Plasmodium falciparum erythrocyte membrane protein 1 mediating sequestration of P. falciparum-infected erythrocytes in patients suffering from severe malaria. Soluble EPCR (sEPCR) inhibits binding of P. falciparum to EPCR in vitro and increased levels of sEPCR have been associated with the H3 haplotype of the EPCR encoding PROCR gene. It has been hypothesized that elevated sEPCR levels, possibly linked to the PROCR H3 genetic variant, may confer protection against severe forms of malaria. This study determined the frequencies of PROCR haplotypes H1–4 and plasma levels of sEPCR in a Tanzanian study population to investigate a possible association with severe malaria.Endothelial protein C receptor (EPCR) was recently identified as a key receptor for PROCR gene were determined by post-PCR ligation detection reaction-fluorescent microsphere assay.Study participants were children under 5 years of age admitted at the Korogwe District Hospital (N = 143), and diagnosed as having severe malaria , uncomplicated malaria (N = 24), or an infection other than malaria (N = 67). In addition, blood samples from 71 children living in nearby villages were included. The SNPs defining the haplotypes of Individuals carrying at least one H3 allele had significantly higher levels of sEPCR than individuals with no H3 alleles (P < 0.001). No difference in the frequency of H3 was found between the non-malaria patients, malaria patients or the village population (P > 0.1). Plasma levels of sEPCR differed between these three groups, with higher sEPCR levels in the village population compared to the hospitalized patients (P < 0.001) and higher levels in malaria patients compared to non-malaria patients (P = 0.001). However, no differences were found in the distribution of H3 (P = 0.2) or levels of sEPCR (P = 0.8) between patients diagnosed with severe and uncomplicated malaria.PROCR haplotypes were in concordance with other African studies. The PROCR H3 allele was associated with higher levels of sEPCR, confirming earlier findings, however, in this Tanzanian population; neither PROCR haplotype nor level of sEPCR was associated with severe malaria, however, larger studies are needed to confirm these findings.Frequencies of SNPs determining  Plasmodium falciparum infection [P. falciparum erythrocyte membrane protein 1 (PfEMP1) variants mediating sequestration of infected erythrocytes in the most severe forms of malaria, including severe anaemia and cerebral malaria [In 2012, there was an estimated 165 million cases of malaria in Africa, leading to over half a million deaths [ malaria , 4.EPCR, also known as activated protein C (APC) receptor plays a key role in the regulation of coagulation, inflammation and endothelial cell integrity –9. It isPROCR gene, encoding EPCR. PROCR variants have been studied mostly in association with venous thrombosis [EPCR is also found as a soluble form in the plasma (sEPCR) where it binds APC in competition with cellular bound EPCR , 17. Elerombosis –21. Fourrombosis –20. The rombosis , 22, 23 rombosis , 21, altrombosis , 20. Conrombosis . The H4 rombosis .PROCR haplotype, sEPCR plasma levels and susceptibility to malaria [PROCR gene and susceptibility to severe malaria in a Ghanaian study [Studies investigating the association between  malaria –28 have  malaria , whereasan study . In a stStudy participants were enrolled at the Korogwe District Hospital and from two villages, Kwamasimba and Mkokola, in northeastern Tanzania. From Korogwe District Hospital, children under the age of 5 years were enrolled, if presenting with any symptoms of malaria, after obtaining informed consent from a parent or legal guardian. For diagnostics and research, blood samples were collected and treatment was initiated if the patients were positive by a malaria rapid diagnostic test (mRDT), following national guidelines. Children were considered as having severe malaria if haemoglobin levels were <5 g/dl (severe anaemia), assessed with a Blantyre coma score below 3 , showing clinical signs of respiratory distress, or reaching a parasitaemia level above 200.000 asexual stages/µl (hyperparasitaemia). Children not fulfilling the above criteria were defined as having uncomplicated malaria, as previously described . FurtherP. falciparum by blood smear microscopy. The study area has previously been described by others [As part of an ongoing, annual, malariometric, cross-sectional study in the study communities, blood samples were collected from volunteers residing in the villages of Mkokola and Kwamasimba, Korogwe district, northeastern Tanzania in 2007 who tested negative for y others , 33.After collection of whole blood, the plasma and buffy coat were each separated from the erythrocytes and stored at −80 and −20 °C, respectively. DNA was extracted from the buffy coat with the NucleoSpin Blood Kit (Machery–Nagel) according to manufacturer’s instructions.PROCR gene on chromosome 20 and consists of four exons and three introns [PROCR gene which define the H1, H3 and H4 haplotypes: the T(4414)C and C(4868)T SNPs in intron 1, and the A(6936)GSNP in exon 4. H1, H3 and H4 were defined based on earlier publications [2O, 2.5 µl 3 µM primermix, and 5.0 µl TEMPase Hot Start Master (Ampliqon). The reaction was performed in a 96-well PCR plate  in a VWRi Duo Cycler .EPCR is encoded by the  introns , 35. Priications , 19, wheications , 19. Siz2O, 0.15 µl 2 µM LDR primermix, 1.5 µl ligase reaction buffer, and 0.05 µl (2 units) Taq DNA ligase . The reaction was performed in a 96-well PCR plate  in a VWRi Duo Cycler (VWR/Bie & Berntsen). The conditions of the following microsphere assay were modified on a few parameters; 12 µl TMAC , 3 mM EDTA (pH 8.0), 0.1 % SDS)/1.2 µl Streptavidin-R-phycoerythrin conjugate  (10 µg/ml) solution was added to each well in the second hybridization; 75 of each microsphere were analysed. All primers were designed based on the GenBank accession number AF106202 and are shown in Table Following the PCR reactions, the products were mixed, and 2 µl used in the succeeding analysis; a ligase detection reaction-fluorescent microsphere assay (LDR-FMA). The LDR-FMA was developed similar to a previously described method  to analyThe levels of sEPCR in the patient plasma samples were determined by enzyme-linked immunosorbent assay (ELISA)  according to manufacturer’s instructions.Differences in mean age and distribution of gender between the groups were analysed with Mann–Whitney and Chi square test, respectively. The genotype distributions for each SNP were analysed for deviations from the Hardy–Weinberg Equilibrium with The Court Lab Calculator . Chi squEthical clearance for the study was granted by the Tanzania Medical Research Coordinating Committee and Ministry of Health and Social Welfare, Tanzania. Informed consent was obtained from parents or legal guardians.A retrospective selection of samples was collected from children under the age of 5 years, admitted at the Korogwe District Hospital. Seventy-six children were diagnosed with malaria based on mRDTs and positive blood smears, 52 with severe malaria, defined as hyperparasitaemia (N = 12), severe anaemia (N = 19), cerebral malaria (N = 17) or respiratory distress (N = 4). Sixty-seven children were admitted with infections other than malaria . Blood samples from 71 children without malaria based on mRDTs from two villages in the district, Kwamasimba and Mkokola, were also collected. The demographic characteristics of the study population are presented in Table The specificity of the LDR-FMA methodology was confirmed with control DNA samples from individuals with known genotypes of the SNPs used to determine the four haplotypes. The three SNPs were correctly identified in the control samples, with positive read-outs easily distinguished from the background read-outs in negative control samples. Results were furthermore confirmed by sequencing of DNA from selected patients (N = 24).2 = 0.34, P = 0.6; 4868: Χ2 = 0.25, P = 0.6; 6936: Χ2 = 0.32, P = 0.6). From the SNP alleles, the four haplotypes were constructed for each individual C, T(4868)C and A(6936)G), were determined for 213 individuals and the overall distributions for the alleles were found to be in Hardy–Weinberg equilibrium . To determine if high sEPCR plasma levels was associated with malaria, sEPCR levels were compared between the three study groups  or in sEPCR levels (P = 0.8). The cerebral malaria patients (N = 17) were then separated from the other severe cases  to determine if these patients differed in sEPCR level. No difference was found between the two groups (P = 0.7).The ia Table . No diffFinally, to investigate if there was any difference in sEPCR level during malaria and 3 weeks post-treatment, a sub-set of 28 children was included in a follow-up study (Fig. PROCR gene and their possible association with the level of sEPCR and disease outcome in Tanzanian patients infected with falciparum malaria. Based on earlier findings, it was hypothesized that the H3 haplotype, resulting in higher levels of sEPCR, could confer a protective advantage against severe malaria.This study investigated the four defined EPCR haplotypes resulting from SNPs of the PROCR gene have also been studied without defining these haplotypes in Ghana, Benin and Thailand [The haplotype distribution in this Tanzanian population differed from a Caucasian population , with a Thailand –27. In GThailand , 26. LasPROCR haplotypes are not a determining factor for severity of the disease, which is in concordance with findings from the study in Ghana, investigating over 40 SNPs, where none of the investigated SNPs was associated with severe malaria [In this study, the haplotypes were studied in three groups: malaria patients, patients with other infections and a village