for retrospective PCR analysis to detect sub-microscopic infections. EDTA anti-coagulated plasma was collected and cryopreserved at −20°C.P. falciparum FVO, comprising amino acids 25–545, expressed in the yeast Pichia pastoris [Escherichia coli (MSP-119) [The following antigens were used to assess humoral responses: (i) the ectodomain of apical membrane antigen (AMA)-1 of pastoris ,15; (ii)pastoris ; (iii) fpastoris ; (iv) thMSP-119) ; and, , 0.5 μg/ml (MSP-3) or 0.25 μg/ml (CSP). Plates were incubated overnight at 4°C, washed and blocked with 200 μl of 5% milk powder in PBS for one hour. Due to the expected high inter-individual variation in antibody titers, plasma samples were assayed at two different dilutions (1:500 and 1:50) to ensure that at least one of the two would fit into the range of the standard curve. Fifty μl of diluted plasma samples were added in duplicate and incubated at room temperature for three hours. Plates were washed four times between steps. Polyclonal goat anti-human IgG-HRP  diluted 1:40,000 was added to antigen-coated plates. Bound secondary antibodies for total IgG were quantified with ready-to-use TMB  substrate for 30 min. Fifty μl of 0.2 M H2SO4 were used to stop the reaction. The plates were read out at the spectrophotometrical absorbance of 450 nm.Microtitre plates  were coated with 50 μl of antigen diluted in PBS at a concentration of 1 μg/ml (AMA-1), 0.5 μg/ml (GLURP-R0), 0.18 μg/ml  . Opticalet al. [et al. [P. falciparum was used. PCR was performed according to the original protocol except that the quantity of template used in the N1 reaction was increased from 1 to 5 μl. In every set of PCR conditions 5 μL template was used in the N1 reaction and 1.5 μl of product in the N2 reaction. For a more detailed overview of primer sequences and product sizes, and PCR cycling conditions, see Additional file P. falciparum NF54 cultured in Nijmegen, The Netherlands were run on every PCR plate as a positive control, alongside a negative water control. The positive control was diluted to the extent that both N1 and N2 fragments were sufficiently amplified so that both amplicons could be visualized on agarose gel. N1 and N2 products were mixed and 10 μl was visualized on 0.8% agarose gel by electrophoresis in 0.5× Tris-acetate-EDTA buffer .DNA was extracted from filter papers using the Chelex protocol as described by Walsh et al. . The 18S [et al.  targetinStatistical analysis was performed using GraphPad Prism v5 software and STATA version 12.0 . Differences in responses among different subject groups  were analysed by non-parametric measures. Mann–Whitney U test was used for two separate groups, Kruskal-Wallis with Dunn’s multiple comparison post-hoc test for more than two groups and Wilcoxon matched-pairs signed rank test for paired analysis of two time points. Associations between age and humoral responses were assessed by Spearman correlation; associations between the number of antigens recognized (AU > 10) and age in categories , parasite status at enrolment  and ‘protection’  was determined by Poisson regression where the number of antigens recognized was analysed as count variable. Trends in the proportion of individuals recognizing ≥1 or ≥3 antigens and age in categories or parasite status were determined by logistic regression. The association between experiencing clinical malaria episodes or asymptomatic malaria and the density of antibody responses was further assessed by logistic regression models with antibody densities as categorical variables . For analyses where ELISA outcomes were analysed on a continuous scale, AU were log-transformed (log10) and analysed by linear regression. All associations were adjusted for age where appropriate. A p-value of < 0.05 was considered statistically significant.P. falciparum infections was highest during the July cross-sectional visit at the beginning of transmission season, with a frequency of slide positive children of 27.6% (27/98), and a proportion of parasite-positive children by PCR of 78.6% (77/98)  Table . Both th8) Table . The numP. falciparum malaria antigens were assessed in the cohort at enrolment, the beginning of the transmission season (baseline), during and after the transmission season. At all time points there was a positive correlation between age and antibody titres for AMA-1, MSP-3, CSP and GLURP-R0, while responses to MSP-119 were not associated with age  of the hyperimmune reference serum  per individual, was stable over the time of follow-up, as evidenced by the strong correlation between this readout at enrolment (December 2011) and the beginning , peak  and end of the transmission