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Objective: To determine the effect of oral administration of calcium on plasma and ionized free calcium concentration in healthy adolescent pregnant women.Methods: In a double blind randomized controlled clinical trial were recruited 48 healthy adolescent pregnant women, 24 (50%) received 600 mg of elemental calcium and 24 (50%) received 600 mg of lactose placebo. At the inclusion time the plasma and intracellular free calcium concentrations were measured by standardized techniques. One month later the plasma and intracellular free calcium concentrations in both groups were measured.Results: At the inclusion time and one month after treatment both groups were comparable for sociodemographic characteristics and the basal intake of calcium (p=0.92, p=0.62). Calcium supplementation did not modify the concentrations of plasma ionized calcium (1.19+0.04 micromol/l vs. 1.23+0.02 micromol/l, p=0.56) and the free intracellular calcium concentration (micromol/l vs. 89.7 micromol/l, p=0.91); similar effects were observed with the placebo treatment (1.20+0.05 micromol/l vs. 1.19+0.03 micromol/l p=0.86; 116.2 micromol/l vs 137.5 micromol/l, p=0.16, respectively).Conclusions: Oral administration of 600 mg of elemental calcium in adolescent pregnant women did not induce changes in the plasma and intracellular ionized free calcium concentrations and could explain in part the lack effect of this only supplementation in preeclampsia prevention. [ABSTRACT FROM AUTHOR]

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Synthetic polypeptides encompassing the non-repeated regions of the circumsporozoite protein (CSP) of Plasmodium falciparum are very immunogenic in mice and are recognized by sera from donors living in regions where malaria is endemic, both in Africa and South America. Long polypeptides, encompassing the N- or C-terminal regions, have now been used to demonstrate peptide-specific T cells in donors living in an endemic area of Colombia. Although the N-terminal peptide (22-125) was recognized almost exclusively by donors from the endemic area, the patterns of recognition of the C-terminal peptide (289-390) in donors from endemic and non-endemic areas were similar and like the pattern with smaller peptides. The availability of the long polypeptides made it possible to compare T-cell responses to the non-repeated regions of the CSP with the presence of peptide-specific antibodies. No correlation was found and no antibodies were detected in donors from non-endemic regions. The long polypeptides also elicited strong antibody and T-cell responses in owl monkeys ( Aotus lemurinus ). The antibodies generated against the synthetic peptides in such monkeys also recognized sporozoites, the natural infective form of the parasite. The results emphasise the potential of the peptides tested as malaria-vaccine candidates. Not only are they recognized by humans at both the B- and T-cell level but they also elicit strong responses in monkeys and encompass several distinct T-cell epitopes, thus overcoming the limitations of specific, major-histocompatibility-complex restriction. [ABSTRACT FROM AUTHOR]

The development of immune responses to malarial infection in inhabitants of endemic areas differs according to the level of exposure to the parasite. Adults living in a region where the level of malaria transmission is low (Colombia) have been shown to exhibit a similar response to each of the three regions of the circumsporozoite protein (the central repeated NANP region, and the flanking N- and C-termini). Conversely, donors exposed to a frequent sporozoite challenge in areas of high malaria transmission (Mali) exhibit antibodies predominantly to the NANP repeated domain. Malaria in the people of Zacarias, a community on the Pacific Coast of Colombia where malaria transmission is low and unstable, was the subject of the present study. Within a 9-year period, a negative correlation between rainfall and documented malaria cases was recorded for this area. Thick smears of blood samples of 319 individuals revealed that 8.5% had malarial infections. As most (67%) of the smear-positive cases were asymptomatic, it seems that, despite the low prevalence of malaria in this area, the establishment of clinical symptoms is attenuated, probably because of the acquisition of premunition. Within this region, the most commonly found Anopheles species (representing 61.1% of the mosquitoes caught) and that giving the highest monthly biting rate (4.0 bites/man) was An. neivai . Most (90%) of the human sera tested possessed antibodies to blood-stage forms of Plasmodium falciparum , and 18% had antibodies to sporozoites. More than half (58%) of the adults had been in contact with hepatitis B virus, 7.2% carried hepatitis B surface antigen, and syphilis was common but no subject was found to be seropositive for HIV. A better understanding of the dynamics of the different elements influencing malaria in areas of low, unstable transmission, such as the one described here, is essential for the design of new malaria-control strategies. [ABSTRACT FROM AUTHOR]

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341., (© 2024. The Author(s).)

Colombia aims to eliminate malaria by 2030 but remains one of the highest burden countries in the Americas. Plasmodium vivax contributes half of all malaria cases, with its control challenged by relapsing parasitaemia, drug resistance and cross-border spread. Using 64 Colombian P. vivax genomes collected between 2013 and 2017, we explored diversity and selection in two major foci of transmission: Chocó and Córdoba. Open-access data from other countries were used for comparative assessment of drug resistance candidates and to assess cross-border spread. Across Colombia, polyclonal infections were infrequent (12%), and infection connectivity was relatively high (median IBD = 5%), consistent with low endemicity. Chocó exhibited a higher frequency of polyclonal infections (23%) than Córdoba (7%), although the difference was not significant (P = 0.300). Most Colombian infections carried double pvdhfr (95%) and single pvdhps (71%) mutants, but other drug resistance mutations were less prevalent (< 10%). There was no evidence of selection at the pvaat1 gene, whose P. falciparum orthologue has recently been implicated in chloroquine resistance. Global population comparisons identified other putative adaptations. Within the Americas, low-level connectivity was observed between Colombia and Peru, highlighting potential for cross-border spread. Our findings demonstrate the potential of molecular data to inform on infection spread and adaptation., (© 2023. The Author(s).)

