Plasmodium falciparum rapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients’ IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stage  Plasmodium falciparum malaria causes million clinical cases and hundreds of thousand deaths worldwide1, although the global disease toll probably exceed these numbers3. Most deaths occur in sub-Saharan Africa (90%) and in children under 5 years old (70%)1. In non-endemic areas, malaria could also be a public health threat due to international tourism and migration to and from endemic areas, which has led to the occasional re-emergence of this parasitic disease as imported infection4.Endemic 5, but the mechanisms that underlie this immunity remain poorly understood6. Landmark studies in the 1960s showed that purified IgG from malaria-immune adults transferred to acute-malaria infected children leads to reduction of both fever and parasitemia7, thus indicating that antibodies against P. falciparum proteins play a critical role in controlling the blood stage of the infection. However, it is still unknown which of the 5,300 proteins presently annotated in the P. falciparum genome  elicit the production of protective antibodies8. In addition to allelic variations of MHC molecules, the degree of antigen exposure, antigen abundance and host immunodominance impede that all possible antigens could be recognized by natural immune responses9. With regard to host immune detection of the P. falciparum parasite, pioneer studies showed that a large number of antigens are recognizable and dispersed amongst a large fraction of the proteome11. Among these, specific epitopes that do induce a response upon immunization with whole native antigens are defined as dominant12. Although most of the putatively exposed parts of the Ag surface could be subject of recognition by antibodies, the selection mechanism by which only certain antigen regions become B-cell epitopes is not fully understood13. The accurate identification of B-cell epitopes constitutes a basis for development of antibody therapeutics14, peptide-based vaccines16, and immunodiagnostic tool17.In-housing vector control, elementary clinical diagnosis and therapeutic drugs are the only available management strategy for both malaria prevention and treatment in endemic regions. As a necessary step towards malaria elimination neither vaccine nor diagnostics of subclinical reservoirs are widely available. After repeated exposure to the parasite, those who survive malaria early in life eventually acquire resistance to the diseaseP. falciparum-specific antibodies. These studies determined antibody reactivity against either a few selected proteins available after cloning18 or a wider selection of the P. falciparum proteome (up to 23%) by means of microarray technology11. More recently, protein microarray has been shown to be an useful tool to correlate plasmodial antigens with protective immunity20 and to discover new immunogenic epitopes from known vaccine candidates and hypothetical proteins21. Scanty information is available about the actual immunodominant B-cell epitopes from the identified antigens. Based on the spatial structure, epitopes can be categorized as a continuous  and discontinuous ; in the latter case amino acid residues are in close contact due to the three-dimensional conformation22. The amount of amino acid sequence in native proteins required for the correct folding of a discontinuous epitope of B cells is always greater than 20 amino acids and can reach up to 400 amino acids. Using a less stringent definition for continuity, it has been proposed that the majority of discontinuous epitopes (over 70%) are composed of 1–5 linear segments of lengths of 1–6 amino acids23. Currently, the Immune Epitope Database (http://www.iedb.org/home_v3.php) contains above 700 epitopes from 63 antigens of P. falciparum. Most of them have been identified in commonly identified antigens, i.e. MSP proteins26, circumsporozoite protein (CSP)28 or ring-infected erythrocyte surface protein (RESA)29 among others. Notwithstanding all these data, correlation between IgG response for the outlined P. falciparum proteins and malaria immunity have not been firmly established, suggesting that antibodies against these proteins might not play a role in protective immunity or, more likely, that IgG against single-parasite proteins are insufficient to confer protection. In addition, the identification of B-cell epitopes can also be used in the development of diagnostic tests by immunodetection approaches30. This diversity of Plasmodium antigens found in different scenarios highlights the interest of identifying other repertoires of antigens and epitopes that are expressed during Plasmodium infection by different strategies. Thus, identifying more P. falciparum antigens can be instrumental to find links between disease state and its progression as well as in advancing in the design of effective diagnosis systems and vaccines.Previous studies in areas endemic for malaria have been carried out to identify a correlation between malaria immunity and P. falciparum antigens and immunodominant epitopes, we first analyzed immunoproteomic profiles shown in sera from patients with imported malaria. These patients living in a non-endemic region were not periodically re-infected after other continuous P. falciparum infections, but occasionally when they visit transiently their home country of endemic malaria. Thus, their humoral immune status may hypothetically reflect a selection for pre-immunogenic anti-P. falciparum IgG antibodies, eventually directed against epitopes that suppressed acute symptoms. The identification of IgG-reactive antigens in sera from imported malaria patients with a single recent restricted infection but with different levels of parasitemia would therefore be particularly informative, as this could allow for the identification of immunoreactive antigens that favour subclinical malaria and therefore may be relevant for both immunisation and diagnosis. In this work, the humoral response in imported malaria patients was analyzed for the identification of potential highly immunoreactive antigens and mapping of immunodominant epitopes using peptide microarrays. Subsequently, the specific IgG response to a selection of these epitopes in adult and child sera from a malaria endemic Ghanaian population was quantified, validating the antigens found and suggesting a possible correlation with the stage of development of the infection.In an effort to expand the search of P. falciparum infection through parasitological and serological analyses. Five of these patients showed clinical symptoms and were classified as imported clinical malaria (ICM) with high parasitemia. The remaining 14 patients were identified during routine medical checks and were classified as imported subclinical malaria (ISC), since no clinical symptoms of malaria were observed in addition to a submicroscopic parasitemia. Four additional sera from individuals who had never been in malaria-endemic regions were used as control. Mean values of specific anti-P. falciparum IgG antibodies quantified by indirect ELISA did not show significant differences between both patient groups, although very large intragroup variability was observed in the ISC group .The sera of 19 imported malaria patients showed p Figure . No specP. falciparum intraerythrocytic proteome obtained in 2D-PAGE from a clinical isolate, originally causing subclinical malaria31, showed 862 ± 14 spots within the range at 3–11 isoelectric point and 3–260 kDa molecular weight , the remaining 34 spots , five of metabolism, four of cell surface or organelles and only one of transport. Although some differences were observed for antigen reactivity, none of the identified proteins appeared to be significantly associated with clinical or subclinical stages. However, with the expectation of characterizing potential differences in epitope recognition, five antigens were selected based on relative immunodominance (four of which showed a positive identification of >50% by patient sera) and computational analysis of antigen function and recognition by T and B cells34: i) Functions that are likely key for survival according to gene ontology terms of the proteins; ii) Secreted or bound to cell membranes; iii) Global antigenicity above 1.01 as determined by the method of Kolaskar & Tongaonkar35; and iv) Immunogenicity considered as potential for recruiting antigen specific Th cells and determined as density of peptides predicted to bind HLA-DR molecules using RANKPEP36.The quantification of signals from all samples allowed the selection of 83 immunoreactive protein spots that were present in at least 30% of the complete set of Westerns blots developed with the imported malaria patients’ sera n = 19). These spots were excised from the reference 2D gel and subjected to MALDI-TOF/TOF analysis and subsequent search in s Figure  were suc9. These 37.These analyses resulted in the following five proteins: Putative protein kinase (Q8I2Q0), Heat shock protein 70 (HSP70-2), Heat shock protein 60 (HSP60) and Star-related lipid transfer protein (START), and the Protein disulfide isomerase 8 (PDI8). Among the five selected proteins, only HSP60 and PDI8 had been reported as antigenic in previous studiesP. falciparum parasitemia and IgG content, and subsequently HLA genotyped (see Table S4). The sera were classified into two groups: adults with subclinical malaria (ASC) with low parasitemia level (<0.01%); and adults with no parasitemia (ANP), and thus no having malaria. Both groups were further subdivided by their specific anti-P. falciparum IgG contents into high (>60 μg mL−1) or low (<60 μg mL−1) IgG. A list of the 38 samples used is shown in Table If any of the antigens detected in imported malaria cases are relevant