population. Malaria patients were divided into sub-groups based on severity of the disease and disease manifestation. No differences in H3 frequency were found between any of the groups. This indicates that the studied  malaria . Both th malaria . Applyin malaria . The stuThe present study also examined the association between sEPCR plasma level and malaria symptoms. Non-malaria patients were found to have the lowest levels of sEPCR, whereas the village population had the highest, and no difference was seen between uncomplicated, severe or cerebral malaria patients. Increased sEPCR levels were found in individuals who had recovered from a malaria episode. These findings differ from the study in Benin, where high levels of sEPCR were associated with cerebral malaria and death , and surThese discrepancies may be related to different disease state and ethnicity of patients, as well as the opposing effects that sEPCR binding to maturing circulating parasites , sEPCR sPROCR gene was sequenced and thoroughly examined to identify new SNPs [As observed in previous studies , 23, 25 new SNPs . HoweverPROCR expression. Thus, a good starting point would be larger studies of sEPCR levels in malaria-exposed populations to examine if elevated sEPCR levels do serve as a mechanism of protection against malaria.PfEMP1 binds EPCR with high specificity and affinity, and with a 3D structure preserved across sequence diversity . This inPROCR gene were studied in relation to malaria and sEPCR level in a Tanzanian study population and hospitalized patients. Frequencies of SNPs determining PROCR haplotypes were in concordance with other African studies. PROCR H3 was associated with higher levels of sEPCR, confirming earlier findings, however, in this Tanzanian population; neither PROCR haplotype nor level of sEPCR was associated with severe malaria.The four defined haplotypes of the


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups  and they contain conserved arrangements of functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria.var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others from laboratory isolates. Antibodies from Papuan patients to these parts of multiple PfEMP1 proteins were measured.Parts of expressed Patients with uncomplicated malaria were more likely to have antibodies that recognized PfEMP1 of Group C type and recognized a broader repertoire of group A and B PfEMP1s than patients with severe malaria.These data suggest that exposure to a broad range of group A and B PfEMP1s is associated with protection from severe disease in Papua, Indonesia.The online version of this article (doi:10.1186/s12936-016-1296-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum expressed on the surface of the infected erythrocyte (IE). Adherence of this molecule to host receptors expressed on endothelial cells, uninfected erythrocytes and placental syncytiotrophoblasts facilitates sequestration of IE in vascular tissues, avoiding destruction in the spleen [var multigene family [var gene variants and switching between single, transcribed var genes leads to changes in cytoadhesive phenotype as well as clonal antigenic variation and immune escape. var gene repertoires differ among isolates [PfEMP1 is the immunodominant antigen of the malaria parasite e spleen . PfEMP1 e family . Individisolates  and immuisolates –8. Immunisolates  and non-isolates  is acquiisolates , 12.var genes are also classified using their upstream sequence into groups A, B, C [var gene repertoire [var gene called var2csa has a different upstream sequence (ups E) and is only involved in malaria during pregnancy [PfEMP1s contain combinations of Duffy binding-like domains  and cysteine rich inter-domain regions  . Some DB A, B, C , 17 whicpertoire ; the uniregnancy .var genes [var genes has been associated with clinical, but not specifically severe malaria in Papua New Guinea (PNG) [var genes.The expression of particular subtypes of DBLα domains in severe malaria suggests severe disease may be preferentially caused by a restricted subset of ar genes , 20. Incea (PNG) , 22 and ea (PNG) . Cerebraea (PNG) , 25 or gea (PNG)  var genevar genes appear to be widely expressed by parasites that infect non- or semi-immune individuals. Antibodies from older children preferentially recognized PfEMP1s encoded by Group A var genes, indicating previous exposure [var genes dominated infection of a naive individual [Consistent with its having a role in severe malaria, PfEMP1s encoded by group A and B exposure . Group Adividual , and mordividual .var genes also encode adhesion phenotypes associated with severe disease. In Africa the adhesion phenotype of rosetting is associated with severe malaria [var genes [Group A and B  malaria  and incrar genes , 21, 25.ar genes , 31, andar genes . IE adhear genes , clinicaar genes  or inverar genes .var genes [Another phenotype associated with severe disease is adhesion to endothelial protein C receptor, EPCR , 37. Parar genes , 38. DC8ar genes , and conar genes , 37. Serar genes , 40.var genes. DC5 PfEMP1s bind platelet-endothelial cell adhesion molecule 1 (PECAM1) [var genes containing DC5 do encode EPCR-binding CIDRα1.5 domains although these domains are not part of the DC5 cassette [DC5 PfEMP1s, which are nearly all group A, are recognized by sera from semi-immune children in a similar manner to other severe malaria associated isolates , and DC5(PECAM1)  and IE a(PECAM1) , 45. How(PECAM1)  and DC5 (PECAM1) , 40. A mThus several promising candidates have emerged as members of the restricted population of PfEMP1s responsible for severe malaria, but the relative contributions of group A and B, and of DCs 8, 13 and 5 remain unclear. In particular very little is known about PfEMP1s in severe disease in the Asia Pacific region. Determining whether conserved PfEMP1sequences elicit protection from severe malaria disease globally is a priority for vaccine research. In this study plasmas from Papuan patients with severe or uncomplicated malaria were analysed for their reactivity with PfEMP1 polypeptides representative of the different groups and DCs including several expressed by parasites causing cerebral malaria.® (Life Technologies), incubated at 37 °C for 5 min and then stored at −80 °C. Venous samples were collected from patients with severe (n = 28) and uncomplicated (n = 35) malaria attending a healthcare facility in Timika, Papua Province, Indonesia. Plasma was separated from blood by centrifugation and stored at −20 °C. This area has unstable malaria transmission with estimated annual parasite incidence of 450 per 1000 population and symptomatic illness in all ages [Two ml of venous blood was collected from a 5-year old female patient with cerebral malaria in PNG. The IEs were separated from white blood cells by Plasmodipur column filtration (Europroxima) as per the manufacturer’s instructions. The IEs were lysed in TRIzolall ages . Severe all ages . Twenty-all ages . Thus inInformed consent was provided by all participants. The study was approved in Indonesia by the Eijkman Institute Research Ethics Commission (project number 46), in Australia by the Melbourne Health Human Research Ethics Committee (project number 2010.284) and Human Research Ethics Committee of the NT Department of Health & Families and Menzies School of Health Research, Darwin, Australia (HREC 2010–1396), and in PNG by the Government of PNG Medical Research Advisory Committee (MRAC no. 11.12).® were thawed at 37 °C, chloroform (1/5th of the TRIzol® volume) was added and vortexed 15 s, the solution was then subjected to centrifugation at 12,000×g for 30 min at 4 °C and the aqueous supernatant was aspirated and mixed with an equal volume of 70 % ethanol in RNase free water. This solution was then directly applied to RNeasy mini columns (QIAGEN), and RNA purification was performed with on-column DNasing as per the manufacturer’s instructions. Eluted RNA was oligo dT purified and used to generate a 65 bp paired end RNAseq library using the standard Illumina protocol. The library was sequenced on an Illumina GAII at Geneworks (Adelaide).Erythrocytes in TRIzolvar genes that were previously described in seven P. falciparum isolates [P. falciparum genome and unmapped reads were extracted to include any var that may have been missed. The reads that mapped to var genes and the reads that did not map to the 3D7 genome were then merged, digitally normalized using khmer [de novo assembly using Cap3 [var genes with BLAST (version 2.2.25). The transcript abundance was determined by aligning the reads again to the assembled transcripts.Alignments were performed using Subreads . Reads wisolates  and mappng khmer , and assng khmer . Assembling Cap3  to assemProteins for Luminex assays were expressed in baculovirus-transfected insect cells as previously described . ProteinPfEMP1 domains identified by RNAseq as expressed by the parasites infecting the PNG CM patient were amplified from gDNA purified from the patient’s blood for use as ELISA antigens. The sequences were then cloned into the plasmid pEU-E01H-N1   and exprvar gene sequences transcribed by parasites infecting the cerebral malaria patient from PNG. Proteins 1 and 3 were encoded by 352 bp and 123 bp fragments of a contig with homology to the DC5 DBLβ7_D8 of Dd2var4 (ranked 33rd by transcript abundance) (Table _D4_PFCLIN_var24 (ranked 96th by abundance).Antigens used for the ELISA included proteins one to seven that were encoded by var gene Dd2var4. This gene has group A-like coding features, e.g. head structure and ATS, and is the only DC5 with a group B-like upstream sequence, the other 11 all being group A [Proteins 1, 2 and 3 were most closely related to the DC5 domain The Luminex assay was performed as previously described . For eacELISA