Background: Malaria remains a leading public health problem worldwide. Co-infections with other pathogens complicate its diagnosis and may modify the disease's clinical course and management. Similarities in malaria clinical presentation with other infections and overlapping endemicity result in underdiagnosis of co-infections and increased mortality. Thus, the aim of this study was to determine the seroprevalence of viral and bacterial pathogens among diagnosed malaria patients in malaria-endemic areas in Venezuela., Methods: A cross-sectional study was conducted on malaria patients attending three reference medical centres in Ciudad Bolivar, Venezuela. Clinical evaluation and laboratory tests for dengue virus (DENV), chikungunya virus (CHIKV), viral hepatitis [hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV)], and leptospirosis (LEP) were performed by enzyme-linked immunosorbent assays. Previous exposure to these pathogens was defined by the presence of specific immunoglobulin (Ig) G, and co-infection or recent exposure (CoRE) was determined by the presence of specific IgM alone or IgM + IgG. Data analysis considered descriptive statistics. Parameter distribution was statistically evaluated using Kolmogorov-Smirnov test and the necessary comparison tests. Odds ratio (OR) for complications was determined according to CoRE presence with a 95% confidence interval (CI)., Results: A total of 161 malaria patients were studied, 66% infected with Plasmodium vivax, 27% with P. falciparum, and 7.5% harboured P. vivax/P. falciparum mixed infection. Previous exposure to DENV (60%) and CHIKV (25%) was frequent. CoRE was confirmed in 55 of the 161 malaria patients (34%) and were more frequent in P. falciparum (49%) than in P. vivax (29%) and mixed malaria patients (25%) (OR = 2.43, 95% CI: 1.39-4.25, P = 0.018). The most frequent CoRE was DENV (15%), followed by HAV (12%), HBV (6.2%), CHIKV (5.5%), and LEP (3.7%); HCV CoRE was absent. Complicated malaria was significantly more frequent in patients with CoRE (56%) than those without CoRE (36%; OR = 2.31, 95% CI: 1.18-4.92, P = 0.013)., Conclusions: We found high CoRE prevalence in malaria patients as determined by serology in the study region; cases were associated with a worse clinical outcome. Further prospective studies with samples from different infection sites and the use of molecular tools are needed to determine the clinical significance of these findings., (© 2023. The Author(s).)

The currently devastating pandemic of severe acute respiratory syndrome known as coronavirus disease 2019 or COVID-19 is caused by the coronavirus SARS-CoV-2. Both the virus and the disease have been extensively studied worldwide. A trimeric spike (S) protein expressed on the virus outer bilayer leaflet has been identified as a ligand that allows the virus to penetrate human host cells and cause infection. Its receptor-binding domain (RBD) interacts with the angiotensin-converting enzyme 2 (ACE2), the host-cell viral receptor, and is, therefore, the subject of intense research for the development of virus control means, particularly vaccines. In this work, we search for smaller fragments of the S protein able to elicit virus-neutralizing antibodies, suitable for production by peptide synthesis technology. Based on the analysis of available data, we selected a 72 aa long receptor binding motif (RBM 436-507 ) of RBD. We used ELISA to study the antibody response to each of the three antigens (S protein, its RBD domain and the RBM 436-507 synthetic peptide) in humans exposed to the infection and in immunized mice. The seroreactivity analysis showed that anti-RBM antibodies are produced in COVID-19 patients and immunized mice and may exert neutralizing function, although with a frequency lower than anti-S and -RBD. These results provide a basis for further studies towards the development of vaccines or treatments focused on specific regions of the S virus protein, which can benefit from the absence of folding problems, conformational constraints and other advantages of the peptide synthesis production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pratesi, Errante, Pacini, Peña-Moreno, Quiceno, Carotenuto, Balam, Konaté, Diakité, Arévalo-Herrera, Kajava, Rovero, Corradin, Migliorini, Papini and Herrera.)

A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo (adjuvant alone). Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11) (54%, 95% CI 0.25-0.84) and phase IIb (7/11) (64%, 95% CI 0.35-0.92). However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206)., (© 2022. The Author(s).)

Background: Pvs48/45 is a Plasmodium vivax gametocyte surface protein involved in the parasite fertilization process. Previous studies showed that Pvs48/45 proteins expressed in Escherichia coli (E. coli) and Chinese hamster ovary (CHO) cells were highly immunoreactive with sera from malaria-endemic areas and highly immunogenic in animal models. Here the immunogenicity in mice of three different vaccine formulations was compared., Methods: Recombinant (r) Pvs48/45 proteins were expressed in E. coli and CHO, purified, formulated in Alhydrogel, GLA-SE and Montanide ISA-51 adjuvants and used to immunize BALB/c mice. Animals were immunized on days 0, 20 and 40, and serum samples were collected for serological analyses of specific antibody responses using ELISA and immunofluorescence (IFAT). Additionally, ex-vivo transmission-reducing activity (TRA) of sera on P. vivax gametocyte-infected human blood fed to Anopheles albimanus in direct membrane feeding assays (DMFA) was evaluated., Results: Most immunized animals seroconverted after the first immunization, and some developed antibody peaks of 10 6 with all adjuvants. However, the three adjuvant formulations induced different antibody responses and TRA efficacy. While GLA-SE formulations of both proteins induced similar antibody profiles, Montanide ISA-51 formulations resulted in higher and longer-lasting antibody titers with CHO-rPvs48/45 than with the E. coli formulation. Although the CHO protein formulated in Alhydrogel generated a high initial antibody peak, antibody responses to both proteins rapidly waned. Likewise, anti-Pvs48/45 antibodies displayed differential recognition of the parasite proteins in IFAT and ex vivo blockade of parasite transmission to mosquitoes. The CHO-rPvs48/45 formulated in Montanide ISA-51 induced the most effective ex vivo parasite blockage., Conclusions: Three out of six vaccine formulations elicited antibodies with ex vivo TRA. The CHO-rPvs48/45 Montanide ISA-51 formulation induced the most stable antibody response, recognizing the native protein and the most robust ex vivo TRA. These results encourage further testing of the vaccine potential of this protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: [Myriam Arevalo-Herrera reports financial support was provided by NIH, NIAID.]., (Copyright © 2021. Published by Elsevier Ltd.)