to the maintenance of acquired malaria immunity, sera from asymptomatic individuals in endemic areas can be expected to contain specific antibodies against epitopes in these proteins. To analyze this possibility, random sampling of sera from patients visiting Breman-Asikuma Hospital (Ghana) for reasons other than malaria symptomatology was undertaken. Blood samples were quantified for P. falciparum IgG and total IgG between the two studied groups  high reactivity with high IgG concentration sera from both ASC and ANP groups; ii) high recognition rate, namely including at least one 15-mer peptide showing signals above de threshold value in, at least, 75% of the high IgG sera challenged with than peptide, and iii) a theoretically calculated acidic or basic isoelectric point to anticipate their solubility. Moreover, the 107–120 region of antigen Q8I2Q0 which showed the largest differences in antibody reactivity in ASC and ANP sera was also included for validation. The sequence of the chosen peptides and their location in the antigens are shown in Table These eight synthesized peptides were tested for IgG reactivity using serum samples from the endemic malaria region. Figure P. falciparum was detected only by PCR of its DNA but not by microscopy , and those who did not show the presence of the parasite by either method (CNP). The samples from the CCM, CSC and CNP groups were defined and split upon analysis since were obtained from children enrolled from clinic visits for any medical reason, and not necessarily malaria suspicion. The results, shown in Fig. Since the persistence of anti-START and anti-PDI8 IgG in the adult population of the malaria-endemic region may be a consequence of repeated exposure to the parasite, it was conceivable that a less exposed population group would show a different pattern. To test this hypothesis, serum samples from children younger than 5 years old in the same endemic area were analyzed to detect specific IgG against peptides 1.1, 1.3, 5.1 and 5.3 that were the most immunoreactive of START and PDI-8 in adults. In this trial, the sera of children with symptoms of malaria or asymptomatic, corresponding to those showing parasites by microscopic screening , those for whom P. falciparum antigenic proteins identified by different approaches40, only a few have been shown to be; albeit partially, relevant to malaria immunity10 or for detection purposes42. Previously, the humoral response to P. falciparum in patients with imported malaria was used to identify immunogenic proteins by western blot 1D analysis.37. Here, we have used a 2D immunoproteomics approach using imported malaria patient sera to increase search resolution, mapped the position of potential epitopes in a selection of five immunodominant antigens, and validated their immunogenicity using serum samples from individuals in a malaria-endemic region. Although imported malaria samples used for immunoproteomics were classified into two groups based on different clinical stage, a clear association between individual antigens and disease stage could not be determined. In fact, no significant differences in total serum anti-P. falciparum IgG levels were found between the two groups, as high antibody levels are associated not to protection but to higher exposure to P. falciparum43.Despite the variety of 10. It is noteworthy that different populations and experimental approaches may yield the identification of different sets of antigens, potentially associated to immune response in endemic areas. Residents in a endemic area of Kenya monitored for reinfection after administration of antimalarial drugs mostly showed in their sera the presence of antibodies against widely known antigens as the liver-stage antigen (LSA), merozoite surface proteins (MSP) or Pf Erythrocyte membrane protein PfEMP1, among others19. In other study using only alpha helicoidal coiled-coil protein motifs, differences were observed in the reactivity of sera antigens between different ethnicity and age20. This supports the notion that broad and diverse experimental approaches are necessary to obtain a more complete range of P. falciparum immunogenic proteins. Chaperones, within the fate category including the heat shock proteins HSP70, HSP70-2, HSP70-3 and HSP60, were the most frequently recognized by sera IgGs. In the metabolic category, IgGs anti-Plasmepsins I and II, (proteases involved in host hemoglobin digestion) were similarly found in sera from both imported malaria groups. Plasmepsin I has been detected in intraerythrocytic young stages whereas Plasmepsin II expression has been associated with mature stages44. A similar distribution was observed in the generation of antibodies against dihydrolipoyl dehydrogenase and dihydrolipoamide acyltransferase, both of them components of the multienzyme pyruvate dehydrogenase complex. Two further metabolism-related proteins from the parasite, ferrodoxin reductase-like protein, involved in fatty acid metabolism, and vacuolar ATP synthase subunit b, likely involved in acidification of the digestive vacuole45, raised antibodies against them in sera from both groups. However, while antibodies against ferrodoxin-reductase-like protein were found mainly in patients with clinical symptoms, the vacuolar ATP synthase subunit b was more antigenic in those individuals without clinical signs of malaria. The most frequently detected antigen was initially identified as a conserved protein of unknown function Uniprot Q8I2Q0, corresponding to gene PF3D7_0925900 in the genome of P. falciparum 3D7. Further analysis by sequence comparison (not shown) revealed that this sequence may correspond to an uncomplete gene duplication of a putative protein kinase Uniprot Q8I2P9, corresponding to gene PFIT_PFI1270W, located 20 kpb downstream PF3D7_0925900. Within the cell surface class, three immunogenic proteins which assist in the process of erythrocyte invasion46 were identified. Two of them, MSP-1 and MSP-9, are located at the merozoite surface47 and RAP2, associated with the low-molecular-weight rhoptry complex in the rhoptry organelles48. Finally, a new antigenic protein, integral membrane STAR-related lipid transport START, raised IgG antibodies in about 55% of the samples from all three groups. Although the humoral response against some of these proteins has previously been reported in other studies using samples from malaria-endemic regions40, antigens such as putative protein kinase, HSP70-2, V-Type proton ATPase subunit B, START, LPD1, PDI8, ferredoxin reductase-like protein and plasmepsins I and II, may be indicative of differences in the specific IgG range shown in individuals with imported malaria caused only by an isolated infection in a partially protected individual. It is therefore conceivable that antibodies against several of these housekeeping proteins are merely markers of exposure rather than part of acquired protective immunity, as previously reported for other malaria antigens49.Eight of the 17 antigens identified by immunoproteomics had not been previously reported, including experimental approaches and computational prediction of the entire proteome32 guaranteeing potential immunogenic contact by the host during the intraerythrocytic cycle 53, while immunity to non-severe malaria is slowly acquired over years of repeated re-infection, often leading to a subclinical course of the disease that can only be detected by PCR amplification54. Remarkably, only P. falciparum-infected children displayed anti-START or anti-PDI8 IgGs, indicating that the presence of these antibodies in adults may be associated with the development of acquired immunity against non-severe malaria.To further explore this possibility, serum samples from children under 5 years old from the same endemic area were analyzed. It is now accepted that resistance to severe malaria is rapidly attained within the first five years of lifeP. falciparum antigens involved in the maintenance of acquired immunity show a wide range of B-cell epitopes of which at least the START and PDI8 epitopes lead to the production of a long-lasting IgG response that is maintained after parasite clearance, which may be indicative of a relevant role of these antigens in naturally acquired immunity. Nevertheless, we cannot discard that the CNP population has been previously infected with malaria. Thus, the low START and PDI8 antibody levels in this group suggest that the IgG response is not long-lived initially. It seems more likely that only after repeated infection(s) do these antibody responses persist. In addition, the presence of anti-START and/or anti-PDI8 IgG in children with subclinical malaria would also be useful as a marker for such undetected reservoir infection. Thus, these epitopes could be used as a rapid method of ELISA immunodetection at large-scale in elimination programs instead of PCR diagnosis.Taken together, these results suggest that Four sets of human sera were used in the present study. The first set (n = 19) was obtained from adult sub-Sahara African patients resident in Spain with imported malaria detected after a recent trip from Africa Table . The secA fourth set of 4 European volunteers never exposed to malaria was used as a control.This study was approved by both, the Ethical Review Board at Research Institute Hospital 12 de Octubre, Madrid (Spain) and the Ethical and Protocol Review Committee from University of Ghana’s College of Health Sciences (Ghana). The study was conducted according to the guidelines laid down by the Declaration of Helsinki, with written informed consent obtained for each adult participant, or in children a parent or guardian of child participant provided written informed consent on their behalf. The samples were specifically obtained for this study including complementary analysis of