season . Next it was investigated whether exposure during the transmission season altered antibody levels for the individual antigens. During the seven months of follow-up, 91/99 children became parasite-positive by either PCR or thick smear during one or more visits. Of these, 47 children remain asymptomatic while 44 experienced one or more episodes of clinical malaria . Since boosting of antibody responses may depend on the strength of the pre-existing response, all exposed children (n = 75) were stratified into low (<1 AU), intermediate (1–10 AU) and high responders (>10 AU) for each antigen. Low early season-responders for AMA-1, MSP-119 and GLURP-R0 showed higher antibody levels after the transmission season, while these titers remained largely unchanged in intermediate early season-responders  or after  the 2012 transmission season, in which clinical malaria was detected (Figure While recognition of individual antigen was therefore no good predictor of protection from clinical disease, children remaining asymptomatic recognized a broader repertoire of antigens compared to those that developed clinical disease Figure . Amongstd Figure D. Again,Immuno-epidemiological studies in multiple settings are needed to further an understanding of the relationship between naturally acquired antibody responses to the parasite and protection from clinical malaria. The key finding of the present study is that in a highly endemic area in Mali, the risk of developing clinical malaria upon exposure was reduced when children showed strong recognition of three or more malaria antigens at any time point during the study, regardless whether those responses were measured before or after the malaria risk was assessed.Studies into antibody correlates of protection can be affected by a number of confounders. A meta-analysis of other studies assessing the association between antibodies and protection from clinical disease found that most studies do not exclude individuals without detected parasitaemia during follow-up from the ‘protected’ group, although there is no evidence that these individuals were even exposed . Such prper se, but the acquisition of specific and strong responses with the degree of cumulative exposure that determines humoral protection.Age, reflecting cumulative malaria exposure, is an important determinant of protection against clinical malaria. In this study, children ranging from age two to 14 years were enrolled. A decreasing risk for clinical malaria , severe An important consideration when interpreting the findings from the current study is the size of the study cohort. In the current study, the number of children from which samples could be analysed was reduced by a number of factors, including commitment to longitudinal assessment and attendance of all cross-sectional visits. This approach was chosen to allow for a single consistent data set to be used for all analysis, as opposed to doing analysis for each time point with a different, only partially overlapping set of participants. While this did not lead to a skewing in the demographic or parasitological parameters compared to the original cohort, the two groups of children remaining asymptomatic or becoming symptomatic upon parasite exposure were too small for several of the results to reach statistical significance. Nevertheless, clear trends were observed throughout time points, justifying future studies in larger cohorts.It was previously highlighted that studies measuring responses at multiple time points are needed to better determine the serological status of an individual and whether the time point of observation may have an impact on the association of the humoral responses with protection ,43. ChanThe apparent decrease in antibody titres over the transmission season in these children might be an artifact caused by asymptomatic parasitaemia at time of plasma sampling: At the beginning of the transmission season the group of children with the highest antibody titres for individual antigens also had the highest frequency of parasitaemia, detected either by thick smear or PCR.That concurrent, even asymptomatic, low-density parasitaemia is associated with elevated antibody titres or prevalence, is in line with previous data ,41,45-4819 was the only one for which reactivity did not increase with age, nor did titers for it differ at all clinical protection in this study. Although antibodies to MSP-119 are generally found to confer protection [19 [19 antigen [Amongst the four blood- and cross-stage antigens examined, MSP-1otection ,51, thistion [19  and alsotion [19 , the rea antigen . Antibod antigen , CSP res