P48/45 is a conserved gametocyte antigen involved in Plasmodium parasite fertilization. A recombinant Plasmodium vivax P48/45 ( Pvs 48/45) protein expressed in Escherichia coli ( E. coli ) was highly antigenic and immunogenic in experimental animals and elicited specific transmission-blocking (TB) antibodies in a previous pilot study. Here, a similar Pvs 48/45 gene was expressed in Chinese Hamster Ovary (CHO) cells and we compared its immunoreactivity with the E. coli product. Specific antibody titers were determined using plasma from Colombian individuals (n=227) living in endemic areas where both P. vivax and P. falciparum are prevalent and from Guatemala (n=54) where P. vivax is highly prevalent. In Colombia, plasma seroprevalence to CHO- rPvs 48/45 protein was 46.3%, while for E. coli - rPvs 48/45 protein was 36.1% ( p< 0.001). In Guatemala, the sero prevalence was 24.1% and 14.8% ( p< 0.001), respectively. Reactivity index (RI) against both proteins showed an age-dependent increase. IgG2 was the predominant subclass and the antibody avidity index evaluated by ELISA ranged between 4-6 mol/L. Ex vivo P. vivax mosquito direct membrane feeding assays (DMFA) performed in presence of study plasmas, displayed significant parasite transmission-blocking (TB), however, there was no direct correlation between antibody titers and oocysts transmission reduction activity (%TRA). Nevertheless, DMFA with CHO rPvs 48/45 affinity purified IgG showed a dose response; 90.2% TRA at 100 μg/mL and 71.8% inhibition at 10 μg/mL. In conclusion, the CHO-r Pvs 48/45 protein was more immunoreactive in most of the malaria endemic places studied, and CHO-r Pvs 48/45 specific IgG showed functional activity, supporting further testing of the protein vaccine potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Arévalo-Herrera, Miura, Cespedes, Echeverry, Solano, Castellanos, Ramirez, Miranda, Kajava, Long, Corradin and Herrera.)

Plasmodium vivax malaria is a neglected disease, particularly during pregnancy. Severe vivax malaria is associated with inflammatory responses but in pregnancy immune alterations make it uncertain as to what cytokine signatures predominate, and how the type and quantity of blood immune mediators influence delivery outcomes. We measured the plasma concentrations of a set of thirty-one biomarkers, comprising cytokines, chemokines and growth factors, in 987 plasma samples from a cohort of 572 pregnant women from five malaria-endemic tropical countries and related these concentrations to delivery outcomes (birth weight and hemoglobin levels) and malaria infection. Samples were collected at recruitment (first antenatal visit) and at delivery (periphery, cord and placenta). At recruitment, we found that P. vivax-infected pregnant women had higher plasma concentrations of proinflammatory (IL-6, IL-1β, CCL4, CCL2, CXCL10) and TH1-related cytokines (mainly IL-12) than uninfected women. This biomarker signature was essentially lost at delivery and was not associated with birth weight nor hemoglobin levels. Antiinflammatory cytokines (IL-10) were positively associated with infection and poor delivery outcomes. CCL11 was the only biomarker to show a negative association with P. vivax infection and its concentration at recruitment was positively associated with hemoglobin levels at delivery. Birth weight was negatively associated with peripheral IL-4 levels at delivery. Our multi-biomarker multicenter study is the first comprehensive one to characterize the immunological signature of P. vivax infection in pregnancy thus far. In conclusion, data show that while TH1 and pro-inflammatory responses are dominant during P. vivax infection in pregnancy, antiinflammatory cytokines may compensate excessive inflammation avoiding poor delivery outcomes, and skewness toward a TH2 response may trigger worse delivery outcomes. CCL11, a chemokine largely neglected in the field of malaria, emerges as an important marker of exposure or mediator in this condition., Competing Interests: The authors have declared that no competing interests exist.

The immune status of women changes during and after pregnancy, differs between blood compartments at delivery and is affected by environmental factors particularly in tropical areas endemic for multiple infections. We quantified the plasma concentration of a set of thirty-one T H 1, T H 2, T H 17 and regulatory cytokines, pro-inflammatory and anti-inflammatory cytokines and chemokines, and growth factors (altogether biomarkers), in a cohort of 540 pregnant women from five malaria-endemic tropical countries. Samples were collected at recruitment (first antenatal visit), delivery (periphery, cord and placenta) and postpartum, allowing a longitudinal analysis. We found the lowest concentration of biomarkers at recruitment and the highest at postpartum, with few exceptions. Among them, IL-6, HGF and TGF-β had the highest levels at delivery, and even higher concentrations in the placenta compared to peripheral blood. Placental concentrations were generally higher than peripheral, except for eotaxin that was lower. We also compared plasma biomarker concentrations between the tropical cohort and a control group from Spain at delivery, presenting overall higher biomarker levels the tropical cohort, particularly pro-inflammatory cytokines and growth factors. Only IL-6 presented lower levels in the tropical group. Moreover, a principal component analysis of biomarker concentrations at delivery showed that women from Spain grouped more homogenously, and that IL-6 and IL-8 clustered together in the tropical cohort but not in the Spanish one. Plasma cytokine concentrations correlated with Plasmodium antibody levels at postpartum but not during pregnancy. This basal profiling of immune mediators over gestation and in different compartments at delivery is important to subsequently understand response to infections and clinical outcomes in mothers and infants in tropical areas., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax . Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4 + T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Background: Malaria remains endemic in several countries of South America with low to moderate transmission intensity. Regional human migration through underserved endemic areas may be responsible for significant parasite dispersion making the disease resilient to interventions. Thus, the genetic characterization of malarial parasites is an important tool to assess how endemic areas may connect via the movement of infected individuals. Here, four sites in geographically separated areas reporting 80% of the malaria morbidity in Colombia were studied. The sites are located on an imaginary transect line of 1,500 km from the northwest to the south Pacific Coast of Colombia with a minimal distance of 500 km between populations that display noticeable ethnic, economic, epidemiological, and ecological differences., Methodology/principal Findings: A total of 624 Plasmodium vivax samples from the four populations were genotyped by using eight microsatellite loci. Although a strong geographic structure was expected between these populations, only moderate evidence of genetic differentiation was observed using a suite of population genetic analyses. High genetic diversity, shared alleles, and low linkage disequilibrium were also found in these P. vivax populations providing no evidence for a bottleneck or clonal expansions as expected from recent reductions in the transmission that could have been the result of scaling up interventions or environmental changes. These patterns are consistent with a disease that is not only endemic in each site but also imply that there is gene flow among these populations across 1,500 km., Conclusion /significance: The observed patterns in P. vivax are consistent with a "corridor" where connected endemic areas can sustain a high level of genetic diversity locally and can restore parasite-subdivided populations via migration of infected individuals even after local interventions achieved a substantial reduction of clinical cases. The consequences of these findings in terms of control and elimination are discussed., Competing Interests: The authors have declared that no competing interests exist.