co-infections and polymorphisms in connection to malaria immunity that are not yet published.P. falciparum isolate UCM731 from West Africa was cultured to high parasitemia (>50%) following the previously described method55. Briefly, the culture media consisted of standard RPMI 1640 (Sigma-Aldrich) supplemented with 0.5% Albumax I (Gibco), 100 μM Hypoxanthine (Sigma-Aldrich), 25 mM 101 HEPES (Sigma-Aldrich), 12.5 μg/mL Gentamicine (Sigma-Aldrich) and 25 mM NaHCO3 (Sigma-Aldrich). Culture at 37 °C with 5% CO2 started by mixing a cryopreserved stock with fresh erythrocytes to achieve 1% hematocrit. Growth was monitored by microscopy in thin blood smears and media changes and synchronization steps following defined criteria55 according to parasite stage and parasitemia.Clinical P. falciparum isolate UCM7 were obtained from three 150 ml culture flasks of infected red blood cells (iRBC) synchronized to mature forms (schizonts) at high parasitemia55. Cultures were harvested and erythrocytes resuspended 1:1 in PBS containing 0.1% saponin to gently vortex during 15 s for lysing RBC. Released parasites were centrifuged at 800 × g during 5 min and the pellet was 3x washed in cold PBS to subsequently centrifuged at 15,000 × g, 10 min. Pellets were solubilized in 50 mM Tris-HCl pH8 containing 50 mM NaCl, 3% CHAPS, 0.5% MEGA 10 and protease inhibitor cocktail from Roche. Resuspension was gently mixed at 4 °C during 15 min followed by 4x freeze-thaw cycles. Cold (4 °C) centrifugation at 15,000 × g during 10 min resulted in a supernatant considered the parasite extract. Protein concentration was quantified using Bradford based Protein Assay from Bio-Rad and total protein lysates stored at −20 °C until use.Protein extracts from P. falciparum IgG were quantified using 0.5 μg/well of parasite protein extract (see above) in the same buffer as coating antigen overnight at 4 °C. From this step onwards, all wells followed same protocol. Plates were blocked with 1% bovine serum albumin (BSA) in Tris-buffered saline solution and diluted human serum was incubated for 1 h at room temperature, using 1:50,000 dilution for total IgG and 1:5,000 dilution for specific IgG quantification. IgG binding was detected with goat anti-human IgG conjugated with horseradish peroxidase (HRP) at 1:75,000 dilution. The enzymatic reaction was developed using 3,3′,5,5′ tetramethyl benzidine (TBM) as enzyme substrate and stopped by the addition of 100 μl of 4 N H2SO4.. Absorbance readings were obtained at 652 nm in a Varian Cary 50 Biospectrophotometer. Serum samples from non-exposed European individuals were used to establish the negative cut-off of the ELISA (mean OD+2 S.D.). Human reference serum from Bethyl Laboratories was used to generate a logistic four-parameter sigmoidal standard curve.Immunoglobin-G (IgG) in sera samples was quantified using indirect IgG ELISA detection kit (Bethyl Laboratories) following the manufacturer’s instructions. For total IgG quantification, microtiter plates were coated with 1 μg/well of anti-human IgG (Bethyl Laboratories) in 100μl carbonate/bicarbonate buffered solution (Sigma C3041) for 1 hour at room temperature; while specific anti-P. falciparum UCM7 extracts were run in 2D-PAGE (300 µg for mass-spectrometry identification / 150 µg for immunoblot) for subsequent Western blot analysis using sera from patients with imported malaria. Protein extract were cup loaded onto IPG strips  previously hydrated overnight with a buffer containing 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1.2%, DeStreak reagent and 2% ampholites pH 3–11. For first-dimensional separation, IEF was performed using the IPGphor 3 IEF system  at 20 °C. Voltage was gradually ramped in a step-and-hold manner to 1,000 V in three steps: 1 h at 120 V, 1 h at 500 V, and 2 h at 500 − 1,000 V. Then, voltage increased to 4,000 V in linear gradient  over the next 9 hours. Run terminated after ≈ 70,000 V/h. The focused strips were equilibrated twice with gentle vertical shaking. The first equilibration during 12 min was performed with a solution containing 100 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 0.5% DTT. In the second equilibration solution, DTT was replaced with 4.5% iodoacetamide and incubation continued 5 min. SDS-PAGE using a Ettan-Daltsix unit at 2 W/gel for 30 min followed by 20 W/gel for 4 h was run on homogeneous 12.5% pre-casted gels.56. Excised spots were washed twice in water, shrunk for 15 min with 100% acetonitrile, and dried in a Savant SpeedVac for 30 min. The samples were then reduced using 10 mM DTT in 25 mM ammonium bicarbonate for 30 min at 56 °C and subsequently alkylated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate for 20 min in the dark. Finally, samples were digested with 12.5 ng/μL sequencing grade trypsin in 25 mM ammonium bicarbonate (pH 8.5) overnight at 37 °C. After digestion, the supernatant was collected, and 1 μL was spotted onto a MALDI target plate and allowed to air-dry at room temperature. Next, 0.4 μL of a 3 mg/mL of α-cyano-4-hydroxytranscinnamic acid matrix in 50% acetonitrile was added to the dried peptide spots and allowed, again, to air-dry at room temperature. MALDI time-of-flight (TOF) MS analyses were performed in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer  operated in positive reflector mode, with an accelerating voltage of 20,000 V. Mass spectra were then acquired for peptide mass fingerprinting (PMF). Proteins were identified by comparing the trypsin-digested peptide masses with the data provided in two databases (NCBI) residues and UniprotKB-SwissProt v.56.6) separately using MASCOT 1.9 (http://www.matrixscience.com) through the Global Protein Server v3.5 from Applied Biosystems. All of the identified proteins fulfilled the criterion of being significant (p < 0.005) according to probability based on the Mowse score57. Statistical analyses were performed using the Statgraphics Plus 5.1 software package.To visualize and further excise and identify protein spots, the 2D-gels loaded with 300 µg were fixed in 10% methanol/7% acetic acid for 30 min and overnight stained with Sypro Ruby (Sigma). Two 2D-gels for staining and protein-spot excising (300 µg) and 20 replicated 2D-gels for Western-blot (150 µg) were run in parallel, in group of 6, under identical voltage conditions to warrant replica. The 2D-gels loaded with 150 µg were transferred to polyvinylidene difluoride (PVDF) membranes. Transfer efficiency was confirmed by: (i) gel staining with Sypro Tangerine before transfer; and (ii) membrane staining with Sypro Ruby after transfer. Stained gels or blots were scanned in a Typhoon 9400 variable mode imager . For immunodetection with individual patient serum, the transferred PVDF membranes were blocked for 2 hours in 10% non-fat skimmed milk (w/v) in PBS containing 0.05% Tween-20 followed by overnight incubation with patient serum at 1: 10,000 dilution. Then, goat anti-human IgG HRP linked (Amersham Bioscience) as secondary antibody was incubated for 1 h at room temperature at a 1:10,000 dilution. Antigen-antibody reaction was visualized using the SuperSignal chemiluminescent substrate (Pierce) and exposure to X-ray film. Human sera from Europeans never exposed to malaria were used as negative controls. Gel and immunoblot images were analyzed using PDQuest software, version 8.0.1 , and spots of interest were manually excised from the 2D gels, in-gel reduced, alkylated, and then digested with trypsin as described elsewhere58 using a layout with 12 identical sectors per slide. Each sector contained 6630 peptide fields. The surface coating on the slides was made by incubation of Nexterion E slides (Schott AG) with a 2% w/v linear copolymer of N,N′-dimethylacrylamide and aminoethyl methacrylate as described59. Each sector of the arrays was incubated with a different serum diluted 1:200 in buffer  for 2 hours in after blocking. Then, after washing in same buffer, the slides were incubated for 2 hours at room temperature with 1 µg/mL Cy3-conjugated goat anti-human IgG (H+L) (Sigma) and washed again. The slides were centrifuged in a slide spin-dryer and scanned at 1 µm resolution in an InnopSys 900 laser scanner using an excitation wavelength of 532 nm. The images were analyzed using the PepArray analysis program (Schafer-N) and the fluorescent signal in each field was calculated as the weighted average of the pixel intensities in that field.Sera samples from subjects from Breman-Asikuma were tested for the recognition of peptides in arrays. They were synthesized on glass slides using photolithographic peptide array−1 solution in 0.05 M Carbonate-Bicarbonate buffer, pH 9.6. After overnight incubation at 4 °C, plates were washed with 50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0 buffer five times, 200 µL of blocking buffer  were added and incubated at room temperature for 30 minutes. After one more wash, 100 µL of serum diluted 1:450 in wash buffer was added. After incubation at room temperature during 60 min, the supernatant was removed, the wells were washed once more and 100 μL of goat Anti-IgG-Fc HRP conjugated at 1 mg mL−1 (Bethyl) were added at 1:15000 dilution. HRP activity was detected as indicated above. Mann-Whitney non parametric test was used for statistical analysis of IgG binding signal between groups.Specific IgG binding in serum samples to selected peptides was tested by ELISA. Microtiter plates  were coated in duplicate with peptides by adding 100 µL of a 2.5 µg mLStatistical analysis of quantitative variables was performed using either parametric Student’s t-test  or Mann-Whitney non-parametric test , as required, to determine significant differences between groups, if any. All statistical analyses were carried out using OriginPro software . A P value ≤ 0.05 was considered significant.Supplementary Information.