antigen -57, but  antigen .P. falciparum antigens to samples from the present and similar studies with carefully defined, protected and non-protected cohorts will be a useful tool to extend these findings and add to the first few existing studies to identify novel antigens and panels thereof associated with protection from clinical disease [When analysed as single antigens, only antibody responses to AMA-1 were found to be associated with a reduced risk of clinical malaria. In contrast, high responses for a combination of at least 3/5 malaria antigens showed a relatively consistent protective effect across all time points analysed. These findings are in line with previous studies showing that protection against malaria may not be associated with the humoral responses to one antigen in isolation, but instead with recognition of a panel of antigens -10,59,60 disease . Based o disease -63, whicThe present study provides immune-epidemiological evidence from a limited cohort of Malian children that protection from clinical malaria is associated with high antibody titres for three or more malaria antigens. Future studies should confirm these findings for other settings and study periods with study populations that are adequately powered to detect more subtle associations between antibody recognition, age and clinical malaria.


Several studies have reported an association between malaria infection of the placenta and the risk of malaria in young children in the first year of life, but it is not known if this is causal, or influenced by malaria control measures during pregnancy. This paper compares the incidence of malaria in infants born to mothers who received either intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp-SP) or screening with a rapid diagnostic test and treatment with artemether–lumefantrine (ISTp-AL) during their pregnancy.From July 2011 to April 2013, 988 infants of women enrolled in a trial of IPTp-SP versus ISTp-AL in the Kassena-Nankana districts of northern Ghana were followed to determine the risk of clinical malaria during early life, and their risk of parasitaemia and anaemia at 6 and 12 months of age. In addition, the incidence of clinical malaria in infants whose mothers had malaria infection of the placenta was compared with that in infants born to women free of placental malaria.The incidence of clinical malaria was 0.237 and 0.211 episodes per child year in infants whose mothers had received ISTp-AL or IPTp-SP, respectively. The adjusted incidence rate ratio and the adjusted rate difference were 0.94  and 0.029  cases per child year at risk respectively. The incidence of clinical malaria was similar in infants born to women with placental malaria (0.195 episodes per child year) and in infants of women without placental malaria (0.224 episodes per child year) .Infants born to women managed with ISTp-AL during pregnancy were not at greatly increased risk of malaria compared with infants born to women who had received IPTp-SP. The incidence of malaria in infants was similar whether or not their mother had had placental malaria.The online version of this article (doi:10.1186/s12936-016-1094-z) contains supplementary material, which is available to authorized users. Malaria infection during pregnancy is a major public health concern because of the risks malaria poses not only to the mother but also to her infant . Some stPlasmodium falciparum is still sensitive to SP .The study was approved by the Navrongo Health Research Centre Institutional Review Board, the Committee on Human Research, Publication and Ethics of the Kwame Nkrumah University of Science and Technology and Komfo Anokye Teaching Hospital, both in Kumasi, Ghana as well as by the London School of Hygiene and Tropical Medicine Ethics Committee. Written informed consent was obtained from all women on behalf of their offspring. The conduct of the trial was guided by the Data Safety and Monitoring Board. The clinical trial of ISTp-AL A total of 988 infants were enrolled  and 54 of the 495 in the IPTp-SP group 12.2 %) experienced at least one passively detected episode of clinical malaria  . At 12 months of age the overall prevalence of parasitaemia was 10.8 and 9.5 % in the ISTp-AL and IPTp-SP groups, respectively, prevalence ratio 1.18 . The prevalence of anaemia was 68.0 % in the ISTp-AL group and 73.1 % in the IPTp-SP group at 6 months of age, prevalence ratio 0.94  and 84.7 and 83.1 % in the ISTp-AL and IPTp-SP groups, respectively, at 12 months of age, prevalence ratio 1.02 . Similar results were found for the ATP2 population  whose mothers’ PM status was known, the incidence of malaria and