To evaluate whether recovery from complicated malaria follows a common trajectory in terms of immunological mechanism or, rather, is highly individualized for each patient, we performed longitudinal gene expression profiling of whole blood. RNA sequencing (RNAseq) was performed on blood samples obtained from eight patients on four consecutive days between hospital admission and discharge. Six patients were infected with Plasmodium falciparum , and two with Plasmodium vivax; one patient was a pregnant woman infected with P. falciparum , who was hospitalized for several weeks. The characterization of blood transcript modules (BTM) and blood informative transcripts (BIT) revealed that patients' responses showed little commonality, being dominated by the balance of gene activity relating to lymphocyte function, inflammation, and interferon responses specific to each patient. Only weak correlations with specific complicated malaria symptoms such as jaundice, thrombocytopenia, or anemia were observed. The differential expression of individual genes, including transcripts derived from the human leukocyte antigen (HLA) complex, generally reflected differences in the underlying immune processes. Although the results of this pilot study do not point to any single process that might provide a target for complicated malaria treatment or prevention or personalized medical strategies, larger patient series and more extensive blood sampling may allow the classification of patients according to their type of response in order to develop novel therapeutic approaches.

Clinical immunity to malaria is associated with the acquisition of IgG specific for members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of clonally variant antigens on the surface of infected erythrocytes (IEs). The VAR2CSA subtype of PfEMP1 mediates IE binding in the placenta. VAR2CSA-specific IgG is normally acquired only after exposure to placental parasites. However, it was recently reported that men and children from Colombia often have high levels of functional VAR2CSA-specific IgG. This potentially undermines the current understanding of malaria immunity in pregnant women, and we thus conducted a study to assess further the levels of VAR2CSA-specific IgG in pregnant and nonpregnant Colombians. Plasma IgG against two full-length recombinant PfEMP1 proteins (one of the VAR2CSA type and one not) produced in baculovirus-transfected insect cells was detected frequently among Colombian men, children, and pregnant women with acute or previous malaria exposure. In contrast, IgG reactivity to a homologous full-length VAR2CSA-type protein expressed in Chinese hamster ovary (CHO) cells was low and infrequent among the Colombian plasma samples, as was reactivity to both corresponding native PfEMP1 proteins. Moreover, human and rabbit antibodies specific for Plasmodium vivax Duffy-binding protein (PvDBP), a protein with some homology to PfEMP1, did not react with VAR2CSA-type recombinant or native proteins, although the mouse monoclonal and PvDBP-specific antibody 3D10 was weakly reactive with recombinant proteins expressed in baculovirus-transfected insect cells. Our data indicate that the previously reported Colombian IgG reactivity to recombinant VAR2CSA is not malaria specific and that the acquisition of VAR2CSA-specific IgG is restricted to pregnancy, in Colombia and elsewhere., (Copyright © 2018 Lopez-Perez et al.)

Almost invariably, humans become ill during primary infections with malaria parasites which is a pathology associated with oxidative stress and perturbations in metabolism. Importantly, repetitive exposure to Plasmodium results in asymptomatic infections, which is a condition defined as clinical tolerance. Integration of transcriptomics and metabolomics data provides a powerful way to investigate complex disease processes involving oxidative stress, energy metabolism and immune cell activation. We used metabolomics and transcriptomics to investigate the different clinical outcomes in a P. vivax controlled human malaria infection trial. At baseline, the naïve and semi-immune subjects differed in the expression of interferon related genes, neutrophil and B cell signatures that progressed with distinct kinetics after infection. Metabolomics data indicated differences in amino acid pathways and lipid metabolism between the two groups. Top pathways during the course of infection included methionine and cysteine metabolism, fatty acid metabolism and urea cycle. There is also evidence for the activation of lipoxygenase, cyclooxygenase and non-specific lipid peroxidation products in the semi-immune group. The integration of transcriptomics and metabolomics revealed concerted molecular events triggered by the infection, notably involving platelet activation, innate immunity and T cell signaling. Additional experiment confirmed that the metabolites associated with platelet activation genes were indeed enriched in the platelet metabolome., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The present article examines the impact of the current limitations of the microcephaly definition in the context of the Zika virus outbreak. It highlights its dependence on the method used for determining gestational age and other anthropometric parameters, and includes original results of prevalence of microcephaly in four countries from two different continents (Mozambique, Brazil, Guatemala and Colombia). Alternative definitions of microcephaly are proposed to allow the identification of true cases of microcephaly in a more accurate manner., (© The Author [2017]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Rapid urbanisation in difficult socio-economic conditions such as inadequate housing infrastructure, lack of public services, improper sanitation, and poor water drainage systems in vegetation-rich areas lead to ecological conditions that are conducive to the breeding of mosquitoes and transmission of malaria, in semi-urban and urban settings., Objectives: This study aimed to describe the cases of malaria that were reported in the peri-urban areas of Pereira (Colombia), between 2008 and 2015., Methods: A retrospective study was conducted using data from the Malaria Surveillance System 2009-2015 and an outbreak study (between December 2008 and March 2009). Frequency distributions and summary measures, as well as univariate analysis were performed for all the variables in consideration. The annual parasite index (API) was calculated., Findings: Data on 214 cases were obtained from the surveillance system. A majority of the cases were reported in men (63.1%), followed by in children < 15 years (23.8%), and were caused predominantly by Plasmodium vivax (86.0%), with most of the infection occurring in the urban areas (52.8%) of Pereira. The API, by sex and age group, was higher among men ≥ 80 years. The outbreak study reported 14 cases of malaria in rural/peri-urban neighborhoods, and it was observed that the anopheline breeding sites were in close proximity to the houses in these areas. This population did not use protective measures against mosquitoes and chemical control was conducted through residual and spatial insecticide spraying., Main Conclusions: This study suggested the presence of autochthonous malaria transmission, in Pereira, between 2008 and 2015, most of which were cases of P. vivax. A greater intensity was observed between 2008 and 2009 when malaria was possibly reintroduced to the region. During the years of the study, a gradual decrease in the number of reported cases of malaria was observed in Pereira, except for the time period between 2008 and 2009 when a spike was noted (estimated using the API); this was most likely caused by an outbreak. Interventions that are more aggressive in nature are required to prevent further malarial transmission and dissemination.