Malaria during pregnancy leads to serious adverse effects on mothers and the fetus. Approximately 25 million pregnant women in sub-Saharan Africa live at risk of malaria. This study would help to achieve Sustainable Development Goals (SDGs) by improving programs that deal with the prevention of malaria. Therefore, this study aimed to assess the prevalence and associated factors of malaria among pregnant women.p-value < 0.2 were included in the final multivariable model. Variables having p-values < 0.05 from the multivariable model were considered to be significantly associated with the dependent variable. The adjusted odds ratio with its 95% confidence interval (CI) was used as a measure of association.A community-based cross-sectional study was conducted from July to August 2018 in Sherkole district, West Ethiopia. A multi-stage sampling technique was used to select 504 pregnant women. The interviewer-administered semi-structured questionnaire was used for data collection. Malaria was also diagnosed using a rapid diagnostic test. The data was entered using EPI info version 7.2.2.2 and transferred to SPSS version 20 for analysis. Descriptive statistics were done using frequency and percentages. Both bivariable and multivariable logistic regression models were employed. Variables having Plasmodium falciparum and Plasmodium vivax, respectively. Decreasing Age (Adjusted Odds Ratio (AOR) 0.78; 95% CI 0.67–0.911), not using insecticide-treated bed net (ITN) , lack of consultation and health education about malaria prevention , being on second-trimester pregnancy , gravidae II  were found to be significantly associated with malaria during pregnancy.Of the total 498 pregnant women who participated in this study, 51 were found to have malaria. Of these, 46 (90.2%) and 5 (9.8%) were caused by Malaria is still a public health problem among pregnant women in the Sherkole district. Age, ITN use, gravidity, gestational age, and health education had a significant association with malaria. Screening pregnant women for asymptomatic malaria infection and educating and consulting on the appropriate malaria preventive methods shall be provided. There are five different human malaria species such as P. falciparum, P. vivax, P. malariae, P. knowlesi and P. ovale. In 2016, an estimated 216 million cases of malaria and 445,000 deaths occurred worldwide [Malaria is caused by parasites of the P. falciparum malaria infection [P. falciparum [Malaria during pregnancy is a serious public health problem in sub-Saharan Africa. It is estimated that each year approximately 25 million pregnant women in sub-Saharan Africa live at risk of nfection . Two insnfection  and 7.7%lciparum . Studieslciparum  also sholciparum –11.Malaria infection during pregnancy causes an enormous risk to the mother, fetus, and neonates . Indeed Different risk factors for malaria among pregnant women were identified by previous studies. These include educational status , 16, ageIn Benishangul Gumuz regional state, almost all districts (98%) of the landmass are malarious areas and 97% of the population are at risk for malaria infection. Despite the high risk of malaria transmission in the area, there is limited evidence about the burden and risk factors of malaria among pregnant women which can be used for reducing maternal and child mortality due to the disease. Therefore, this study aimed to assess the prevalence of malaria and its associated factors among pregnant women in Sherkole District, Benishangul Gumuz regional state, West Ethiopia.The study was conducted in Sherkole district, Benishangul Gumuz Regional state (BGRS) from July 20 to August 30, 2018. Sherkole district is one of the 21 BGRS administration districts which is found 756 km to the West of Addis Ababa, the capital city of the country, and 96 km far away from region city, Assosa. The district is found at a latitude of 13.169308 and longitude of 39.987117 and the altitude of the district is 680–800 m above sea level. The climatic condition of the district is hot and the annual temperature is estimated to be between 25 °C and 41 °C. The Annual range of rainfall in the district is 900–1200 mm. In this district, all kebeles are malarious with 39,373 populations at risk of the disease. In 2016/2017, the annual malaria incidence rate in the district was 263 cases per 1000 population. There were 1243 pregnant women, 1196 under 1 year, and 6370 under 5 years old of children in the district ; obstetric factors ; malaria prevention measures  use, personal protective measures, and ITN utilization); health service use .The outcome variable for this study was malaria infection which was assessed using RDT and pregnant mother with any type of The interviewer-administered Semi-structured questionnaire was used to collect the required information. For those pregnant women who were on ANC followup, the data collector reviewed their antenatal followup cards to cross-check the information given by them. Card information checked includes; gravidity, parity, and gestational age at first ANC visit. Following the interviews, blood was obtained from the third finger of women’s left hand. First, the tip of the finger was wiped with a piece of cotton wool lightly soaked in alcohol. Then piercing with sterile lancet was done and the blood allowed to flow freely without squeezing the finger. Then, 5 μl (μl) blood was collected and a single small drop was added on the CareStart RDT to examine the presence or absence of malaria and to differentiate its species. The RDT read and determine the species qualitatively after 15–20 min of putting the blood to the kit. Ten percent of the randomly selected negative slides were rechecked and reread. Eight trained diploma nurses and midwives collected the data and they were supervised by two health professionals with a qualification of BSc degree. The questioner was pretested and one-day training was given for supervisors and data collectors on the basic technique of the data collection.p-value ≤0.2 from the bi-variable model were included in the final model. Variables having a p-value < 0.05 from the multivariable model were considered as having a statistically significant association with the outcome. Adjusted Odds ratio with 95% CI was used as a measure of association. The model goodness of fit was assessed using the Hosmer lemisho test.The data were entered using EPI-Info 7.2.2 and then transferred to SPSS version 20 statistical package for further analysis. Data cleaning and management were done. Descriptive statistics  were done to explain the study population in relation to relevant variables. The Chi-square assumption was checked for all categorical independent variables and multicollinearity was also checked using the Variance inflation factor (VIF). Both bi-variable and multi-variable logistic regressions were used to assess the association between outcome and explanatory variables. Factors with A total of 498 pregnant women participated in this study with a response rate of 98.8%. The majority, 20841.8%), of the pregnant women were in the age group of 25–29 years. Concerning the educational status, more than three fourth, 384(77.1%), of the mothers had no formal education. Almost all, 482 (96.8%), study participants were farmers and traditional gold miners. About 478 (96%) of respondents owned at least one mosquito bed net, and 405 (81.3%) of them sleep under mosquito nets in the previous night. Almost all, 485 (97.8%), of the households had Indoor Residual Spray (IRS) in the last 12 months. All women 431 (86.5%) who had ANC follow-up were given health education about the prevention methods of malaria infection during their ANC follow-up. The majority, 323 (64.9%), of the study participants were multi-gravida, and more than half, 292 (58.6%), of the study participants were in their third trimester of pregnancy  were P. vivax cases. From the total confirmed cases, the majority, 35 (68.8%) were asymptomatic.In our study, the prevalence of malaria was found to be 10.2% (95% CI: 7.72–13.24). Of these, 46(90.2%) were From the bi-variable logistic regression, malaria was significantly associated with all of the variables at a significance level of 0.2. However, from the multivariable logistic regression model only age, ITN utilization, consultation about malaria prevention methods during ANC, trimester of pregnancy, and gravidity were significantly associated with malaria infection during pregnancy. For 1 year increase in the age of the pregnant women, the odds of malaria infection was decreased by 22%. The odds of malaria infection was 14.98 times higher among pregnant women who did not utilize ITN compared to their counterparts . Pregnant women who had no education about malaria prevention methods during their ANC follow up had 7.15 times increased odds of malaria infection compared to their counterparts . Women who were in their first trimester of pregnancy had 23.33 times increased odds of having malaria infection compared to mothers on their third trimester . Women who are in their second trimester of pregnancy also had 7.78 times increased odds of having malaria infection compared to mothers on their third trimester . The odds of malaria infection was 5.87 times higher among women who had their second pregnancy compared to multi gravid women   , rural d3% , ruraP.falciparum species. This result was in line with the study conducted in tropical Africa which showed 80–95% of malaria infections are caused by P. falciparum [P. falciparum causes the most severe form of the disease and it can cause devastating complications not only for the mother but also for the fetus. This result also implies that there is a need for early screening of pregnant women for early detection and treatment of the cases to prevent possible complications. On the other hand, the proportion of malaria cases caused by P.falciparum in our study was lower than the WHO malaria 2017 report which revealed over 99% of malaria cases were due to P.falciparum [In this study, 90.2% of the cases were caused by lciparum . Howeverlciparum . This hiIn this study mothers with an increased age were found to have lower odds of developing malaria infection. This is in line with studies conducted in different tropical African countries , 17 whicPlasmodium-infected erythrocytes sequester/arrest in the maternal placenta by producing surface antigens mainly variant surface antigens that adhere to chondroitin sulphate-A (CSA) receptors expressed by syncytiotrophoblasts in the placenta. These antibodies are associated with protection against placental infection. Therefore, primigravidae and secundigravidae mothers lack these anti-adhesion antibodies against CSA binding parasites, which develop only after successive pregnancies and this makes them more susceptible to infection [According to our study, pregnant women who were in the second trimester of pregnancy were at increased odds of developing malaria infection compared to mothers in the third trimester. Besides, and women who were gravidae II have increased odds of malaria infection compared to the multi gravid. Similar results were found from studies done in sub-Saharan Africa countries , 11, 18,nfection .In our study, getting a consultation and health education about malaria preventive methods during ANC follow up significantly decreased the odds of developing malaria infection during pregnancy. A similar association was found in studies conducted different parts of Ethiopia , 28. HeaIn this study, not using ITN increases the odds of developing malaria infection during pregnancy. Indeed, WHO, MoH, and presidents malaria initiatives (PMI) have advocated for a three-pronged approach to tackle malaria and one of the strategies is the use of ITN , 25, 29.Since our study used a cross-sectional study design, it does not show a direct temporal relationship. Though using PCR and blood film microscopy may have higher sensitivity, we could not do these tests because the study is done in rural areas and there is no electricity in the area. Therefore, the result of this study could be affected by the inherent performance of the RDT utilized.P. falciparum is the most predominant Plasmodium species in the area. Age of respondents, ITN use, gravidity, gestational age, and health education about malaria prevention methods during ANC had a significant association with malaria infection. Health professionals should give health education about malaria prevention methods during ANC and they should also give special attention to those pregnant women with the identified risk factors. Besides, further research is recommended by using more sensitive diagnostic methods like PCR and blood film microscopy for the diagnosis of malaria.The prevalence of malaria infection among pregnant women was relatively low in Sherkole district and