other study endpoints was similar among infants whose mothers had PM and infants whose mothers did not. The incidence of clinical malaria episodes in the PM-negative and PM-positive groups, respectively, were 0.22 per year and 0.20 per year, rate ratio 0.86 . The respective incidence rates in the PM-negative and PM-positive groups for non-malarial fevers were 0.90 per year and 0.73 per year with a rate ratio of 0.85 . For anaemia, respective incidence rates in the PM-negative and PM-positive groups were 0.16 per year and 0.20 per year, with an overall rate ratio of 0.99  (Table Nelson-Aalen plots of cumulative incidence of clinical malaria among infants of mother’s PM positive or negative and gravidity status also showed similarity in the incidence malaria Figs. , 5, respThe findings of this study suggest that children born to women who were managed with ISTp-AL during ANC visits did not experience an elevated risk of clinical malaria compared to those born to mothers who were given IPTp-SP. The incidence of all febrile illnesses, non-malaria fever and anaemia was also similar in the two groups of children. This finding may be attributable to the randomization of a large number of women to IPTp-SP or ISTp-AL groups successfully balancing factors related to exposure of the mother, and consequently the exposure of the infant, and because the two interventions performed similarly well in preventing PM.The risk of malaria in infancy was not increased in the infants of women who had PM, agreeing with some previous studies that investigated the risk among primigravidae  or in alThis study had a number of limitations. As there was a delay in obtaining the resources needed to conduct the study, not all infants were recruited immediately after birth and the time period during which the one-year period of follow-up took place varied from nought–12 to 5–17 months of age. However, the age pattern of follow-up was very similar in the two groups. In addition, 18.7 % of women in the initial cohort and their infants were not enrolled for follow-up in this study, primarily because some mothers who had delivered in the main study could not be located when the infant follow-up began. Randomization of infants was dictated by the study group of their mothers and not by randomization of the infants themselves, but the baseline characteristics of the infants in each study group were well balanced. The sample size of the infant population was driven by the number of women enrolled into the pregnancy trial and could not be adjusted for the purpose of this follow-up study. The study was planned to have sufficient power to exclude a relative increase in the incidence of malaria of 20 % between the two groups, assuming the malaria incidence rates previously found in the study area. However, the much lower than expected incidence of malaria meant that it was not possible to rule out differences of this magnitude between the ISTp-AL and IPTp-SP groups.P. falciparum is still sensitive to SP. However, if ISTp-AL is to be deployed in the future in any specific epidemiological situation, the results of this study suggest that infants will not be put at any major increased risk.There are currently no grounds for changing the well-accepted WHO policy of administration of SP at each routine ANC attendance in the study area, or in other areas where


Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels.Malaria is a public health problem in parts of Thailand, where P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals.Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others.Of 219 samples from the village, qPCR detected 25 (11.4%) These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.The online version of this article (doi:10.1186/s12936-015-0611-9) contains supplementary material, which is available to authorized users. Plasmodium species that cause human disease  and dknowlesi . A majorknowlesi ,5.In Thailand, malaria control efforts have been highly effective in curbing the infection nationwide . NonetheP. falciparum and P. vivax proteins.In light of this, and of reports on high prevalence of subpatent asymptomatic infections in other regions -19, the n = 558) detected one (0.17%) positive infection with P. falciparum. The predominant malaria vectors are Anopheles dirus, Anopheles maculatus and Anopheles minimus. Five malaria species that cause human infection are found in Thailand, but P. falciparum and P. vivax are vastly predominant [The study was conducted in the northwestern Province of Tak in Thailand, on the bank of Moei River, bordering with Myanmar. The study sites are located 51 km apart: community samples were collected in the hamlet Mae Salid Noi , and malaria clinic samples were collected in the town of Mae Tan . The climate in this region is tropical. Average temperature ranges from 20.2°C in December to 29.3°C in April . Rainy sdominant ,21,22.Firstly, active case detection (ACD) surveys were conducted in Mae Salid Noi, and resident’s health status history was recorded weekly during a five-month period, from March to July 2012. For the molecular evaluation of parasite prevalence study, whole blood samples were collected during a community mass blood survey (MBS) in May 2012 from individuals aged >10 years old (range 11 to 95), resulting in 219 samples. This represented 39.2% of the population of the hamlet, from a total of 558 residents. This number of samples enabled us to detect 6.5% margin of error, using alpha 0.05. Secondly, 61 whole blood samples were collected from individuals >15 years old presenting at the malaria clinic of Mae Tan in May 2012, where passive case detection (PCD) is routinely conducted. All sample donors gave written informed consent. The study was approved by the Institutional Review Boards of the Pennsylvania State University protocol (number 34319); University of California Irvine (number 2012–9123); Thai Ministry of Health (number 0435.3/857), and EC of the Department of Disease Control Ministry of Public Health, Thailand protocol number 7/54-479.Plasmodium DNA by qPCR, and presence or absence of symptoms at the time of blood collection. Samples with qPCR result positive for Plasmodium DNA were classified as from 1) asymptomatic malaria, if donors presented no symptoms; or 2) symptomatic malaria, if donor presented symptoms as described below. Samples with negative qPCR result were classified as from 3) healthy individuals, if no symptoms were present; or 4) non-malaria illness, if symptoms were present. Malaria symptomatology was 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 presence or absence of From each study participant, approximately 300 μl of whole blood was collected from a finger prick into a Microvette CB300 capillary blood collector with Lithium-Heparin , 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, aliquoted and stored at −80°C until use. Total genomic DNA was isolated from the pelleted cellular fraction using DNeasy Blood and Tissue kit , and further purified with Genomic DNA Clean and Concentrator , according to manufacturer’s instructions. Purified genomic DNA samples were kept at −20°C until use.P. falciparum (Pf) and P. vivax (Pv) parasites in blood samples was performed by qPCR in the 219 MBS and 61 PCD samples using a SYBR Green detection method as described in Rougemont et al. [P. falciparum and P. vivax 18S rRNA gene: for P. falciparum, the forward primer sequence was 5′-AGTCATCTTTCGAGGTGACTTTTAGATTGCT-3′ and the reverse was 5′-GCCGCAAGCTCCACGCCTGGTGGTGC-3′; for P. vivax, the forward primer sequence was 5′-GAATTTTCTCTTCGGAGTTTATTCTTAGATTGC-3′ and the reverse was 5′GCCGCAAGCTCCACGCCTGGTGGTGC-3′. Amplification was performed in 20 μl reactions containing 2 μl of genomic DNA, 10 μl 2XSYBR Green qPCR Master Mix , and 0.5 μM of each primer, in a CFX96 Touch Real-Time PCR Detection System . After initial denaturation at 95°C for 3 min, 45 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min were followed by a final step of 95°C for 10 sec. This was then followed by a melting curve from 65°C to 95°C with 0.5°C increments for 5 sec. Samples were tested in triplicates and a given sample was considered positive if the lower 95% confidence interval for adjusted Ct value was greater than 0. The detection limit of this method was 50 parasites/mL of blood.Field microscopy is performed by local trained staff who provides the first result, and positive cases were treated per national malaria treatment guidelines. Expert microscopy was performed later at Mahidol University by an expert microscopist, with over three decades of experience , who prot et al. . SpeciesP. falciparum and 515 P. vivax polypeptides printed as in vitro transcription translation (IVTT) reactions was manufactured as described previously [in vitro reactions spotted, as determined by detection of N- and C- terminal polyhistidine and influenza haemagglutinin epitope tags, respectively. Protein amount was consistent between multiple subarrays, with signal intensity of anti-6xHis tag probing showing a minimal R2 = 0.78 and a maximal R2 = 0.93 between subarrays and slides. For large proteins printed on the microarray as overlapping polypeptides or individual exons, the exon position relative to the full molecule and the segment of the ORF