Introduction: Inborn errors of metabolism (IEM) represent an important public health problem due to current diagnosis and treatment limitations, poor life quality of affected patients, and consequent untimely child death. In contrast to classical methods, tandem mass spectrometry (MS/MS) has allowed simultaneous evaluation of multiple metabolites associated with IEM offering higher sensitivity, low false positive rates and high throughput., Aims: Determine concentration levels for amino acids and acylcarnitines in blood of newborns from Colombia, to establish reference values for further use in diagnosis of IEM., Methods: Implementation of a method to determine amino acids, acylcarnitines and succinylacetone in newborn dried blood spots using MS/MS, and its application in a cross-sectional study conducted in 891 healthy neonates from Cali and Quibdo cities is described., Results: fifty-seven analytes that allow the diagnosis of more than 40 different pathologies were tested. The method showed to be linear, precise and accurate. Healthy neonates 1-18 days of age were included, 523 from Cali and 368 from Quibdo; 52% male and 48% female. Age-related differences on the concentration levels of amino acids and acylcarnitines were observed whereas no significant differences by gender were found., Conclusion: The study has contributed to reveal the usual concentration levels of amino acids, acylcarnitines and succinylacetone that could be used as reference for the establishment of a newborn metabolic screening program in Colombia.

Background: Complicated malaria remains an important public health problem, particularly in endemic settings where access to health services is limited and consequently malaria fatal outcomes occur. Few publications describing the clinical course and outcomes of complicated malaria in Latin America are found in the literature. This prospective study approached the clinical and laboratory characteristics of hospitalized patients with complicated malaria in different endemic areas of the Colombian Pacific Coast with the aim to provide epidemiological knowledge and guide to further reducing malaria severity and mortality., Methods and Findings: A prospective, descriptive hospital-based study was conducted in 323 complicated malaria patients (median age 20 years) enrolled in Quibdó, Tumaco and Cali between 2014 and 2016. Clinical evaluation was performed and laboratory parameters were assessed during hospitalization. Plasmodium falciparum was the most common parasite species (70%), followed by P. vivax (28%), and mixed malaria (Pf/Pv; 1.9%). Overall, predominant laboratory complications were severe thrombocytopenia (43%), hepatic dysfunction (40%), and severe anaemia (34%). Severe thrombocytopenia was more common in adults (52%) regardless of parasite species. Severe anaemia was the most frequent complication in children ≤10 years (72%) and was most commonly related to P. vivax infection (p < 0.001); whereas liver dysfunction was more frequent in older patients (54%) with P. falciparum (p < 0.001). Two deaths due to P. vivax and P. falciparum each were registered. Treatment provision before recruitment hindered qPCR confirmation of parasite species in some cases., Conclusions: The study identified a high prevalence of complicated malaria in the Pacific Coast, together with more frequent severe anaemia in children infected by P. vivax and hepatic dysfunction in adults with P. falciparum. Results indicated the need for earlier diagnosis and treatment to prevent complications development as well as more effective attention at hospital level, in order to rapidly identify and appropriately treat these severe clinical conditions. The study describes epidemiological profiles of the study region and identified the most common complications on which clinicians must focus on to prevent mortality.

Our understanding of the structure and regulation of Plasmodium vivax genes is limited by our inability to grow the parasites in long-term in vitro cultures. Most P. vivax studies must therefore rely on patient samples, which typically display a low proportion of parasites and asynchronous parasites. Here, we present stranded RNA-seq data generated directly from a small volume of blood from three Cambodian vivax malaria patients collected before treatment. Our analyses show surprising similarities of the parasite gene expression patterns across infections, despite extensive variations in parasite stage proportion. These similarities contrast with the unique gene expression patterns observed in sporozoites isolated from salivary glands of infected Colombian mosquitoes. Our analyses also indicate that more than 10% of P. vivax genes encode multiple, often undescribed, protein-coding sequences, potentially increasing the diversity of proteins synthesized by blood stage parasites. These data also greatly improve the annotations of P. vivax gene untranslated regions, providing an important resource for future studies of specific genes.