Bacillus thuringiensis israelensis (Bti) or community education and mobilization (CEM), or with both interventions in the context of integrated vector management (IVM).Malaria prevention in Africa is mainly through the use of long-lasting insecticide treated nets (LLINs). The objective of the study was to assess the effect of supplementing LLINs with either larviciding with Bti (arm 2); LLINs and CEM (arm 3); and, LLINs combined with Bti and CEM (arm 4). Between January 2013 and December 2015, CDC light traps were used to sample adult mosquitoes during the second, third and fourth quarter of each year in 10 houses in each of 16 villages at each of the three study sites. Larvae were sampled once a fortnight from potential mosquito-breeding habitats using standard plastic dippers. Cross-sectional malaria parasite prevalence surveys were conducted involving a total of 11,846 primary school children during the 3-year period, including 4800 children in Tolay, 3000 in Malindi and 4046 in Nyabondo study sites.The study involved a factorial, cluster-randomized, controlled trial conducted in Malindi and Nyabondo sites in Kenya and Tolay site in Ethiopia, to assess the impact of the following four intervention options on mosquitoes and malaria prevalence: LLINs only (arm 1); LLINs and Bti, community education and mobilization. The two other sites did not reveal significant reduction of prevalence as a result of combining LLINs with any of the other supplementary interventions.Baseline relative indoor anopheline density was 0.11, 0.05 and 0.02 mosquitoes per house per night in Malindi, Tolay and Nyabondo sites, respectively. Nyabondo had the highest recorded overall average malaria prevalence among school children at 32.4%, followed by Malindi with 5.7% and Tolay 1.7%. There was no significant reduction in adult anopheline density at each of the three sites, which could be attributed to adding of the supplementary interventions to the usage of LLINs. Malaria prevalence was significantly reduced by 50% in Tolay when using LLINs coupled with application of Bti and CEM further reduced malaria infection in a low prevalence setting in Ethiopia, but not at sites with relatively higher prevalence in Kenya. More research is necessary at the selected sites in Kenya to periodically determine the suite of vector control interventions and broader disease management strategies, which when integrated would further reduce adult anopheline populations and malaria prevalence beyond what is achieved with LLINs.Combining LLINs with larviciding with  After more than two decades of malaria being associated with an estimated one million global deaths annually prior to 2000, the disease cases declined by 40–60% in many countries between 2000 and 2015 –5, amongUnfortunately, as different countries pursue the goal of malaria elimination, encouraged by the gains experienced since 2000, serious concerns have been expressed at national and international level regarding sustainability of the primary interventions, particularly those targeting vector control. In the first instance, there has been a looming threat posed to malaria control programmes by widespread vector resistance to the insecticides commonly used in IRS and LLINs , 10. SecBacillus thuringiensis israelensis (Bti) [However, research evidence is largely unavailable to support the perception of greater impact of IVM on malaria when compared to relatively non-integrated vector control in different eco-epidemiological settings. Towards this end, several studies have been conducted to assess whether adding a secondary anti-vector intervention in a setting where the primary vector control method is the usage of LLINs can lead to further site-specific decline of malaria prevalence towards elimination. The secondary methods most generally evaluated include IRS, screening of houses with fine wire mesh to reduce mosquito entry and consequently reducing vector-host contact, and larval source management, particularly involving biolarviciding with is (Bti) –18. The is (Bti) .Bti in potential mosquito breeding sites, and the promotion of proper use of LLINs [IVM for malaria control has previously been promoted in areas where the present studies were conducted in Kenya and Ethiopia through collaborative research and non-research developmental activities . The IVMof LLINs –24. An eof LLINs , 25.Bti and community education and mobilization (CEM) onto the frontline intervention of LLINs. It specifically aimed at verifying under field conditions if adding the former two interventions, herein, also referred to as supplementary interventions, either individually or combined can further reduce indoor malaria vector density and malaria prevalence in the community compared to when LLINs alone are used.The objective of this study was therefore to determine the impact of adding larviciding using The studies were conducted from January 2013 to December 2015 simultaneously in three different geographic locations: the Nyabondo plateau in the western part of Kenya, Malindi Sub-County at the Kenyan coast, and the Tolay region in southwestern Ethiopia , about 30 km northeast of Lake Victoria, at an altitude between 1520 and 1670 m above sea level. An estimated 34,000 people live in homesteads spread out in the area but administratively grouped into villages, each of up to about 200 houses. The main livelihood activities include subsistence farming, mainly of maize, small-scale livestock rearing and brick-making. Water accumulation in shallow ground pits from the brick-making activity contributes to mosquito breeding . Malariaabiensis .An. arabiensis, Anopheles merus and Anopheles funestus [Malindi Sub-County is located in Kenya’s coastal area about 108 km north of Mombasa  at an altitude of between 40 and 400 m above sea level. This study was conducted in a rural area of Malindi with approximately 50,000 people. Subsistence farming, fishing and trading are the major economic activities in this area. Malaria is one of the key causes of morbidity and mortality within the Sub-County with an average prevalence of 4–8% [An. arabiensis [Tolay is a rural semi-arid, agro-climatic zone located in the southwestern part of Ethiopia  at an altitude of 1100–1600 m above sea level. The community is mainly engaged in small-scale mixed agriculture and trade at village level. The major food crops in the region are maize, sorghum and teff. Malaria is unstable and seasonal, with a prevalence of about 2% or less [abiensis . Common Bti) and one social intervention (community education and mobilization) were added either individually or in combination. Survey information available retrospectively regarding household LLIN ownership at the beginning of the baseline data collection in 2013 indicates household net ownership of 83% in Nyabondo area, 84% in Malindi and 64% in Tolay [All the three project sites were in areas with universal coverage with LLINs, distributed by the countries’ national malaria control programmes (NMCPs) via multiple channels, including free mass and continuous distributions mainly through antenatal and immunization services , 4, 28. in Tolay , 29.Bti granules  was done once every 2 weeks to all potential Anopheles breeding habitats, identified in advance at a particular study site where this intervention was to be implemented. All habitats found to have anopheline mosquito larvae within a study village were treated. Also included were those habitats within a 200-m buffer zone from the last house in the village in order to mitigate potential spillover effects in entomological assessments due to mosquito flight range [Bti was applied by hand, by a team of 6–8 people, half of whom were project field staff and the other half members of the community, commonly referred to as mosquito scouts [Bti granules were broadcast by hand within the manufacturer’s recommended dose of about 3.0 kg/ha [Application of ht range . The Btio scouts , 25, who.0 kg/ha .The community education and mobilization intervention entailed conducting intensive community educational activities deliberately planned to surpass the awareness creation that normally accompanies LLIN distribution by the Ministry of Health (MOH) of either country. In all three project sites, resident project field staff of about 2–3 persons conducted the CEM activities on regular monthly basis assisted by about 6–10 mosquito scouts. The scouts had previously been trained by the research team in basic aspects of IVM including: visually identifying presence of mosquito larvae in aquatic habitats, larval source management like draining of stagnant water and filling up of temporary pools of water with soil or levelling them wherever appropriate, proper care and usage of mosquito nets, adult mosquito identification, and community participation in malaria prevention and control. The following methods of community education and mobilization were applied: door to door campaigns, engagement of primary school pupils through school health and environment clubs, distribution of information, education and communication (IEC) materials, neighbourhood clean-up campaigns with emphasis on elimination of mosquito breeding and resting habitats, and community meetings on mosquito and malaria control activities.Bti; Arm 3 comprised LLIN use and community education and mobilization; and, Arm 4 comprised LLIN use, application of Bti and community education and mobilization. In each village at each site, 10 houses were randomly included in entomological assessment using indoor adult anopheline mosquito relative density as the proxy indicator of malaria transmission risk. A total of 38 schools located within the study sites  were randomly sampled for cross-sectional malaria parasitological surveys among school children, using parasite prevalence as the measure of epidemiological impact. The overriding hypothesis was that integrating usage of LLINs with CEM and larviciding with Bti (arm 4) has greater impact in reducing populations of anopheline mosquitoes and malaria prevalence than when LLINs are used alone or integrated with only either one of the other two supplementary interventions.A cluster-randomized, controlled trial with a factorial design , 33 was The study villages at each study site were randomly selected from a larger pool of villages, which were purposively chosen from the entire list of villages in each study site, and fulfilled most or all of the following criteria: ecologically