are indicated when applicable [P. falciparum polypeptides spotted on the microarray, the distribution of parasite life stage of maximum expression, based on information from PlasmoDB according to Le Roch et al. [P. vivax proteins at this time.A protein microarray displaying 500 eviously . Gene aceviously ,27. The eviously -30 and uh et al. , is as fPlasmodium infection, a subset of samples were selected for Pf/Pv500 microarray probing, using the following criteria: i) being 15 years or older in age, and ii) had recorded lack of malaria symptoms (as defined above) during household visits two months prior and two months after blood collection. This yielded 93 samples. Sixty plasma samples from patients from the malaria clinic were also probed . Twelve plasma samples from unexposed donors from the United States, with no travel history to malaria endemic regions, were used as controls for serology comparisons. Probing of plasma samples on the microarray was previously described in Baum et al. [From the 219 whole blood samples from the community MBS screened by qPCR for m et al. . The rawno DNA control spots) were subtracted from each plasma’s raw values of antibody binding measured as the mean signal intensity of spots of printed polypeptides; negative or zero values after background subtraction were assigned a net value of 1; (ii) net values were log2-transformed for data normalization. Normalized data was used for statistical analyses and for figure representations of the data; (iii) to determine which polypeptides were considered seroreactive by plasma from the Thai study cohort, Significance Analysis for Microarrays (SAM) [n = 153) and unexposed controls from the USA (n = 12). The test was performed using MeV 4.8.1, with the following parameters: median and 90th percentile of False Discovery Rate, 0.15% and 0.92%, respectively; median and 90th percentile of Number of False Significant Genes, 0.66 and 4.19, respectively. This resulted in 458 polypeptides being considered significantly seroreactive in exposed Thai plasma and all further analyses considered only this set. (iv) 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. For analysis of intensity of response, (v) ANOVA testing with post hoc Tukey-Kramer  was used for pairwise comparison of the mean signal intensity amongst the plasma groups using JMP9.0; significance tests were 2-sided and set at the 0.05 level for type I error. (vi) Z-scores of signal intensity were calculated as the number of standard deviations above or below the mean signal intensity of the unexposed group. (vii) ROC analysis was performed using ROCR package for R to obtain AUC (area under the curve) values and Mann–Whitney U test values (Benjamini-Hochberg-corrected) for each seroreactive protein, in comparisons of intensity of antibody binding between samples from asymptomatic malaria (MBS) (n = 13) and symptomatic malaria cases (PCD) (n = 26) to identify serological markers significantly associated with asymptomatic infections.For analysis of antibody binding to Pf or Pv polypeptides on the microarray the following steps were taken: (i) the mean background signal of antibody binding to 24 control spots of IVTT reaction without DNA template (ys (SAM)  was perfPlasmodium DNA in 25, revealing 11.4% (95% CI 7.3 - 15.5%) malaria infections amongst villagers . However, microscopy was significantly less sensitive to detect P. vivax infections than qPCR (p = 0.03).Of 61 samples collected from symptomatic patients at the malaria clinic PCD, 27 (44.3%) (CI 32.6 – 56%) n = 93) and the clinic PCD (n = 60) were probed on a protein microarray displaying P. falciparum and P. vivax polypeptides. Of the community samples, 80 belonged to the healthy group and 13 to asymptomatic malaria group . Of the malaria clinic samples, 34 were from non-malaria illness cases and 26 were from symptomatic malaria cases . Infection status of samples probed was kept blind until data analysis of array results.A subset of plasma samples from the community cross-sectional MBS  of antibody binding by exposed Thai plasma was 8.65 (CI 8.61 – 8.69), while the mean SI produced by unexposed control plasma was 3.50 (CI 3.37–3.63) (Mann–Whitney U p < .0001). The names and gene IDs of the 458 seroreactive targets of antibody response are listed in Additional file Of the targets present on the microarray, 281 n = 153) were reactive to several polypeptides on the microarray, evidence of exposure to Plasmodium parasites in all sample donors. The median number of proteins recognized by a plasma sample was 297, and ranged from 54 to 457 antigens.Figure P. vivax MSP-10, which was recognized by 100% of