was performed as previously described . ELISA pU-test and with gender using a Fisher’s exact test. RU values for individual proteins were compared by Mann–Whitney U-test. To compare between patients with severe and uncomplicated malaria for antibody responses to proteins belonging to a single domain cassette or PfEMP1 group, patients were categorized according to whether their plasma sample lay above or below the median concentration of RU for that antigen: those above or equal scoring 1, or 0 if below. To derive a single quantitative score for each plasma for all the proteins belonging to a single DC or PfEMP1 group, the plasma’s scores for each antigen in that DC or group that were determined by either Luminex or ELISA were summed.The association between disease severity with age and parasitaemia was assessed using a Mann–Whitney Any individual plasma with a score of 1 for any protein within a DC or PfEMP1 group was classified as a responder to that DC or PfEMP1. Individual plasma samples with a combined score of 0 for all proteins within a DC or group were classified as non-responders.U- tests. This indicated differences in the breadth of the response to PfEMP1s within that group. The patients that did not respond to any protein within the group, i.e. had a score of zero were not included to remove any biases associated with large frequencies of zero values in non-parametric comparisons [Differences in the proportions of severe and uncomplicated malaria patients whose plasma responded to a DC or PfEMP1 group were compared by contingency tables using Fisher’s exact test. Differences between severe and uncomplicated malaria patients in the number of proteins within a DC or PfEMP1 group to which patients responded were compared only for patients who responded to at least one protein in the DC or PfEMP1 group using Mann–Whitney parisons .var sequences transcribed by parasites infecting this patient we used Illumina RNAseq to generate 65 bp paired-end short reads. Reads that mapped to the 399 full length var gene sequences available [P. falciparum 3D7 strain genome nor Homo sapiens, and the merged reads were subjected to two rounds of de novo assembly to generate 623 contigs that included 362 contigs with homology to var genes. The total length of assembled contigs was 200,158 bp, N50 423 bp, maximum contig length 4083 bp. The short contig length allowed assembly of only a few, full domains. However, the contigs could be aligned by BLASTN  to individual domains from the 399 full-length var genes and orthologs of high identity to annotated domains and DC types were identified  but which were transcribed at more than three times their level of representation in the seven sequenced genomes included DBLγ6, 4, 9, and 10, DBLδ5, DBLβ6 and 7, CIDRα2.4, CIDRb2 and DBLε1 and 6  see “” sectionvar genes may have been abundantly expressed by the parasites infecting this patient however these domains are also found at least as frequently in non-DC8 PfEMP1s. The DC13 is characterized by the tandem array of a DBLα1.7-CIDRα1.4 but neither of these domains were abundantly transcribed in this patient.The most promising candidates for causing severe malaria are the EPCR-binding CIDRα1 domains of DC8 and DC13 PfEMP1s. The most abundant CIDRα1 capable of EPCR binding  in this Var1csa sequences were the 7th, 12th and 45th most abundant transcripts; expression of this gene was not previously observed to be elevated in severe disease [var genes encoding the adhesion phenotype [var2csa was the 43rd most abundant transcript. No other DCs were identified in the 50 most abundant transcripts. disease  and it i disease , 60, atyhenotype , 62. Thehenotype , 64. DBLP. falciparum parasites/µl 41,220, IQR 8260-334,273; 61 % male) and 35 patients with uncomplicated malaria . Patients with severe malaria tended to be older than those with uncomplicated malaria (p = 0.0599), but there was no significant difference in P. falciparum density (p = 0.7288) and gender (p = 0.7981). Twenty-three patients with severe malaria had a single diagnostic criterion (WHO) [Antibody reactivity with PfEMP1 was assessed in plasma from 28 patients with severe malaria  , includiTo examine the reactivity of Papuan patient plasma with different PfEMP1 groups and DCs previously associated with severe or uncomplicated disease we tested 10 PfEMP1 DCs and the non-PfEMP1 proteins GLURP and MSP3 by a Luminex multiplexed bead assay; and twelve recombinant, partial PfEMP1 domains by ELISA. The proteins tested included groupings previously associated with severe disease  Fig. . Individp = 0.055). The MSP3 data suggests that patients with uncomplicated malaria may have had more prior exposure to P. falciparum infection than the patients with severe malaria.The DC domain constructs used in the Luminex assay were derived from the 3D7, ItG and HB3 isolates. Seven of the proteins used for ELISA were derived from sequences transcribed in the PNG cerebral malaria patient described above and included abundantly transcribed (by RPK) representatives of DC5 DBLβ7, DC5 DBLδ5, DC8 DBLγ6 and DBLγ10 Table b. By Lump > 0.054) in antibody response between plasma from patients with severe and uncomplicated malaria. These proteins included three severe malaria-associated PfEMP1s, two DC5, and one DC8  Fig. a. IntereDC8 Fig. a.p < 0.05). A non-significant trend in the same direction (p < 0.10) was observed for a single DC5 and a single DC8.In the ELISA, patients with uncomplicated malaria had significantly higher levels of antibody than patients with severe malaria to two group C PfEMP1s that were from lab isolates and a single group B DBLγ from the cerebral malaria patient  and to PfEMP1s that were not DC5, nor 8, nor 13 (p = 0.008) . It is possible that analysing larger numbers of proteins in these groupings on a single platform may have detected differences.The combined results for the ELISA and the Luminex assays revealed that a greater proportion of the patients with uncomplicated malaria than with severe malaria had antibodies to group C PfEMP1s (p = 0.031, p = 0.044 and p = 0.014, respectively, Fig. Amongst patients with an antibody response to at least one protein of a cassette or group, plasma from patients with uncomplicated malaria recognized more PfEMP1s overall, and more group A and group B PfEMP1s than did plasma from patients with severe malaria (var repertoire in human malaria. Although entire genes could not be assembled, advances in the phylogenetics of var sequences [var contigs to be separated into useful classifications. Previous studies that implicated parasites expressing group A PfEMP1s [var gene upstream sequences or by sequencing short DBLα tags.Sequencing the transcriptome of parasites causing CM in a single patient enabled assembly of a snapshot of the transcribed equences  allowed  PfEMP1s , 23–25 a PfEMP1s  in severvar genes in pathogenesis and abundantly expressed var genes identified included DC5 and possibly DC8, the DBLβ3 domain subtype and individual domains including CIDRα1.5 and DBLβ3. Recombinant proteins derived from the latter two domain subtypes have been shown to bind EPCR and ICAM-1 respectively [var genes in severe disease in future studies.The RNAseq of parasites causing CM in this PNG patient was consistent with previous studies of ectively , 36. Othvar repertoire was consistent with a previous report of cerebral malaria in Africa [var transcripts were in the 20 most abundant var contigs that between them represented 27 domains. Thus the quantitative nature of RNAseq revealed a hierarchy of var transcript abundance in this patient’s peripheral blood that would be difficult to detect using the non-quantitative, nested RT-PCR approaches available to this previous study [var transcripts expressed by parasites causing cerebral malaria in a single patient are probably restricted in number.The diversity of the transcribed n Africa . Howeverus study . This suPatients with uncomplicated malaria more commonly had antibodies to PfEMP1s that were from Group C or were not from DC5 nor DC8 nor DC13 than patients with severe malaria (Fig. var gene expression in both Africa and PNG [Overall, the serology findings in this Papuan adult population are consistent with existing models of infection in African children where parasites expressing severe malaria-associated group A and B PfEMP1s infect naive individuals and elicit antibodies , 66. Sus and PNG –26.var gene expression in severe disease [var gene sequences.In Papuan adults severe malaria is associated with a lack of antibodies to non-DC5 and 8 and group C PfEMP1s in general, and with antibodies to a narrower repertoire of group A and group B PfEMP1s than in patients with uncomplicated malaria. These findings from Papua are consistent with reports from Africa of elevated group A and B  disease , 23–26 a disease , 29. Thi