similar including, among other features, the presence of natural or man-made potential mosquito breeding habitats; location in an area with reported universal MOH-LLIN coverage; accessibility throughout the year; availability of a local primary school; existence of a local health facility such as dispensary or hospital. During selection of the 16 villages, a minimum distance of approximately 0.5 km was maintained between study households in one village and those in the next in order to mitigate potential spillover of intervention effects , 34.Mosquito larvae were sampled once every 2 weeks from all the study villages. All stagnant water bodies within and about 200 m from the last house in a village were inspected for mosquito larvae using a standard dipper (11.5 cm diameter and 350 ml capacity)  and theiAnopheles gambiae sensu lato (s.l.) species complex and other Anopheles species [An. gambiae s.l. were analysed for sibling species composition using recombinant deoxyribonucleic acid (rDNA)-polymerase chain reaction (PCR) technique [Adult mosquitoes were sampled indoors once every 3 months from a total of 160 houses at each of the three project sites. The total number of houses comprised of 10 houses from each of the 16 villages, initially selected using simple random sampling. These same houses continued to be used for the rest of the study for ease of field logistics. Consent of the house owners was sought in order to make the houses accessible for the duration of the study. Mosquitoes were collected indoors using standard Centers for Disease Control (CDC) light traps . In each selected house, the trap was hung about 1.5 m from the floor, at the rear end of a regularly used bed, with the occupant protected with a bed net . Mosquit species . Finallyechnique –40. The Cross-sectional malaria parasite surveys in children aged between 6 and 12 years were conducted at each project site to assess the local prevalence of malaria. A total of 38 public primary schools were used in the study, including 12 schools in Nyabondo, 10 in Malindi and 16 in Tolay. The schools were intended to be matched with the study villages at a ratio of one school per village. However, this was possible in Tolay but not in Malindi and Nyabondo. For this reason, some of the study arms at the latter two sites had fewer than four schools included in the prevalence assessments in spite of them having four study villages. In each school, 20 children of equal number of males and females were randomly selected from each of classes 2–6 based on the children present that day, and using computer-generated random number tables. This sample size of 100 children per school sought to estimate a 5% change in prevalence of infection across the years, assuming a power of 80% and test size of 5%. Oversampling of 20% was considered to take care of any contingencies such as drop-outs, data entry error, non-response, and damaged samples.Plasmodium falciparum prevalence (PfPR) [Plasmodium infection in the peripheral blood. The sample collection was done by trained MOH technicians. All children who were found to be positive were treated with artemisinin-based combination therapy (ACT), artemether-lumefantrine, by the MOH staff according to the national guidelines for the diagnosis, treatment and prevention of malaria in Kenya and Ethiopia [e (PfPR)  was estaEthiopia , 4.P. falciparum infection in school children as assessed by the school-level cross-sectional surveys; and, (3) estimate the magnitude of the intervention effects over time for the four intervention groups on malaria transmission risk and P. falciparum infection prevalence.The primary objective of the statistical analysis was to: (1) determine the malaria transmission risk as measured by the relative vector density each year of the survey; (2) determine the prevalence of The following key entomological indicators for measuring vector characteristics were calculated: (1) adult mosquito relative density defined as the number of adult mosquitoes per house per night; and (2) mosquito larval density defined as the number of mosquito larvae per dip. Vector density was estimated using negative binomial regression and adjusted for the number of habitats  and household clusters (for adult mosquitoes) with the number of trap-nights included as an offset to account for differences in collection effort in the mosquito collection method. The effect of the treatment interventions was estimated using generalized estimating equations (GEE) on vector density, allowing for within-subject correlation using robust variance estimator to calculate standard errors (SEs). From the GEE model, the incidence rate ratios (IRRs) were reported and herein described as density ratios (DR), the control intervention (LLINs only) was used as the reference against the other interventions.Plasmodium falciparum infection was defined as a positive RDT result [P. falciparum infection varied significantly among the treatment arms. The covariates included in the model were treatment interventions, age, gender, and study site. Additionally, multivariable mixed effects logistic regression model at three levels was separately fitted, individuals nested within schools selected within treatment groups and finally within study site with age and gender retained as fixed terms in the model and reporting the adjusted odds ratio (aOR). The results from the first and second model were compared and found no significant difference between the two models. Hence, the results of the latter model only were reported here.T result –43. Propggplot package implemented in R [All statistical analyses were performed using STATA version 14.1 . Graphs were developed using the ted in R .2 = 101.85, p = 0.090) and culicine  mosquito densities did not vary significantly in the treatment intervention groups. Similarly, the baseline larval anopheline and culicine mosquito densities did not vary significantly in the treatment intervention groups.Table Anopheles captured included An. gambiae , An. funestus , and other Anopheles species . Over half  of all the culicine mosquitoes collected were from Nyabondo site, followed by Malindi  and Tolay . On the other hand, the majority of all the anopheline mosquitoes collected were from Malindi site , followed by Nyabondo  and Tolay .Overall, 24,763 adult anopheline and culicine mosquitoes were collected over the three years in all the three study sites with majority being culicines . The Anopheles arabiensis was the predominant An. gambiae sibling species in all the sites being more than 99% in Tolay and Nyabondo where the number of specimens identified by PCR were 94 and 100, respectively. The composition of An. gambiae s.l. in Malindi as determined from 207 specimens identified by PCR was An. arabiensis (64%) and Anopheles merus (36%). The sibling species An. gambiae sensu stricto of the An. gambiae complex was virtually absent at the three project sites.The overall adult anopheline mosquito density was 0.07 with site-specific anopheline density of 0.20, 0.03 and 0.02 for Malindi, Tolay and Nyabondo sites, respectively. The least adult anopheline density was observed in arm 4 (0.04) and arm 3 (0.06) of the study. Notably, arm 4 of the study showed least adult anopheline density in all the three sites , and also significantly reduced adult culicines density by nearly two-thirds . In all the three study sites, arm 4 non-significantly reduced both the adult anophelines and culicines density  followed by Malindi (0.55) and Nyabondo (0.08) Fig. . SimilarAnalysis of the effect of the treatment interventions on larval vector density showed that in overall arm 2 of the study significantly reduced the larval anophelines density by nearly a third , and also significantly reduced the larval culicines density by nearly 12% . In two study sites, Malindi and Nyabondo, arm 2 significantly reduced the larval anophelines density by 85 and 20% in the two sites, respectively. However, the larval culicines density was significantly reduced in arm 2 and both arms 2 and 3 only in Nyabondo and Malindi study sites, respectively  and highest prevalence in arm 4 of the study (19.3%). However, arm 3 of the study showed least prevalence in two study sites, Malindi and Nyabondo, with arm 4 showing the least prevalence in Tolay site only.Figure The results demonstrated that children under 5 years of age  as well as those aged 6 to 10 years  were at significant risk of malaria infection when compared to older children (over 10 years). However, male children were at non-significant risk of malaria infection compared to females, aOR = 1.1, p = 0.635.Bti, and intensive CEM, on malaria vector populations and malaria prevalence at three rural sites, namely Nyabondo and Malindi in Kenya, and Tolay in Ethiopia. While LLINs and Bti are the actual mosquito control methods, CEM is a social intervention which is necessary for their implementation and sustainability. By analysing the individual and combined effects of two technical and one social interventions, the study aimed at a better understanding of their respective contributions in the set-up broadly defined as IVM strategy [The study assessed the impact of integrating the usage of LLINs with larviciding with strategy , 13, 45.Bti application and CEM (arm 4) significantly reduced by half the adult anopheline density. Nonetheless, at individual site level, no significant reduction in adult anopheline density was observed in any of the three sites.The relative density of anopheline mosquitoes indoors was found to be generally very low at all the three sites, being less than one mosquito per house per night. The overall average malaria prevalence varied considerably across the three sites with the order of magnitude being 19:3:1 for Nyabondo:Malindi:Tolay, respectively. The assessment of the adult anopheline density at the combined three-site  showed that compared to the control arm (use of LLINs only), LLINs coupled with For the adult culicines density, the global analysis revealed that all three study arms  were significantly effective in reducing adult culicines density, with arm 4 showing up to two-thirds reduction. At site-specific level, two sites  did not show significant reduction of adult culicines density in any of the interventions, however, Malindi site showed that arms 3 and 4 were significantly effective in reducing adult culicines density.Similarly, assessment of larval anopheline density at global level revealed that arm 2 was significantly effective in reducing larval anopheline density by up to a third. At site-specific level, all the study arms showed significant reduction of larval anopheline density in Malindi by at least 70%, while in Nyabondo only arm 2 reduced density by 20%, and no study arm showed significant reduction of larval anopheline density in Tolay.Study arm 2 was similarly effective in reducing the overall larval culicines density by about 12%, with the same study arm reducing larval culicines density by nearly a third in Nyabondo site. In Tolay site, both arms 2 and 3 were significantly effective in reducing larval culicines density by at least 30%. However, no significant reductions were observed in any of the interventions in Malindi site.circa 10% though this was not statistically significant. However, it is important to point out that in Tolay site, malaria prevalence was significantly reduced by 50% in arm 4. The two other sites did not reveal significant reduction of prevalence as a result of the use of any of the interventions.At global level, arm 3 reduced malaria prevalence by Bti, had a significant reduction of malaria