Background: The recent scale-up in malaria control measures in Latin America has resulted in a significant decrease in the number of reported cases in several countries including Ecuador, where it presented a low malaria incidence in recent years (558 reported cases in 2015) with occasional outbreaks of both Plasmodium falciparum and Plasmodium vivax in the coastal and Amazonian regions. This success in malaria control in recent years has led Ecuador to transition its malaria policy from control to elimination., Results: This study evaluated the general knowledge, attitude and practices (KAP) about malaria, as well as its prevalence in four communities of an endemic area in northwest Ecuador. A total of 258 interviews to assess KAP in the community indicated that most people in the study area have a basic knowledge about the disease but did not use to contribute to its control. Six hundred and forty-eight blood samples were collected and analysed by thick blood smear and real-time PCR. In addition, the distribution of the infections was mapped in the study communities. Although, no parasites were found by microscopy, by PCR the total malaria prevalence was 7.5% (6.9% P. vivax and 0.6% P. falciparum), much higher than expected and comparable to that reported in endemic areas of neighbouring countries with higher malaria transmission. Serology using ELISA and immunofluorescence indicated 27% respondents for P. vivax and 22% respondents for P. falciparum., Conclusions: Results suggest that despite a great malaria reduction in Ecuador, transition from control to elimination would demand further improvement in malaria diagnostics, including active case detection to identify and treat parasite asymptomatic carriers, as well as community participation in its elimination.

Protein α-helical coiled coil structures are known to induce antibodies able to block critical functions in different pathogens. In a previous study, a total of 50 proteins of Plasmodium vivax erythrocytic asexual stages containing α-helical coiled coil structural motifs were identified in silico, and the corresponding peptides were chemically synthesized. A total of 43 peptides were recognized by naturally acquired antibodies in plasma samples from both Papua New Guinea (PNG) and Colombian adult donors. In this study, the association between IgG antibodies to these peptides and clinical immunity was further explored by measuring total IgG antibody levels to 24 peptides in baseline samples from a longitudinal study of children aged 1-3 years (n = 164) followed for 16 months. Samples were reactive to all peptides tested. Eight peptides were recognized by >50% of individuals, whereas only one peptide had < 20% reactivity. Children infected at baseline were seropositive to 23/24 peptides. No significant association was observed between antibody titers and age or molecular force of infection, suggesting that antibody levels had already reached an equilibrium. There was a strong association between antibody levels to all peptides and protection against P. vivax clinical episodes during the 16 months follow-up. These results suggest that the selected coiled coil antigens might be good markers of both exposure and acquired immunity to P. vivax malaria, and further preclinical investigation should be performed to determine their potential as P. vivax vaccine antigens.

Background: Despite that over 90 million pregnancies are at risk of Plasmodium vivax infection annually, little is known about the epidemiology and impact of the infection in pregnancy., Methodology and Principal Findings: We undertook a health facility-based prospective observational study in pregnant women from Guatemala (GT), Colombia (CO), Brazil (BR), India (IN) and Papua New Guinea PNG). Malaria and anemia were determined during pregnancy and fetal outcomes assessed at delivery. A total of 9388 women were enrolled at antennal care (ANC), of whom 53% (4957) were followed until delivery. Prevalence of P. vivax monoinfection in maternal blood at delivery was 0.4% (20/4461) by microscopy [GT 0.1%, CO 0.5%, BR 0.1%, IN 0.2%, PNG 1.2%] and 7% (104/1488) by PCR. P. falciparum monoinfection was found in 0.5% (22/4463) of women by microscopy [GT 0%, CO 0.5%, BR 0%, IN 0%, PNG 2%]. P. vivax infection was observed in 0.4% (14/3725) of placentas examined by microscopy and in 3.7% (19/508) by PCR. P. vivax in newborn blood was detected in 0.02% (1/4302) of samples examined by microscopy [in cord blood; 0.05% (2/4040) by microscopy, and 2.6% (13/497) by PCR]. Clinical P. vivax infection was associated with increased risk of maternal anemia (Odds Ratio-OR, 5.48, [95% CI 1.83-16.41]; p = 0.009), while submicroscopic vivax infection was not associated with increased risk of moderate-severe anemia (Hb<8g/dL) (OR, 1.16, [95% CI 0.52-2.59]; p = 0.717), or low birth weight (<2500g) (OR, 0.52, [95% CI, 0.23-1.16]; p = 0.110)., Conclusions: In this multicenter study, the prevalence of P. vivax infection in pregnancy by microscopy was overall low across all endemic study sites; however, molecular methods revealed a significant number of submicroscopic infections. Clinical vivax infection in pregnancy was associated with maternal anemia, which may be deleterious for infant's health. These results may help to guide maternal health programs in settings where vivax malaria is endemic; they also highlight the need of addressing a vulnerable population such as pregnant women while embracing malaria elimination in endemic countries.

A vaccine to eliminate malaria would need a multi-stage and multi-species composition to achieve robust protection, but the lack of knowledge about antigen targets and mechanisms of protection precludes the development of fully efficacious malaria vaccines, especially for Plasmodium vivax (Pv). Pregnant women constitute a risk population who would greatly benefit from a vaccine preventing the adverse events of Plasmodium infection during gestation. We hypothesized that functional immune responses against putative targets of naturally acquired immunity to malaria and vaccine candidates will be associated with protection against malaria infection and/or poor outcomes during pregnancy. We measured (i) IgG responses to a large panel of Pv and Plasmodium falciparum (Pf) antigens, (ii) the capacity of anti-Pv ligand Duffy binding protein (PvDBP) antibodies to inhibit binding to Duffy antigen, and (iii) cellular immune responses to two Pv antigens, in a subset of 1,056 pregnant women from Brazil, Colombia, Guatemala, India, and Papua New Guinea (PNG). There were significant intraspecies and interspecies correlations for most antibody responses (e.g., PfMSP1 19 versus PfAMA1, Spearman's rho = 0.81). Women from PNG and Colombia had the highest levels of IgG overall. Submicroscopic infections seemed sufficient to boost antibody responses in Guatemala but not antigen-specific cellular responses in PNG. Brazil had the highest percentage of Duffy binding inhibition ( p -values versus Colombia: 0.040; Guatemala: 0.047; India: 0.003, and PNG: 0.153) despite having low anti-PvDBP IgG levels. Almost all antibodies had a positive association with present infection, and coinfection with the other species increased this association. Anti-PvDBP, anti-PfMSP1, and anti-PfAMA1 IgG levels at recruitment were positively associated with infection at delivery ( p -values: 0.010, 0.003, and 0.023, respectively), suggesting that they are markers of malaria exposure. Peripheral blood mononuclear cells from Pv-infected women presented fewer CD8 + IFN-γ + T cells and secreted more G-CSF and IL-4 independently of the stimulus used in vitro . Functional anti-PvDBP levels at recruitment had a positive association with birth weight (difference per doubling antibody levels: 45 g, p -value: 0.046). Thus, naturally acquired binding-inhibitory antibodies to PvDBP might confer protection against poor outcomes of Pv malaria in pregnancy.