Anopheles mosquitoes, generally healthier human populations, better access to healthcare, and higher housing standards. Statistical relationships have been explored at global and local scales, but generally only examining the effects of urbanization on single malaria metrics. In this study, associations between multiple measures of urbanization and a variety of malaria metrics were estimated at local scales.Sub-Saharan Africa is expected to show the greatest rates of urbanization over the next 50 years. Urbanization has shown a substantial impact in reducing malaria transmission due to multiple factors, including unfavourable habitats for Anopheles mosquitoes captured), parasite prevalence and malaria incidence. Associations between measures of urbanicity and malaria metrics were made using negative binomial and logistic regression models.Cohorts of children and adults from 100 households across each of three contrasting sub-counties of Uganda  were followed for 24 months. Measures of urbanicity included density of surrounding households, vegetation index, satellite-derived night-time lights, land cover, and a composite urbanicity score. Malaria metrics included the household density of mosquitoes  had significantly higher urbanicity measures compared to the two rural sites. In Walukuba, all individual measures of higher urbanicity were significantly associated with a lower household density of mosquitoes. The higher composite urbanicity score in Walukuba was also associated with a lower household density of mosquitoes  and a lower parasite prevalence . In one rural site (Kihihi), only a higher density of surrounding households was associated with a lower parasite prevalence . And, in only one rural site (Nagongera) was living where NDVI ≤0.45 associated with higher incidence of malaria .Urbanicity has been shown previously to lead to a reduction in malaria transmission at large spatial scales. At finer scales, individual household measures of higher urbanicity were associated with lower mosquito densities and parasite prevalence only in the site that was generally characterized as being urban. The approaches outlined here can help better characterize urbanicity at the household level and improve targeting of control interventions. Plasmodium falciparum [Plasmodium vivax [Urbanization has generally been associated with a reduction in malaria transmission across spatial scales –5. Lowerum vivax  has beenum vivax , 7. Urbaum vivax , 4, 5 anum vivax , 8. Prevum vivax . No studMalaria remains a major problem in Uganda and many efforts have been made to control it including, but not limited to, provision and/or promotion of long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticides (IRS) . InterveHere, existing cohort studies were utilized in an exploratory analysis to examine associations between various measures of urbanicity and entomological, parasitological and clinical malaria metrics. The studies were conducted in three sites in Uganda with different levels of urbanicity and transmission intensity. Because of the underlying differences in the sites, several physical and infrastructure-based urbanicity metrics were evaluated that had consistent and quantifiable spatial characteristics, including the densities of surrounding households from survey data, and satellite image variables covering land use, night-time lights and occurrence or amount of vegetation.Cohort studies were conducted at three sites in Uganda:1) Walukuba sub-county in Jinja district ≈12.9 sq km  at the shores of Lake Victoria; (2) Kihihi sub-county in Kanungu district ≈186 sq km  in the southwest part of Uganda; and, (3) Nagongera sub-county in Tororo district ≈81 sq km  close to the Uganda–Kenya border. Figure  WalukubaJinja district was historically the industrial capital of Uganda and as such, Walukuba was urbanized in the industrial times of 1950–80s, hence the presence of factory buildings (now deserted for the most part), factory worker estates, more tarmac roads, and wider coverage of hydro-electric power than other sites. In Nagongera, according to a 2009/2010 survey, 2.6 % of households had electricity, 59.8 % of the homes were grass thatched , and lesAll households in the three sub-counties were enumerated in a census survey and geo-located using a Garmin e-Trex Legend H GPS unit . A household was defined as any single permanent or semi-permanent dwelling structure for humans who generally cook and/or eat together. One hundred households were randomly selected from each of the sub-counties and all children aged 6 months to 10 years and one adult primary caregiver from each household were enrolled as previously described . At enroThe cohorts were followed from August 2011 to September 2013, and they were dynamic, such that all newly eligible children were enrolled and study participants who reached 11 years of age were excluded from further follow-up. At enrolment, study participants and their parents/guardians were given a LLIN and underwent a standardized evaluation, including a history, physical examination and collection of blood for haemoglobin measurement and thick/thin blood smear. Cohort study participants received all medical care free of charge at a designated study clinic open every day. Participants who presented with a documented fever (tympanic temperature >38.0 °C) or history of fever in the previous 24 h had blood obtained by finger prick for a thick blood smear. If the smear had malaria parasites, the patient was diagnosed with malaria. Episodes of uncomplicated malaria were treated with artemether–lumefantrine and episodes of complicated malaria or recurrent malaria occurring within 14 days of prior therapy were treated with quinine. Routine thick blood smears were done every 3 months to estimate parasite prevalence.Entomological surveys were conducted monthly from August 2011 to September 2013 in each household using miniature CDC light traps  with the light positioned 1 m above the floor at the foot end of the bed where a cohort study participant slept. Traps were set at 19.00 h and collected at 07.00 the following morning by field workers. Methods used for the processing of mosquito specimens were as described previously .Anopheles mosquitoes captured divided by the number of house-nights of collection. Parasite prevalence was calculated as the number of routine blood smears positive for asexual parasites divided by the total number of routine blood smears done. The incidence of malaria was calculated as the number of episodes of malaria divided by the person years of observation. The malaria metrics used here can be placed on a spectrum that is comprised of three major components: the transmission phase, at the frontline between environment and the human host; the infection phase, an indication of malaria parasites independent of symptoms; and, the clinical phase, which manifests itself as a documented infection in the setting of symptomatic illness. In this study, HDM is in the first phase of this spectrum while parasite prevalence is apportioned to the second phase, one level removed from the environment, and the incidence of malaria is apportioned to the third phase, two levels removed from the environment.Malaria metrics used in this study included the household density of mosquitoes (HDM), parasite prevalence by light microscopy, and the incidence of malaria. The HDM was calculated as the total number of female Many different methods have been used in previous studies to provide consistent measures of either binary urban–rural classifications, or continuous quantification of urbanicity. Urbanicity can be measured in multiple ways and in this study physical-based measures that could be consistently derived between and across sites were measured, including household density, land cover, vegetation amount, and night-time light brightness. For each cohort household, 100-m buffer zones, also known as observational buffers, were created for generating estimates of household level measures of urbanicity by extracting data using ESRI ArcGIS 10.1 software . Cohort households accounted for 0.8, 1.0 and 1.4 % of the total number of households enumerated in the Kihihi, Walukuba, and Nagongera sub-counties, respectively.Household density was defined as the number of households enumerated in the observational buffers around each cohort study household.A supervised classification of Landsat imagery was undertaken, using Google earth  to definNDVI is measured on a scale of −1 to 1 where the extreme negative values represent water; values around zero reflect barren earth surfaces such as rock, sand or snow; medium positive values represent bushes or grassland; and, the higher positive values (≥0.6) represent temperate/tropical rainforest cover . NDVI ofhttp://ngdc.noaa.gov/eog/viirs) and the levels of brightness within the observational buffers were estimated on a relative scale.Urban areas generally display greater rates of electrification than rural ones , 23, andIn addition to the individual urbanicity metrics measured in this study, overall urbanicity at each participating household, referred to as the composite urbanicity score or composite score, was estimated using the values of the individual urbanicity metrics in the observational buffer of the household. A score of 1 was assigned for every instance of a high level of individual urbanicity metric and 0 otherwise Tables , 3, and Anopheles caught per house), with the number of sampling nights included as an offset term in the model. At the individual cohort participant level, logistic regression was used to model the relationships between measures of urbanicity and the odds of malaria infection  at the time of each routine clinic visit, and negative binomial regression was used to model these relationships with the incidence of malaria, with the duration of follow-up included as an offset term. Analyses at the individual level were adjusted for age of the study participants and robust standard errors were used to adjust for clustering of cohort participants living in the same household. Associations using negative binomial regression models were expressed as the incidence rate ratio (IRR) and associations using logistic regression models were expressed as the odds ratio (OR). A p value <0.05 was considered statistically significant.Data collected were analysed using Stata 12 . Lowess smoothing plots were used to explore the relationships between continuous measures of urbanicity and the various malaria metrics. Based on these exploratory analyses, continuous measures of urbanicity for participating households were dichotomized into the following categories for estimating associations with malaria metrics: (1) household density (≤80 vs >80 households per 100-m radius buffer); (2) night-time lights (≤3.0 vs >3.0 units of single band 32 bit pixels); (3) percentage residential low dense urban land cover (≤20 vs >20 %); (4) NDVI (≤0.45 vs >0.45); (5) composite score  compared to Kihihi  and Nagongera . At the level of the individual cohort participants, parasite prevalence in Walukuba (6.1 %) was similar to Kihihi  but significantly lower than in Nagongera . The incidence of malaria in Walukuba (0.36 episodes per person year (PY)) was significantly lower than in Kihihi  and Nagongera .A total of 1167 participants (878 children and 289 adults) in 300 households were enrolled and followed over the 2-year period; 348 in Walukuba, 419 in Kihihi and 400 in Nagongera. Overall, 1087 participants (93.1 %) and 292 households (97.3 %) were followed for at least 6 months. Characteristics of study households and cohort participants are presented in Table Anopheles collected over the 24-month period were 2358 in Walukuba, 10,370 in Kihihi and 100,797 in Nagongera. The most common species was Anopheles gambiae, with 86.3 % of those caught in Walukuba, 99.0 % in Kihihi and 90.3 % in Nagongera. Anopheles funestus, made up 6.7 % of catches in Walukuba, 0.0 % in Kanungu and 9.1 % in Nagongera. Finally, other female Anopheles species caught together made up 7.0 % of catches in Walukuba, 1.0 % in Kihihi and 0.7 % in Nagongera.The totals of female Anopheles collected over the 24-month period were 1256 (53.3 %), 666 (28.2 %) and 436 (18.5 %), respectively.Within the more urban site of Walukuba, individual households with characteristics of greater urbanicity were associated with a lower HDM including a household density >80 per 100 m radius , NDVI ≤0.45 , night-time lights >3 units , and land cover >20 %  , individuals living in households with characteristics of greater urbanicity were associated with lower parasite prevalence, but none of these associations reached statistical significance Table . HoweverNone of the measures of urbanicity, either individually or as a composite score, were associated with a lower incidence of malaria in individual study participants at any of the three sites Table . In NagoEvidence for the impact of urbanicity on reduced malaria prevalence over large scales is clear , 8 but tIncreased levels of urbanicity, specifically defined by higher household density, lower levels of NDVI, low residential urban land cover and higher night-time light brightness were observed to be strongly associated with reduced HDM at the urban site, Walukuba, but not in the other two more rural sites. For Walukuba, parasite prevalence was not found to be significantly associated with individual urbanicity metrics at the spatial scales examined here, but rather with the composite urbanicity score. However, increased urbanicity as measured by household density does appear to be associated with reduced parasite prevalence in one rural site, Kihihi, but does not in the other.Additional observations of disease burden across the three sites indicate that the urban site recorded significant declines in malaria incidence over the study duration . With thWhereas higher parasite prevalence was found in the more rural sites than Walukuba, consistent with multiple studies that have reported similar trends , 24, onlSome aspects of this study limit the ability to draw additional conclusions about urbanicity and malaria transmission. With just 100 households per site, sufficient variability may not have been captured to uncover weaker relationships that may have existed, especially within the more rural sites. These households were, however, randomly selected without bias and are thus likely representative of the populations, and the observational buffers used describe an environment shared by a larger number of households. Another notable limitation is that Nagongera and Kihihi are not so different or diverse in terms of urbanicity, and neither can be described as particularly ‘urban’ overall. High-resolution imagery that could better elicit differences between these sites is not readily available. However, malaria metrics between the two are substantially different and hence do provide a useful contrast in transmission. Factors such as agricultural practices may influence the malaria epidemiology of these rural regions far more than any urban-related factors, but analysis of these were beyond the scope of this current paper.Other measures used to define urbanicity that were not assessed in this study include the principal economic activity of the population. However, in a study of the risk of malaria in these three sites posed by the nature of house of residence, strong associations were found between HDM and type of house or wealth . In furtIn view of these limitations, future work may focus on better understanding the impact of urbanization on malaria through expanded analyses to investigate the role played by other contributory factors, such as rainfall, water bodies, temperature, intervention coverage, as well as nuances of malaria policy disparities across administrative units. Considering that other than space, the temporal aspects of public health are important, additional work should examine the impact of the temporal variations in urbanicity and other factors on malaria. Also, given a stronger understanding of the urbanicity influence on malaria, scaling up analyses to generate local and national risk maps would be a valuable effort for guiding malaria and other public health strategic planning. This, with the integration of mathematical transmission models to bolster guidance for planning for public health and malaria control, would be an important next step. Finally, given previous studies on travel and movement patterns in East Africa, and their perceived, as well as reported correlation with urbanization, proposed future work may incorporate these influences to further establish the contribution of travel and movement coupled with urbanization, to the malaria situation.Given differences in culture/behaviour, population characteristics, socio-economic strata, and environment between urban and rural communities, these results, though exploratory in nature, support the need for more localized approaches to malaria control and treatment, such as urbanicity tailored packaging of strategic planning foci, intervention approaches, impact evaluation, malaria awareness messages and/or public health campaigns. Africa, more than ever, is becoming increasingly urbanized and targeted interventions such as better garbage and drainage management coupled with LSM in urban areas, would likely results in a great depletion of breeding sites. Given the burden of malaria in sub-Saharan Africa, there is a need to understand the broad impacts of urbanicity on malaria at various scales. Whereas previous studies have quantified these impacts chiefly on large scales and used just single metrics for quantifying the relationship between urbanicity and malaria, this study has shown that the situation is much more complex at smaller scales.


Malaria causes a significant burden in highly endemic areas where children and pregnant women are more susceptible to severe disease and death, however, in low transmission settings malaria in pregnant women is less frequent. The aim of this study was to provide information of clinical profile, anti-parasite host immune responses and parasite genotyping of pregnant women with malaria in low endemic areas of Colombia.This was a descriptive study conducted through passive surveillance in 1328 individuals from three endemic areas of Córdoba, Nariño and Chocó departments between 2011 and 2013. Trained physicians confirmed the pregnancy status and recorded clinical and epidemiological information. Haematological parameters, as well as hepatic and renal function, anti-malarial antibodies and parasite genotypes were evaluated.Plasmodium falciparum (n = 24). In 44 % (n = 15) of the women, the infection occurred during the first half of pregnancy. Although uncomplicated disease and parasitaemia ≤20,000 parasites/µL were common (n = 31), three women (8.8 %) infected by P. falciparum were classified as severe cases. Mild to moderate anaemia (68 %) and mild thrombocytopaenia (41 %) were the most frequent blood alterations and in four women acute renal failure was observed. Six women presented a second malaria episode during pregnancy mainly caused by P. vivax (n = 5), although no direct evidence of relapse was found by genotyping. Two out of the six women presenting a second malaria episode had severe malaria. A low prevalence of specific anti-parasite antibodies was found. Microsatellites indicated that all P. vivax infections involved multiple lineages whereas all but one P. falciparum infections harboured single genotypes.A total of 582 women presented with malaria infection, 34 of whom were pregnant (5.8 %), and most were infected by Most malaria infected pregnant women displayed uncomplicated malaria, although a few of them with a second malaria episode presented an increased risk of severe malaria which appeared to be associated with malaria transmission intensity and not with levels of anti-parasite antibodies. The effects of severe malaria in both mother and fetus warrant future studies in low transmission settings.The online version of this article (doi:10.1186/s12936-016-1125-9) contains supplementary material, which is available to authorized users. Plasmodium falciparum is the predominant malaria parasite. Outside Africa, Plasmodium vivax infections are predominant and every year about 90 million pregnant women are exposed to the risk of infection mostly in the Asia–Pacific region only . Malaria parasite species was retrospectively confirmed in all samples by real time PCR, as described elsewhere . AutomatP. vivax and P. falciparum are present in the study areas, sera from all patients were analysed for antibodies to both parasite species. IFA tests were performed using P. vivax and P. falciparum blood stages antigen preparations derived from infected patients or from in vitro cultures respectively as previously described [P. vivax circumsporozoite protein , the merozoite surface protein-1  as well as the response against P. falciparum CS  and MSP-1  as described elsewhere [Specific IgG anti-malarial antibodies in serum were determined both by immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) tests. Because both escribed . Antibodlsewhere . OpticalP. vivax and nine for P. falciparum selected from an extensive pool of choices that have been explored [P. vivax, the following loci were included in the analyses: MS2, MS5, MS6, MS15 [P. falciparum [Whole blood samples collected with EDTA anticoagulant were used for extraction and purification of genomic DNA using the QIAamp DNA Blood kit according to manufacturer’s instructions . The DNA was eluted and stored at −20 °C until use. Genotyping was performed using fluorescently labeled PCR primers. Those with low parasitaemia were amplified by whole genome amplification using the REPLI-g Mini Kit . A set of 10 standardized STRs loci for explored  were useS6, MS15  and 14.1S6, MS15 . Loci POlciparum . Fluoreslciparum .