prevalence when compared to the control arm.The results of parasite prevalence obtained during this study were similar to what has previously been reported for the three respective project sites , 4. In tBti can help to reduce malaria further in settings where prevalence is already low, either naturally or due to conventional implementation of a primary vector control method such as LLINs. As regards the study sites with moderate to high malaria prevalence and more extensive potential anopheline breeding habitats, the results of Bti application corroborated previous assertions of low or no additional impact of larviciding in malaria control, as expressed in a recent systematic review of past studies on the intervention [A logical inference based on the above evidence is that bringing on board larviciding with rvention .According to the results, low adult anopheline densities recorded at all three sites during the 2013–2015 period of the study did not necessarily translate to equally low malaria prevalence across the sites. Thus, malaria prevalence in Nyabondo and Malindi sites in Kenya remained higher than in Tolay, in spite of adult anopheline densities being similarly low at less than one mosquito collected per trap per night indoors. Interestingly, this situation contrasts with that in a follow-up study from 2017 to 2018 in Nyabondo alone, where indoor anopheline densities were higher, and screening of house eaves was found to lower indoor anopheline density as well as malaria parasite prevalence . FurtherBti, i.e., arm 2 and arm 4, compared to those that did not have Bti. The results support previous findings of Bti’s known high efficacy against mosquito larvae under controlled conditions [Bti likely point to operational gaps in the way larviciding was conducted. Thus, some of the potential anopheline breeding sites might have gone untreated due to failure to detect them, especially during the wet season when they are many and spread over a wide area. WHO policy recommendation in response to this particular challenge as it relates to malaria vector control is that larviciding should only be considered, with or without other interventions, when the breeding sites in the targeted area are few, fixed and findable [In Malindi, the study demonstrated significantly lower anopheline and culicine larval populations in the experimental arms that had nditions , Howeverfindable . ConsequIn the case of Tolay study site in Ethiopia, it should be noted that vector control also often involves the use of IRS in addition to LLINs, and this could generally have also contributed to low indoor anopheline vector densities observed in the study site , 51. HowCEM in the study was expected to influence communities’ behaviour leading to a range of anti-vector activities, including improved usage of LLINs and general environmental management. The lack of significant additional impact on adult anopheline density and malaria prevalence in the experimental arm where CEM was added to LLINs could suggest that, in spite of household net ownership being relatively high and meeting universal coverage, net usage, although not monitored during the study, might have been below the desired optimal levels anticipated in universal coverage . ResultsBti and CEM were interventions that essentially operated at community level, while the usage of LLINs is a self-protection measure at personal and household level, in spite of having a communal dimension when scaled up in a village setting. Thus, implementing the former two interventions could potentially have led to unintended spillover effects among the study clusters in spite of randomization efforts. For instance, a school enrolled in the cross-sectional malaria surveys and associated with a particular IVM intervention might have had some of its pupils who were tested for malaria resident in clusters with different interventions, but not recorded as such. Furthermore, the CEM intervention may have been confounded by intermingling of residents of different clusters. The problem of spillover effects is well known and has been a subject of discussion in other studies on educational interventions [Certain challenges were encountered in the study methodology which require that the results are interpreted with caution, but which may also provide a useful basis for refinement of future similar studies. For instance, larviciding with ventions . Moreoveventions , 55–57.Furthermore, information on universal net coverage was not verified through actual household surveys. Nevertheless, congruence of data from other detailed studies and MIS reports lends credibility to net coverage as cited for the different study sites in “Bti and CEM led to further reduction in malaria in a setting of low disease prevalence in Tolay in Ethiopia, but not in settings with relatively higher prevalence in Kenya. The results suggest that reducing malaria beyond what has been achieved with LLINs in the latter settings would require a different IVM configuration, possibly including other anti-vector measures as well as integration with prompt diagnosis and effective treatment of malaria cases. The observations underscore the need to view IVM as an adaptive approach which may need to be calibrated differently for different settings based on prior knowledge of the socio-ecological situation, disease epidemiology and effectiveness and practicality of interventions as is often reiterated by WHO [The study demonstrated that integration of conventional usage of LLINs with larviciding with d by WHO .


Plasmodium falciparum malaria parasites in umbilical cord blood denotes activation of complement and oxidative stress to affect the duration of pregnancy and birth weight.  It is unknown whether the presence of  In a cross-sectional study conducted from January to April 2019 in Bamenda, Cameroon, cord blood samples were collected from 300 women at delivery. Parasitaemia was determined microscopically. Babies' weight and age of gestation were recorded. Plasma levels of complement and oxidative stress were measured by specific tests. 50) and specific classical pathway activity (CPA) in cord blood. CH50 and CPA levels, however, were significantly higher in infected cord blood samples, compared with uninfected cord blood samples. CH50 showed a negative correlation with the birth weight and gestational age in infected cord blood samples. The levels of total oxidative stress (TOS) and total antioxidant defense were significantly lower in infected cord blood than uninfected. TOS displayed a positive correlation with the density of parasitaemia and a weak negative correlation with the birth weight and gestational age in infected cord blood.  Cord blood malaria prevalence was 21.33%. Babies with an infected cord showed a low birth weight and gestation age than those with uninfected cords. More babies with infected cords had LBW (6.25%) compared to the counterparts (5.50%). The levels of parasitaemia and the babies' weight showed a weak positive correlation. The prevalence of preterm and postterm birth was 4.33% and 24.33% respectively, with a weak negative correlation between the age of gestation and the umbilical cord parasitaemia. There was correlation between cord parasitaemia and levels of complement haemolytic activity titter (CH Cord blood infection lowers the complement haemolytic titter, oxygen radicals and total antioxidant defense in neonates. This lowering of complement haemolytic titter and oxygen radical compounds in umbilical cord malaria are associated with low birth weight and preterm birth. Plasmodium genus. The parasites are transmitted to humans through the bite of infected female Anopheles mosquitoes. Malaria remains the first endemic parasitic disease in the world. In 2017, it was responsible for about 219 million clinical cases of sickness and 435,000 deaths worldwide. About 92% of cases and 93% deaths were reported in sub-Saharan Africa (SSA) [Malaria is a disease caused by parasites belonging to the Mother-to-child transmission constitutes in malaria as one of the major problems compromising the foetal development. It represents a risk factor for preterm birth, foetal growth restriction, and low birth weight –6. Low bHowever, the mechanism that leads to low birth weight or preterm delivery in vertical transmission of malaria was not clearly understood. Diverse factors are suggested to be responsible. Anaemia related to immune activities are thought to be associated with malaria-related low birth weight , 6, 10. In the past decades, numerous researches showed a wide-spread involvement of the complement system in a number of placental malaria processes . Thus, mPlasmodium falciparum is responsible for more than 90% of malaria infections [The study took place at the Regional Hospital of Bamenda. Bamenda is the capital of the Northwest Region, Cameroon. Bamenda lies between latitude 5°94′N and 5°98′N and longitudes 10°15′E and 10°18′E . It sitsfections .The study participants were pregnant women who voluntarily consented to permit collection of blood from their cord during labour and satisfied the inclusion criteria. Inclusion criteria were confirmation of the active phase of labour and asymptomatic, nonfebrile parturient and singleton pregnancy without any known congenital anomaly. Exclusion from the study were subjects that refused to participate in the study; presence of any medical illness such as diabetes mellitus, chronic renal disease, haemoglobinopathies, HIV/AIDs, filarial infection, chronic hypertension, and severe pre-eclampsia/eclampsia; and presence of multiple gestation, intrauterine foetal death, and obvious foetal anomalies. There were 300 women recruited having met the inclusion criteria.Women delivering at the health facility, after giving informed consent, were asked a standard series of questions focused on sociodemographic characteristics, history of fever, antimalarial drug use, clinical malaria during pregnancy, and the use of antimalarial chemoprophylaxis. During labour, five millilitres of blood were drawn by venepuncture from the clamped cord immediately after delivery. Neonates and placentas were weighted with an electronic digital scale (±10 grams). The Dubowitz scoring system was used to estimate the gestational age, using findings from physical and neurologic examinations. Scoring by the APGAR index was performed at delivery but was not recorded in this study. Blood samples were separated into two: 1 ml into ethylenediamine tetra acetic acid (EDTA) tubes and the remaining in tubes without EDTA. Serum was collected from the tubes without EDTA.t (F1) [µL), moderate , and high  as reported by Owa et al. [µl of whole blood) were then calculated. All slides were double-read. If the ratio of densities from the first two readings was greater than 1.5 or less than 0.67 or if fewer than 30 parasites were counted with a difference of more than 10 parasites between the two readings, the slide was evaluated a third time. The geometric mean of the parasite density of the two most concordant results of the three readings was taken as the final result.Thick smears of cord blood were prepared from each sample, stained with Giemsa stain, and examined for the presence of malarial parasites using routine microscopy to determine parasitaemia. For positive slides, t (F1) , 16. Par 1,000–4,99/µL, an8 E/ml were opsonized with rabbit anti-sheep haemolysin  by adding an equal volume of a 1 : 160 dilution of haemolysin to the E suspension, and the mixture was, then, incubated at 37°C in a shaking water bath for 30 minutes. The sensitized E were, then, centrifuged at 3,000 rpm for five minutes and washed three times with gelatine veronal buffer (GVB) . Sensitized E were stored in GVBE supplemented with 2.5% glucose, 0.03% NaN3 at 4°C for not more than two weeks. For use, sensitized erythrocytes were washed with GVB containing Mg2+ and Ca2+ (GVB++).Complement titters were based on the classical haemolytic assay (CH50) for lysis of antibody-sensitized sheep E. , 18 In d++ to give a final dilution of 1/320. To each serum dilution, 25 µl of 5% sensitized E was added followed by incubation at 37°C for 60 min. The reaction was stopped by adding 100 µl of GVBE to all wells. Control wells included spontaneous lysis of opsonized E without serum and 100% lysis well in which complete lysis was achieved by adding 1% triton X. The plate was centrifuged at 3000 rpm in a plate centrifuge at 4°C for 5 min to pellet unlysed cells. 