Background: Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization., Methodology/principal Findings: A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8-13 days after CHMI with a mean pre-patent period of 12.8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection., Conclusion: Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria., Trial Registration: Identifier: NCT01082341., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: TLR is a salaried, full time employee of Sanaria Inc., the developer and sponsor of Sanaria PfSPZ vaccine. JT and JO are full time employee of Asoclinic Inmunología LTDA and Centro Médico Imbanaco, respectively. The other authors have declared that no competing interests exist.

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ TH1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings., Competing Interests: HdP is the co-founder of INNOVEX THERAPEUTICS SL which holds proprietary rights to use PvLP1 and PvLP2 as vaccine candidates against vivax malaria; thus, having potential conflicts of interest. There are no other conflicts of interest.

Background: Even though malaria incidence has decreased substantially in Guatemala since 2000, Guatemala remains one of the countries with the highest malaria transmission in Mesoamerica. Guatemala is committed to eliminating malaria as part of the initiative 'Elimination of Malaria in Mesoamerica and the Island of Hispaniola' (EMMIE); however, it is still in the control phase. During the past decade, the government strengthened malaria control activities including mass distribution of long-lasting insecticide-impregnated bed nets, early diagnosis and prompt treatment. This study aimed to determine the prevalence of malaria, including gametocytes, in three areas of Guatemala using active case detection (ACD) and quantitative polymerase chain reaction (qPCR)., Methods: Cross-sectional surveys were conducted in three departments with varying transmission intensities: Escuintla, Alta Verapaz and Zacapa. Blood samples from 706 volunteers were screened for malaria using microscopy and qPCR which was also used to determine the prevalence of gametocytes among infected individuals. Results were collected and analysed using REDCap and R Project, respectively., Results: Malaria was diagnosed by microscopy in only 2.8 % (4/141) of the volunteers from Escuintla. By contrast, qPCR detected a prevalence of 7.1 % (10/141) in the same volunteers, 8.4 % (36/429) in Alta Verapaz, and 5.9 % (8/136) in Zacapa. Overall, 7.6 % (54/706) of the screened individuals were positive, with an average parasitaemia level of 40.2 parasites/μL (range 1-1133 parasites/μL) and 27.8 % carried mature gametocytes. Fifty-seven percent (31/54) of qPCR positive volunteers were asymptomatic and out of the 42.6 % of symptomatic individuals, only one had a positive microscopy result., Conclusions: This study found a considerable number of asymptomatic P. vivax infections that were mostly submicroscopic, of which, approximately one-quarter harboured mature gametocytes. This pattern is likely to contribute to maintaining transmission across the region. Robust surveillance systems, molecular diagnostic tests and tailored malaria detection activities for each endemic site may prove to be imperative in accelerating malaria elimination in Guatemala and possibly across all of Mesoamerica.

Plasmodium vivax is a major public health burden, responsible for the majority of malaria infections outside Africa. We explored the impact of demographic history and selective pressures on the P. vivax genome by sequencing 182 clinical isolates sampled from 11 countries across the globe, using hybrid selection to overcome human DNA contamination. We confirmed previous reports of high genomic diversity in P. vivax relative to the more virulent Plasmodium falciparum species; regional populations of P. vivax exhibited greater diversity than the global P. falciparum population, indicating a large and/or stable population. Signals of natural selection suggest that P. vivax is evolving in response to antimalarial drugs and is adapting to regional differences in the human host and the mosquito vector. These findings underline the variable epidemiology of this parasite species and highlight the breadth of approaches that may be required to eliminate P. vivax globally.

[This corrects the article DOI: 10.1186/s12936-016-1343-1.].

Introduction: Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity., Methods/principal Findings: A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy., Conclusions: We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.

Background: Glucose 6-phosphate dehydrogenase (G6PD) is an enzyme involved in prevention of cellular oxidative damage, particularly protecting erythrocytes from haemolysis. An estimated 400 million people present variable degrees of inherited G6PD deficiency (G6PDd) which puts them at risk for developing haemolysis triggered by several risk factors including multiple drugs and certain foods. Primaquine (PQ) is a widely used anti-malarial drug that can trigger haemolysis in individuals with G6PDd. Intensification of malaria control programmes worldwide and particularly malaria elimination planning in some regions recommend a more extensive use of PQ and related drugs in populations with different G6PDd prevalence. This a preliminary study to assess the prevalence of G6PDd in representative malaria endemic areas of Colombia by measuring G6PD phonotype and genotypes., Methods: Volunteers (n = 426) from four malaria endemic areas in Colombia (Buenaventura, Tumaco, Tierralta and Quibdo) were enrolled. Blood samples were drawn to evaluate G6PD enzymatic activity by using a quantitative G6PD test and a subset of samples was analysed by PCR-RFLP to determine the frequency of the three most common G6PD genotypic variants: A-, A+ and Mediterranean., Results: A total of 28 individuals (6.56 %) displayed either severe or intermediate G6PDd. The highest prevalence (3.51 %) was in Buenaventura, whereas G6PDd prevalence was lower (<1 %) in Tierralta and Quibdo. G6PD A alleles were the most frequent (15.23 %) particularly in Buenaventura and Tumaco. Overall, a high frequency of G6PD A- genotype, followed by A+ genotype was found in the analysed population., Conclusions: G6PDd based on enzymatic activity as well as G6PD A allelic variants were found in malaria-endemic populations on the Pacific coast of Colombia, where most of malaria cases are caused by Plasmodium vivax infections. These infections are treated for 14 days with PQ, however there are no official reports of PQ-induced haemolytic crises. Further assessment of G6PDd prevalence in malaria endemic areas in Colombia is crucial in view of possible mass drug administration for malaria elimination in these regions, as well as implementation of appropriate G6PDd diagnostic methods.