® 2013a . The nominal variables were analysed using descriptive statistics. The Mann–Whitney U test was used to compare two groups and Wilcoxon signed rank test for paired data. Spearman’s rank correlation (rs) was used to assess the correlation between numeric variables. Fisher’s exact test was used to compare proportion differences. A p value <0.05 was considered statistically significant.Study data were collected and managed using REDCap  electronic data capture tool . Data weFrom 1328 subjects infected with malaria, 582 were women and 34 of them were pregnant (5.8 %). Most patients were ≤25 years of age  and a high proportion of them  became infected in the first half of pregnancy, seven of them during the first trimester. Afro-descendants  and mestizos  were the most frequent ethnic groups and the remainder corresponded to indigenous population. In total, 62 % (n = 21) of women had resided in the malaria-endemic region  and 88 % (n = 30) were working at home. Half of the women self-reported previous lifetime malaria episodes and in eight women this happened in the previous year  appeared higher than in P. falciparum infections . Such difference was not statistically significant.All malaria infections were confirmed by real time PCR as P. falciparum and three with P. vivax) reported one or more of the following symptoms associated with pregnancy: reduction of foetal movement, preeclampsia signs or abdominal and pelvic pain (sudden and intense) without signs of preterm delivery.Seven women 21 %) attended at the POC for diagnosis within 48 h after symptoms onset (range 0–22 days) and only four women reported >15 days of illness . Thrombocytopaenia  was found in 41 % (n = 14) of the women, but only three of them had <100,000 platelets/µL. A negative correlation was found between platelets counts and parasitaemia , but it was not associated with parasite species, age or weeks of gestation (data not shown). Mild leukopaenia (18 %), lymphopaenia (15 %) and neutropaenia (5 %) were observed mainly in P. falciparum infected women.There were no significant differences for haematological parameters induced by the two parasite species Table . Mild-toP. vivax infections. Hepatic enzymes (ALT and AST) were altered in 21 % (n = 7) of women, all of them infected with P. falciparum. Proteinuria (61.8 %), ketonuria (29.4 %) and leukocyturia (23.5 %) were observed in women infected with either parasite species. Acute renal failure of pre-renal origin (BUN: creatinine >20) was found in four women, of whom two had also proteinuria and choluria. Using standard criteria in pregnancy [P. falciparum, 6/8 and 4/6, respectively.Median values of renal and hepatic function laboratory parameters were similar in both parasites species Table , althougregnancy , eight wP. falciparum and five with P. vivax) presented with a second malaria episode by the same parasite species, even after receiving treatment for asexual parasite stages in the first episode; however primaquine was not administered for P. vivax due to its contraindication during pregnancy. Since the follow-up was passive, new episodes, complications or self-treatment in some of the women cannot be ruled out.As described above, patients were asked to return to the POC if malaria symptoms recurred. A total of six women  and patients reported similar signs, symptoms, and days of illness before consultation (median 4 days) but lower parasitaemia than in the first episode  were classified as severe malaria during the first episode: two women developed prostration and required hospitalization, and one had severe thrombocytopaenia  without spontaneous bleeding. The three women also presented moderate anaemia (mean Hb 8.7 ± 0.1 g/dL). All women received anti-malarial and supporting treatment according to severity of infection as recommended by Colombian guidelines [Three idelines ..P. falciparum infection  presented hepatic dysfunction with hyperbilirubinaemia  and clinical jaundice, while another with P. vivax (3655 parasites/µL), presented severe anaemia (Hb 4.4 g/dL) with general pallor without co-incidental infections. No deaths were reported.In addition, two 15 year-old women presenting with a second malaria episode during pregnancy, were severe. One woman withP. vivax and P. falciparum blood stages by IFA test respectively and significant differences were observed between antibody specific parasite species (Table PvCS (32.4 %), PvMSP-1, (55.9 %), PfCS (26.5 %) and PfMSP-1 (73.5 %); 8.8 % (n = 3) and 47 % (n = 16) women were double positive for P. vivax and P. falciparum CS and MSP-1 antigens, respectively, however antibody levels displayed low titers  and PfMSP-1 , respectively. In contrast, the RI values for PvCS and PvMSP-1 were similar in P. falciparum and P. vivax infected women . In women who had two P. vivax episodes during pregnancy an increase in the RI for PvMSP-1 during the second episode was observed . No association was found between antibody titers and disease severity.A total of 47.1 % (n = 16) and 52.9 % (n = 18) pregnant women had antibodies to whole es Table . When anP. falciparum (14/24) and P. vivax (8/10) were analysed using a set of physically unlinked microsatellite loci , with six genotypes in 14 samples and only one sample with two genotypes differentiated by one locus. In comparison, P. vivax was more diverse, with all eight samples with two or more genetically distinct lineages.A total of 22/34 parasite samples including lciparum 4/24 and P. falciparum episodes, the same genotype was found in both episodes, whereas the two women who presented two P. vivax episodes had distinct genotypes with differences in at least three loci between episodes  indicated a higher prevalence 9 %–14 %) of MiP , 35, 36 P. vivax , 11. IndP. vivax  and Thai4 % of MiP. falciparum infections [P. vivax is more prevalent in Colombia, most MiP cases reported here were induced by P. falciparum, particularly in Tumaco and Quibdó, two areas where population is Afro-descendant with high prevalence of Duffy-negative individuals [Importantly, younger maternal age (≤25 years) observed in 76 % of patients and early malaria infection (<20 weeks of gestation) in 44 %, appeared associated with clinical presentation and may to increase the risk of intrauterine growth restriction , 38. A sfections . Althougividuals .P. vivax infection [P. falciparum harboured single genotypes regardless of disease severity; those also reported longer residence in the endemic areas. In contrast, all infections with P. vivax involved multiple genotypes. Such differences between P. falciparum and P. vivax have been observed in many malaria endemic areas [P. vivax infection is associated with severe malaria in Colombian patients [P. vivax episode displayed different genotypes suggesting a new infection. However, a relapse cannot be ruled out due to the restriction of using primaquine during pregnancy [P. falciparum genotype was found in the women presenting two episodes. The second episode occurred 27 days after the first one and was a severe case, but the parasite had wild-type allele of the K13 gene associated with artemisinin-based combination therapy resistance [P. falciparum and P. vivax in pregnant women [Previous exposure to malaria infection might have contributed to a higher percentage of uncomplicated cases. Between 47 and 53 % of women had antibodies against whole blood stages, which confirmed self-reported previous exposure to parasite. A previous study demonstrated the development of significant protection from clinical symptoms in young adults from the study regions exposed to experimental nfection , in addinfection . Most woic areas . Althougpatients . In thisregnancy . In contsistance , suggestnt women ; therefoPrompt diagnosis, effective treatment, and partial acquisition of clinical immunity appear to be associated with the high prevalence of uncomplicated MiP. However, anaemia and acute renal failure observed warrant more detailed studies because of the potential deleterious effects on both mother and fetus. This study reinforces the need for a more careful medical follow up during pregnancy, including malaria diagnosis as part of antenatal care for all pregnant women residing in endemic areas, to decrease the risk of complications and to avoid multiple malaria episodes associated with greater malaria severity.