100 µl of supernatant was, then, transferred to a flat-bottomed plate, and the absorbance readings of released haemoglobin were read at 415 nm using a microplate reader. The degree of lysis (Y) was determined from the formula (Cord blood sera (in duplicates) were, then, serially diluted in a 96 micro-well plate (NUNC) in GVBThe functional complement activity in classical and alternative pathways was evaluated using ELISA kits (Sigma) supplied precoated with specific activators for the classical pathway (CP) and alternative pathway (AP). Serum samples were, then, diluted into buffers containing specific blockers of each pathway. Samples were diluted at 1 : 100 and 1 : 18 for the CP and AP, respectively. Positive and negative controls with known activities were used for quality control of the assay and for calculation of the functional activities.µl of each diluted sample and control were added in duplicate to the wells of the respective pathway-specific plates and incubated for 70 min at 37°C. Wells were, then, aspirated and washed thrice with wash buffer. Then, 100 µl of conjugate containing alkaline phosphatase-labelled antibodies to C5b-9 was added to each well and incubated for 30 min at 25°C. Wells were again washed thrice, and 100 µl of substrate ) was added, and the samples were incubated for 30 min at 25°C. Absorbance was, then, immediately read at 405 nm. Functional activity for the respective pathways was, then, calculated using the OD values of the positive and negative controls.In detail, 100 µl) of plasma diluted to 20 times in PBS was dissolved in 1 ml of acetate buffer. 25 µl of working chromogen  solution was, then, added, and absorbance was taken at 505 nm by 6 minutes time-scan in a UV-VIS spectrophotometer. The absorbance values were obtained at 4 to 6 minutes for each sample against blank. The quantity of radical compounds was determined as proportional to the absorbance.Total oxidative stress (TOS) was measured by quantifying alkoxyl (RO∗) and peroxyl (ROO∗) radicals as described by Piyashi et al.  One hundp-phenylenediamine sulphate) by antioxidant compounds in the sample. In detail, 1 ml of acetate buffer (pH = 5.2) taken in a test tube, 25 µl chromogen reagent that contains N,N-dimethyl-p-phenylenediamine sulphate, and 10 microliter FeCl3 solutions are added. Thereafter, 10 µl of 20 times diluted plasma was added, and the absorbance was photometrically read at 505 nm at 37°C. Total antioxidant defense of the sample was considered to be inversely correlated to the absorbance.Total antioxidant defense (TAD) was determined as reported by Piyashi et al.  based onBlood film results were considered to be positive if any asexual-stage parasites were identified and negative if no parasites were seen in 100 high-power fields. Prematurity was defined as a gestational age <37 weeks (≤245 days), normal gestation age 251–280 days, and postterm >280 days as estimated by Dubowitz examination , 20 and t -test. Statistical results were considered significant when the two-sided P value was ≤0.05.Data collected in the study questionnaire were verified and, then, double-entered. Data were analyzed using Graph Pad Prism software v. 5.20 and SPSS. The analysis included data from births of all enrolled participants. Continuous normally distributed data were described by their means and standard deviation. Proportions were compared using the Chi-square test, and normally distributed continuous variables were compared using the Student The study was discussed with health authorities and local leaders to obtain their assent. The study was reviewed and approved by the Faculty of Health Science ethical committee, University of Bamenda. Informed consent was obtained after the consent document was read to the women in the local language. For illiterate mothers, the informed consent discussion process was witnessed by an impartial individual.Data were collected from three hundred women, with a majority (88%) aged between 20 and 35 years. Amongst these women, 98.33% were taking fansidar, and only 8 (2.66%) had clinical malaria during pregnancy .The mean value of the gestational age of women who participated in this study was 275.27 days (±16.33), with a majority delivering at the normal time with normal babies' birth weight. The mean (SD) placenta weight was 606.66 g (±116.69), with a birth/placenta weight ratio of 5.38 .Out of the 300 cords, there were 64 (21.33%) positive blood films. The density of parasitaemia varied from low to high density. There were 18 (28.12%), 36 (56.25%), and 10 (15.62%) of the cords that showed low, moderate, and chronic infection, respectively .P ≤ 0.05). Relative to the density of parasites, this showed that the gestational age had a positive correlation (r = 0.14), while the birth weight showed a negative correlation (r = −0.16). However, these relationships were not significant  and specific complement classical and alternative activities of umbilical cord serum. The Levels of CH50 were significantly higher (P < 0.0001) in malaria-infected cords (INF), (CH50 = 21.41 ± 7.35 UEq/mL) than in noninfected cords (UNF), (CH50 = 14.25 ± 2.06 UEq/mL). In specific pathways, only the activities of the classical pathways showed a significantly higher difference at P ≤ 0.0001 (% activity = 60.91 ± 40.06%) in INF than in UNF (% activity = 19.91 ± 22.24%). In linear regression, the parasitaemia density showed a significant and negative correlation with total haemolytic complement .50 was observed to correlate positively with the gestational age and the baby's birth weight, while in umbilical cord malaria-negative women, it was positively and negatively correlated with the gestational age and the baby's birth weight, respectively. Comparative analysis showed that, in relation to the gestational age, either the delivery was premature, normal, or postterm, and the CH50 was high in women with infected cords than in counterpart controls. In contrast, the specific classical pathway activity was low in umbilical cords without parasites than in infected cords for any category of delivery  lower in case of INF compared to the corresponding UNF . When evaluating the TAD, optical density was significantly (P < 0.0001) high in case of INF as compared to UNF cords  and total antioxidant defence , with a positive correlation.Determination of the total oxidative stress (TOS) and total antioxidant defence (TAD) was based on the optical density, whose values are known to be positively and negatively correlated with total oxidative stress and total antioxidant defence. Here, in relation with the TOS of the cord blood serum, data showed that the optical density value was significantly  with the TOS, while it was negatively correlated  with TOS in uninfected cords  and positively  with TOS in the umbilical cord with malaria and without malaria, respectively , a high optical density characterizing a low serum TAD was observed in uninfected cords compared to infected cords. However, in case of low birth weight, the optical density indicating the serum TOS was high in infected cords. Furthermore, data showed that the birth weights correlated negatively  based on fansidar. Thus, cord malaria should be given greater attention in malaria control strategies that might include changes of actual drugs, fansidar. Clinical malaria of a mother during pregnancy has been shown not to be associated with cord infection. Thus, asymptomatic malaria of pregnant women should be given greater attention in the prevention of vertical transmission of malaria.µL) as previously reported [In the current study, the frequency of cords with high density of parasitaemia was higher than the frequency of cords of women with clinical malaria during pregnancy. This demonstrated that vertical transmission of malaria is not a matter of having a clinical malaria in the mother during pregnancy  was significantly higher in the infected cord than their counterpart control uninfected. The presence of malaria parasite in the cord might activate the complement system, and subsequently, it results in low CH50. In a specific pathway functional screen test, data showed that the cord blood plasma of infected cords had high specific classical pathways activities than those of noninfected cords. This suggests that the classical pathway activation, a strict antibody-dependent pathway, might not be affected as the alternative pathway which was low in infected cords. The complement system is a fundamental element of the normal host defence against malaria infection [In the present study, the cord blood plasma haemolytic titter of the complement system (CHnfection . A generFurthermore, plasma levels of cord total oxidative stress and total antioxidant defence were significantly lower in malaria-infected than in noninfected women. This finding raises the important question of whether the presence of parasites in cord blood results in oxidative stress in neonates. Oxygen radicals have been demonstrated to be important for the clearance of parasites , 26. The50 was observed to correlate positively with the gestational age and the baby's birth weight in a malaria-infected cord, while in noninfected cords, it was negatively correlated with the baby's birth weight. Placental malaria was also suggested to be the main determinant of low birth weight [50, birth weight, and gestational age, as observed in this study, suggests that the activation of complement resulting in low CH50 might be the cause of low birth weight. This is in line with recent observations that showed that soluble terminal complement complex levels were higher in placental plasma samples of newborns weighing less than 2700 g than in heavier newborns [50 can result in high soluble terminal complement complex levels.The complement system has been linked to many disease symptoms and complications, including poor pregnancy outcomes , 27, 28.h weight . The posnewborns , 28 as tFurthermore, the total oxidative stress was positively correlated with the gestational age, but negatively correlated with the birth weight. Low radical oxygen as observed in malaria-infected cords in consequence of a low complement system subsequent to infection might induce preterm birth or low birth.Findings of this study revealed that cord malaria remains prevalent in Bamenda, Cameroon, with a high rate of asymptomatic carriage of malaria parasites. The level of cord infection significantly influenced the birth weight and gestational age. In addition to the cord blood parasitaemia that constituted a source of preterm delivery and low birthweight, the low complement activities, low total oxidative stress, and antioxidant defence of cord blood during vertical transmission might reveal an additional interesting cause of low birth weight and preterm delivery in malaria endemic areas.