qPCR-positive and >90% of qPCR-negative samples, from both MBS and PCD. The most frequently recognized P. falciparum proteins were ETRAMP-2, ETRAMP-5, heat shock protein 70, MSP-2 and MSP-4, Plasmodium exported protein PHISTc, ring exported protein 1 REX1, sexual stage specific protein precursor Pfs16 and conserved Plasmodium proteins PF3D7_1014100, PF3D7_0516400. For P. vivax proteins, the most frequently recognized antigens were ETRAMP, major blood stage surface protein Pv200, MSP-8 and MSP-10, sexual stage antigen s16 (putative), transmission blocking target antigen Pfs230 (putative), liver stage antigen (putative), endoplasmin precursor (putative) and hypothetical proteins PVX_092070, PVX_090110, and PVX_095185.Seroprevalence rates for the seroreactive polypeptides are presented in Additional file n = 380, 83%). The P. falciparum antigenic markers of exposure that were similarly recognized by all plasma cohorts, including plasma from qPCR-negative donors, were: apical membrane protein 1 (AMA1), several members of the early transcribed membrane protein family , erythrocyte binding antigens 175 and 181, LSA1 and LSA3, several members of the MSP family , heat-shock protein 70, duffy binding-like merozoite surface protein (DBLMSP), chromosome assembly factor 1 (CAF1), and several hypothetical Plasmodium proteins without known function, amongst others  3, 4 and 5, amongst others, were equally seroreactive amongst plasma cohorts  of their signal intensity of antibody binding to the 458 seroreactive proteins on the array, there was a homogenous response (HSD p > 0.05) to the majority of those targets , the intensity of antibody response to falciparum antigens was identical between asymptomatic (MBS) and symptomatic (PCD) cases (HSD p = 0.99); however, samples from asymptomatic carriers had significantly higher levels of antibody binding to vivax proteins than the clinic patients (HSD p = 0.001).For P. vivax (green bars), antibody binding to falciparum proteins was identical between cohorts (HSD p = 0.94); however, to vivax proteins there was significantly higher antibody response in symptomatic (PCD) cases than in asymptomatic (MBS) carriers (HSD p < .001).For individuals infected with P. vivax (green bars) or mixed-species (purple bars) infections in most comparisons  showed equal or higher antibody responses than those with s Figure A. Mixed-The breadth of antibody response observed amongst the different plasma groups is shown in the box-whisker plot in Figure Plasmodium infections.Receiver operating characteristic (ROC) analysis can be used to compare the sensitivity versus specificity of antibody binding to individual antigens for their ability to distinguish between two diagnostic groups. The antibody responses in asymptomatic and symptomatic malaria cases were compared using ROC analysis to determine which plasmodial proteins elicited higher antibody responses in asymptomatic infections and could be associated with protection from clinical disease in Pf MSP2 protein, a well-known marker of clinical disease protection [P. falciparum and P. vivax of unknown functions. Finally, two P. falciparum proteins of putative functions, the chaperone of unfolded proteins or heat shock protein (DnaJ protein) [Table otection ,35, follprotein) , and theprotein)  were alsP. falciparum and P. vivax proteins simultaneously [According to World Health Organization, Thailand is at the pre-elimination phase of malaria control . The Govaneously ,32,43-47Thailand’s highest malaria burden regions, including the Tak Province where the present study was conducted, have experienced a drastic reduction in malaria transmission recently . ParasitP. falciparum/P. vivax mixed infections as single-species – mixed-species infections represented almost 26% of confirmed infections in patients at the malaria clinic. The underestimation of mixed-species infections in malaria patients by microscopy was previously documented [et al. found that submicroscopic infections may contribute to 20-50% of transmission in areas where slide prevalence is 4% and below, but contribute considerably less in areas of high transmission intensity [In our study, the sensitivity of microscopy was higher for blood smears from symptomatic individuals from the malaria clinic. However, microscopy misdiagnosed all cumented ,48-51, acumented ,53. Simicumented . The Malntensity -16,19 asntensity , whereasWith the serological profiling using the protein microarray, the study’s objectives were two-fold: 1) to broadly describe the profiles of naturally acquired antibody responses of the study populations for the first time in such