Plasmodium ovale infection with molecular confirmation in Central America, which occurred in a Guatemalan soldier that had been deployed in Africa. The obstacles for its diagnosis using the standard microscopy technique and the need to improve its detection are discussed.The Mesoamerican Ministers of Health have set 2020 as the target for malaria elimination to be achieved in the region. Imported malaria cases are a potential threat to countries attempting elimination or working to prevent resurgence. We report the first imported  Plasmodium falciparum or P. vivax. However, P.ovale infection has been established as a relevant cause of imported malaria in these groups, resulting in challenges for accurate diagnosis and treatment [One factor in the worldwide increase of imported malaria cases has been the presence of armed forces and United Nations (UN) peacekeeping missions in malaria-endemic areas and their subsequent return to their countries of origin . As expereatment .P. ovale is mainly distributed in sub-Saharian Africa and the islands of western Pacific with prevalence between 3-10% depending on the region, population and type of diagnostic tests used [et al. demonstrated there are two non-recombining sympatric species of P. ovale, named P. ovale curtisi (classic type) and P. ovale (variant type) [P. ovale cases have occurred and there have been just a few reports of imported P. ovale infections [sts used . Recentlnt type) . In Latifections -8.P. vivax and P. falciparum are the etiologic agents of malaria, with the last case of P. malariae reported in 1971 and no P. ovale cases ever documented in Guatemala [Guatemala is a Central American country, with approximately 80% of its territory considered a malaria transmission setting . P. vivauatemala . In the uatemala  to 3,292uatemala .P. falciparum malaria in 150 Guatemalan soldiers returning from the Democratic Republic of the Congo (DRC) [P. vivax /P. falciparum malaria carriers are conducted annually among returning contingents from the DRC as part of a program established between the Guatemalan National Reference Laboratory, Universidad del Valle de Guatemala and Military Healthcare and Social Services. Here we present the first P. ovale case detected in this population. To the best of our knowledge this is the first report of molecularly-confirmed imported P. ovale infection in Central America. The challenges that this case presented for an accurate diagnosis and the need to incorporate more sensitive methods will be discussed.Since 2004, Guatemala has deployed military personnel to contribute to UN peacekeeping missions. In 2010, an epidemiological investigation detected 12 cases of imported go (DRC) ,14. SincP. vivax malaria. Three months previously he had returned from a 9-month deployment in Africa, as part of a Guatemalan Army operation to aid the UN Organization Stabilization Mission in DRC.A 38-year-old male Guatemalan soldier presented to the facilities of the Military Hospital on June 29, 2012, to confirm suspected P.vivax/P.falciparum PCR. Microscopic and molecular results were negative for the entire contingent.The soldier reported that during his time in DRC he stayed in the towns of Dungu, Bangadi and Bunia. He travelled for a short period of time to Entebbe and Kampala (Uganda) and Mombasa (Kenya) as part of his rest and recuperation time. During the entire mission, he used an untreated bed net and received mefloquine prophylaxis (250 mg base/week). He reported high compliance with the drug, although he admitted to skipping some doses during his period in Uganda and Kenya. Upon his return to Guatemala, he and all deployed personnel were screened by malaria microscopy and P. falciparum/P. vivax  includes chloroquine [Two months after his return to Guatemala, the patient travelled to Puerto de San José, Escuintla  and then returned to his residence in Jalpatagua, Jutiapa (eastern region). Malaria is endemic in both of these regions. He reported fatigue and fever, and malaria was clinically diagnosed by a private physician without any laboratory confirmation. He was given chloroquine diphosphate injections (no available dosage data) because the standard treatment in oroquine .P. vivax infection. The patient was transferred to Guatemala City to be evaluated in the Military Medical Center.The patient reported an improvement and returned to military duties. Three weeks later, he again presented with fever and fatigue. Malaria microscopy conducted at the Jutiapa Malaria Vector Control Program erroneously diagnosed a P. vivax, although concerns about the morphology of some of the observed parasites were raised by the hospital microbiologist. A complete blood count at admission revealed thrombocytopenia  and anemia (haemoglobin 12 g/dl). A rapid dengue diagnostic assay showed a current or recent infection. Other laboratory tests results were within reference values.Venous and capillary blood was taken for laboratory testing. A thin blood film identified Plasmodium species. A P. ovale mono-infection was identified on both the blood film and whole blood of the patient using a Plasmodium genus-specific nested PCR  analysis revealed 95% similarity with P. ovale ssrRNA gene. The strain was classified as the classic type, P. ovale curtisi, based on its nucleotide polymorphisms [Based on the travel history of the patient and the doubts arisen as to its species identity, molecular testing was conducted to confirm the R Figure  and a SYR Figure . SequencP. vivax infections, as the results of the molecular analyses were not available at the time of treatment initiation. The patient was discharged after his clinical condition and hematological results improved. Follow up specimens were taken at day 1, one and two months after treatment completion, confirming parasite clearance. Also, all family members in the household and one neighbor provided blood samples, with negative results for Plasmodium spp. As a control measure, the household was sprayed with deltamethrin.For malaria treatment he was prescribed 1,500 mg of chloroquine base given over 3 days and 15 mg of primaquine base over 14 days, following national guidelines for the treatment of Plasmodium ovale case in Guatemala. Based on the patient’s travel history, he could have acquired the infection in any of the African countries he stayed in, as P. ovale is present in all of them [Here we present the first imported  of them ,18. Lack of them . The pat of them .P. ovale as a cause of imported malaria and to include its detection, as well as P. malariae, in imported malaria screening programs. This patient presented particular diagnostic challenges that resulted in a failure of early detection and diagnosis of P. ovale infection. Distinguishing P. ovale from P. vivax by microscopy, already difficult due to the species’ morphologic similarity [P. ovale’s non-indigenous status. Since this event, all returning military contingents are being tested with a pan-Plasmodium real time PCR assay. In addition to malaria screening programs, clinical diagnoses should not automatically exclude consideration of non-endemic species of malaria. Regular training of microscopists for the accurate identification of all malaria parasites, including non-indigenous species, should be mandatory [This report highlights the need to consider milarity , was furandatory .Plasmodium species [In addition to mitigating the diagnostic challenges of imported malaria, monitoring imported cases into an already malaria-endemic area is important because the presence of viable vectors could lead to secondary autochthonous cases and the spread of drug-resistant strains, thus hampering malaria control and elimination efforts in regions, such as Central America that remains one of the few areas of the world where no antimalarial drug resistance has been reported . An obse species .P. vivax or P. ovale infections in long-term Army deployments which are significantly exposed to these parasites [Considering the long latency of these infections, further strategies should be considered to improve prevention. For instance, the use of presumptive anti-relapse therapy could be an effective approach to prevent relapses of arasites .Imported malaria cases could lead to the introduction of drug resistant strains in Central America, as well as atypical malaria species, especially in areas where competent hosts could harbor these parasites and transmit them . An inteWritten informed consent was obtained from the patient for publication of this Case Report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


To examine the associations between objectively measured physical activity and sedentary time with pulse wave velocity (PWV) in Brazilian young adults.g), time spent in moderate-to-vigorous physical activity (MVPA – min/day) and sedentary time (min/day) were calculated from acceleration data. Carotid-femoral PWV (m/s) was assessed using a portable ultrasound. Systolic and diastolic blood pressure (SBP/DBP), waist circumference (WC) and body mass index (BMI) were analyzed as possible mediators. Multiple linear regression and g-computation formula were used in the analyses.Cross-sectional analysis with participants of the 1982 Pelotas (Brazil) Birth Cohort who were followed-up from birth to 30 years of age. Overall physical activity (PA) assessed as the average acceleration  compared with the lowest quartile. Participants in the highest quartile of sedentary time had 0.39 m/s higher PWV  than those in the lowest quartile. Individuals achieving ≥30 min/day in MVPA had lower PWV . Mutually adjusted analyses between MVPA and sedentary time and PWV changed the coefficients, although results from sedentary time remained more consistent. WC captured 44% of the association between MVPA and PWV. DBP explained 46% of the association between acceleration and PWV.Physical activity was inversely related to PWV in young adults, whereas sedentary time was positively associated.Such associations were only partially mediated by WC and DBP. These individuals have thereafter been followed on several occasions throughout their life-course . Further details about the methods of the cohort are available elsewhere Between June 2012 and February 2013, when participants were on average 30 years, we tracked the entire cohort using multiple strategies to locate cohort members. All participants were invited to visit the research clinic for interviews and a clinical examination.2.2g .Physical activity was measured using the GENEActiv accelerometer . The monitor was worn on the non-dominant wrist. The GENEActiv activity monitor is waterproof and measures acceleration in three axes  with a sample frequency of 85.7 Hz. Data are stored directly as sampled from the MEMS chip and provided in units of Individuals received the device during their visits to the research clinic. Participants who were disabled, living in others cities , with labor activity that did not allow the accelerometer use  were excluded from the measurements . Women who were pregnant during the clinical visit were not eligible and invited after delivery to wear the