Background: During the last decade, Colombia presented a significant decrease in malaria clinical cases and associated mortality. However, there is a lack of reliable information about the prevalence and characteristics of complicated malaria cases as well as its association with different Plasmodium species. A description of the epidemiological and clinical aspects of complicated malaria in Colombia is presented here., Methods: A descriptive study was conducted using data collected between 2007 and 2013 by the Public Health Surveillance System (SIVIGILA). Demographic and clinical features were described. Frequency of complicated malaria cases, annual parasite index (API) and annual percent change (APC) for trend modelling by gender and age were also calculated., Results: A total of 547,542 malaria cases were recorded by SIVIGILA during the study period, of which 2553 (0.47 %) corresponded to complicated cases with similar distribution by Plasmodium vivax and Plasmodium falciparum species. Mixed infections were found in 153 cases (6.0 %). Trend modelling of the API for complicated malaria for all parasite species showed a non-significant increase throughout the years (APC 14.4 %; 95 % CI -4.3 to 36.6 %). Complicated malaria individuals were mostly males (62.2 %) and young adults (median age of 23 years). Notably, 72.4 % of the patients attended for malaria diagnosis >72 h after symptoms onset and 17 % reported malaria episodes in the last 30 days. All patients received anti-malarial treatment, but only 40 % received the first-line as recommended by the Colombian guidelines. Overall, hepatic and renal complications were the most common severe manifestations (63.6 %). Whereas hepatic and pulmonary complications were more common in P. vivax infections, renal and cerebral complications were significantly more frequent in patients with P. falciparum. In contrast with mono-infected patients, severe anaemia and shock were more frequent in patients with mixed infection., Conclusion: In contrast with the malaria-decreasing trend over the last years, the complicated malaria trend showed a non-significant annual increase. Therefore, in addition to existing national policies on early diagnosis and prompt anti-malarial treatment, more efforts have to be committed addressing the delayed diagnosis and inadequate treatment found in this study. Improving malaria notification forms, medical assistance skills, and capacity should be prioritized.

Background: Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared., Methods: Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential., Results: Nine non-synonymous substitutions (E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, K418R) and three synonymous substitutions (I73, T149, C156) that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033-0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions (E353Q: 41.9 %, K250N: 39.5 %) were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure., Conclusions: There is limited sequence polymorphism in pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as transmission-blocking vaccine candidate.

Background: Acquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection., Methodology/principal Findings: Serum samples from naïve (n = 7) and semi-immune (n = 9) volunteers exposed to P. vivax sporozoite-infected mosquito bites were probed against a custom protein microarray displaying 515 P. vivax antigens. The array revealed higher serological responses in semi-immune individuals before the challenge, although malaria naïve individuals also had pre-existing antibodies, which were higher in Colombians than US adults (control group). In both experimental groups the response to the P. vivax challenge peaked at day 45 and returned to near baseline at day 145. Additional analysis indicated that semi-immune volunteers without fever displayed a lower response to the challenge, but recognized new antigens afterwards., Conclusion: Clinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens.

Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied.

Background: Malaria control programmes rely on confirmation of parasite presence in patients' blood prior to treatment administration. Plasmodium parasites are detected mostly by microscopy or rapid diagnostic test (RDT). Although these methods contribute significantly to malaria control/elimination, they are not suitable for detecting the significant proportion of asymptomatic subjects harbouring low levels of parasitaemia, which endure untreated as potential reservoirs for transmission. Malaria prevalence was assessed in endemic regions of Colombia over a 4-year follow-up., Methods: A series of cross-sectional surveys were conducted between 2011 and 2014 in low to moderate malaria transmission sentinel sites (SS) of Tumaco, Buenaventura and Tierralta municipalities of Colombia. A census was performed and a random sample of houses was selected from each SS prior to each survey. Inhabitants were asked to answer a questionnaire on clinical, epidemiological and demographic aspects, and to provide a blood sample for malaria diagnosis using microscopy and quantitative real time polymerase chain reaction (qPCR)., Results: A total of 3059 blood samples were obtained from all SS, 58.5 % of which were from women and displayed a malaria prevalence ranging from 4 % (95 % CI 3-5 %) to 10 % (95 % CI 8-12 %) in the 4 years' study period. Almost all malaria cases (n = 220, 97 %) were sub-microscopic and only detectable by qPCR; 90 % of the cases were asymptomatic at the time of blood collection. While Buenaventura and Tierralta had a decreasing tendency during the follow-up, Tumaco had a rise in 2013 and then a decrease in 2014. Plasmodium vivax accounted for the majority (66-100 %) of cases in Tierralta and Buenaventura and for 25-50 % of the cases in Tumaco., Conclusions: This study demonstrates an important prevalence of asymptomatic malaria cases not detectable by microscopy, which therefore remain untreated representing a parasite pool for malaria transmission. This demands the introduction of alternative strategies for diagnosis and treatment, especially for areas of low transmission to reduce it to appropriate levels for malaria pre-elimination efforts to start.