amplitude, correlating these findings with the epidemiology of malaria in the region; and, 2) to identify antigen-specific responses that could be serological correlates of protection from symptomatic manifestation during infection.P. falciparum and P. vivax was detected in all the samples tested, including non-parasitemic individuals. Non-infected individuals exhibited overall similar levels of antibodies against plasmodia as individuals infected with P. vivax or mixed infections, indicating previous exposure to malaria parasites and possible maintenance of humoral immunity to infection. It is important to note that our cohort included only adults , and their serological profiles may reflect exposure to plasmodia from weeks to months past. Indeed, it is interesting that despite relatively higher prevalence of P. vivax in the region, antibody responses to P. falciparum were broader and more intense than to P. vivax. It is possible that the biology of falciparum infections may generate a stronger antibody response than vivax malaria, as seen in the P. falciparum + samples in this study (Figure P. falciparum in adult donors, which was relatively more prevalent in the region up to the mid-1990’s than P. vivax [P. falciparum throughout those earlier years, which is maintained by “boosts” of occasional P. falciparum infections, or alternatively by P. vivax infections and re-activation of cross-reactive epitopes between the two species. The longevity of antibodies against plasmodia varies amongst antigens [. falciparum and P. vivax was not addressed in this study due to the complexities of antigen-specific longevity of antibody responses, and the co-existence of these two species in the region, with the high likelihood of individuals having been infected with both at some point. Similarly, cross-reactivity between P. ovale, P. malariae and P. falciparum, P. vivax was not assessed.Firstly, the serological survey with a microarray showed surprisingly little differences in antibody responses amongst the four plasma groups tested, both in terms of antigen-specific responses and intensity or breadth of response. Seroreactivity to y Figure A. It is P. vivax . Perhapsantigens ,56 and hantigens -59 and lantigens -62 compoP. falciparum MSP2 showed the greatest ability to distinguish individuals with immunity to malaria disease from those who suffer symptoms when infected, confirming previous findings [Secondly, of the over 1,000 antigens analysed on the microarray, less than 8% of proteins elicited differential antibody responses amongst the plasma groups tested. Of those, only six showed significantly high responses associated with asymptomatic carriers and not with those who became sick. Once again, this likely resulted from studying solely the responses of adults, who have mounted a broad antibody repertoire throughout multiple exposures. Nonetheless, antibody responses to findings ,35.The main limitation of the serological study was the exclusion of plasma samples from children and adolescents. The inclusion of individuals younger than 15 years old in the serological survey would provide a better indicator of recent and current parasite prevalence, as they have not yet built a persistent antibody repertoire and possibly reflect more accurately the present picture of parasite exposure in the region. For the same reason, their serological profiles would possibly provide more distinguishing features for determining correlates of disease immunity . AnotherOverall, the present findings suggest that low blood slide positivity rates in the community in Tak obtained by public health surveys should be interpreted cautiously in terms of malaria prevalence in the region, and that it may be imperative to include high-throughput molecular screening methods for malaria infection surveillance to identify infectious reservoirs or for evaluation of intervention program efficacy in the community. Although blood smear examinations by microscopy have lower cost for malaria detection, the use of sample pooling for PCR screening can bring the cost per sample down so that it can be considered for mass survey screening ,64, withMicroscopic diagnosis grossly underestimates malaria exposure in Tak, Thailand. Our findings based on serological and qPCR surveys suggest that parasite prevalence is higher than currently estimated by local authorities based on microscopic screening of blood smears from community mass blood surveys or patients in clinics. As transmission levels drop in Thailand, it will be imperative to employ high-throughput methods with higher sensitivity for parasite detection in the phase of malaria elimination.