accelerometer. Physical activity was assessed between four and seven days, including at least one weekend day using a 24-h protocol. Participants who started their measurements on Mondays, Tuesdays or Wednesdays were monitored until the following Monday and, those who started their measurements on Thursdays, Fridays or Saturdays, were monitored until the following Wednesday. The first 10 h were excluded because this was the maximum period observed between initialization and attachment of the monitors.http://cran.r-project.org). The average magnitude of wrist acceleration over the measurement period normalized to a 24-h period after exclusion of invalid data segments was the main measure used. The signal processing scheme as carried out by GGIR included the following steps: verification of sensor calibration error using local gravity as a reference The accelerometers were set up and downloaded in the GENEActiv software. The accelerometer data in binary format were analyzed with R-package GGIR . Results are presented in milli-g (1 mg = 0.001 g) for readability reasons. A time window (60-min with 15-min moving increments) was classified as non-wear time if, for at least two out of the three accelerometer's axes, the standard deviation was less than 13 mg and the value range was less than 50 mg.Files were considered as valid if data were present for every 15-min period in a 24-h cycle (even when scattered over multiple days) and with calibration error lower than 0.02 g based on 5-s epoch data and 10 min bout durations in the minimum, and <20% of the data points below this threshold. Sedentary time was defined as the time spent below an intensity threshold of 50 mg, excluding the hours between 11:00 p.m. and 7:00 a.m. – assumed as sleeping period, measured in minutes/day The summary measure ENMO was used as an indicator of average magnitude of dynamic wrist acceleration over the measurement period. Time spent in moderate-to-vigorous physical activity (MVPA) per day was estimated using an intensity threshold of 100 m2.3®  in the supine position and measurements were taken in the right side. An electrocardiogram was registered at the same time. Duration of the examination was 10–15 min for each participant. The distance of pulse wave transit was measured by a flexible tape as the distance from suprasternal notch to femoral point of application of the tonometer and the distance from carotid point of tonometer application and the suprasternal notch. PWV was calculated by the software as the distance between the measurement sites divided by transit time delay between femoral and carotid pulse wave. The mean of measurements was used in the data analysis.The carotid-femoral PWV (meters/second) was examined twice during the clinical visit using a portable ultrasound, SphygmocorTraining for PWV assessment was carried out in two days using volunteers. PWV was calculated by the software as the distance between the measurement sites divided by transit time delay between femoral and carotid pulse wave. The software evaluated the quality of each measurement according to the format of wave and synchronism with wave from electrocardiogram. This protocol followed recommendations from expert consensus document on arterial stiffness and the Research Applications Manual of SphygmoCor 2.4Other characteristics evaluated in this study were: sex, skin color (self-reported), family income at birth, socioeconomic status  ® scale with a precision of 0.01 kg. Waist circumference was measured using a flexible tape  with an accuracy of 0.1 cm at the narrowest part of the trunk, identified as the midpoint between the lowest rib margin and the iliac crest. The measurement was taken after a gentle expiration twice. If the difference between the two measures was greater than 1 cm, two additional measures were taken. Blood pressure was measured twice, at the beginning and at the end of the anthropometric measurements, in the sitting position using a digital sphygmomanometer Omron model HEM-705CPINT  on the left arm. The means of the two readings were used in the present analysis.Possible mediators evaluated in this study were: body mass index (BMI), waist circumference (WC), systolic (SBP) and diastolic blood pressure (DBP). BMI was calculated by dividing the weight in kg by the square of height in meters. Standing height was measured to the nearest 1 mm with barefooted subjects using a wooden stadiometer. Weight was assessed using the BodPodDaily energy intake  was estimated using a semi-quantitative food frequency questionnaire (FFQ) in a self-administered and digital version with 88 food items especially created for this cohort. Frequency of food consumption was asked in <once/month; 1–3 times/month; once/week; 2–4 times/week; 5–6 times/week; once/day; 2–4 times/day and ≥5 times/day. After, using photos of standard servings based on previous 24-h dietary recall (50 percentile), individuals answered if they usually ate a serving equal, greater or lower than shown in the picture. Because FFQs usually overestimate the food consumption, the lowest frequency chosen was considered for each food. Frequency was multiplied by 0.5, 1 or 1.5 if serving chosen was lower, equal or greater than the standard serving, respectively. Daily food frequency was obtained dividing the information in years by 365.24 days/year. Based on references available to estimate the macronutrients 2.5Data analysis was carried out using Stata 12.0 . Description of the sample with complete data of PWV was described in proportion or mean and standard deviation, according to type of variable. Linear regression models were used in the crude and adjusted analyses using all exposures in quartiles to assess possible dose-response. Statistical significance was obtained using two-sided Wald's tests. Possible confounders were: sex, skin color, family income at birth, socioeconomic status and current smoking. The association of MVPA and sedentary time with PWV was also mutually adjusted between them. We evaluated whether the associations were modified by sex. Mediation analyses for BMI, WC, SBP and DBP were carried out using the g-computation formula 3At 30 years of age, 3701 individuals were located and examined representing a follow-up rate of 68.1% (following exclusion of 325 known deaths). Due to practical reasons  PWV was measured in 1576 participants (42.6% of those interviewed) and data on physical activity were available in 2740 participants. In total, 1241 participants provided information on both physical activity and PWV and were included in the present analyses. Because there was no evidence of interaction with sex in the association between accelerometry and PWV , all analyzes were performed sex combined adjusting for sex as a confounder. Acceleration was inversely related to PWV - subjects in the highest quartile of acceleration had on average −0.37 m/s [β = −0.37 m/s ] of PWV than those in the first quartile. Individuals in the third and fourth quartile of time spent in MVPA had similar reductions in PWV. Conversely, participants in the highest quartile of sedentary time had on average 0.36 m/s higher PWV [β = 0.36 m/s ] than those in the lowest quartile. Association between MVPA in quartiles and PWV remained after adjustment for sedentary time (p = 0.046), although beta coefficients reduced for −0.18 m/s in the two highest quartiles [β = −0.18 m/s ], and 95% confidence interval was not statistically significant in the fourth quartile. On the other hand, PWV was substantially higher in the highest quartile of sedentary time, even after adjustment for MVPA [β = 0.28 m/s ] .4In an analysis involving 1259 young adults from Brazil, we observed that subjects who were more physically active had lower PWV. Subjects who spent more than 30 min/day in MVPA had a lower PWV. In addition, PWV was positively associated with sedentary time. Coefficients from association between MVPA and sedentary time and PWV changed in mutually adjusted analyses and were more consistent for results using sedentary time. WC and DBP were important mediators in the association of MVPA and sedentary time with PWV.Similarly to our study, an inverse association between physical activity and the central augmentation index has been observed among adults aged 55 years, on average Recently, sedentary behavior has been recognized as a risk factor for obesity, diabetes and cardiovascular diseases, independent of MVPA There is debate in the literature whether the health effects of sedentary time are really independent of those of physical activity Training positively affect aortic PWV Waist circumference captured part of the relationship between MVPA and PWV. This can be due to the effect of physical activity on weight loss and in visceral fat High levels of sedentary time can coexist with high levels of MVPA Possible limitations of our study include losses to follow up, particularly because due to operational reasons, we were unable to measure PWV and physical activity from accelerometry in all those followed up at 30 years. However, since this is an association study, these losses do not affect the relevance of our findings. In addition, though with statistical significance, the differences found showed very similar distribution of the individuals in the variables investigated. Moreover, the amount of days/time measure was variable depending on the day of the week the person was included. In addition, some had more weekdays, others less. Since the amount of activity varies between weekdays and weekend, this could have influenced the results. Another issue to be considered is that since only a couple of days/hours were collected, this may not represent routine life. Finally, since the changes in PWV are a chronic process, and the amount of activity may change over the years, it may well happen that the PWV measured today reflects the history of physical activity over the last years, which may not be related to today's activity.The most important strengths of the current study are the large and representative sample of young adults who had their levels of MVPA and sedentary time objectively evaluated by the use of accelerometer. In addition, early vascular disease was objectively assessed. Moreover, the availability of information to perform a mediating analysis helped to elucidate the pathways that connect physical activity and sedentary time to PWV.In conclusion, the findings showed an inverse association between objectively measured physical activity and PWV, as well as a positive association between sedentary time and PWV. Such associations were only partially mediated by WC and DBP. Reducing sitting time and promoting physical activity is essential to help prevent the incidence of cardiovascular diseases across the lifespan.None declared.


Plasmodium falciparum and 161,236 suspected infections reported in 2012. At low prevalence levels, passive surveillance measures, which rely primarily on reports from health systems, becomes less appropriate for capturing annual malaria incidence. To improve understanding of malaria transmission in Haiti, participants from the Ouest and Sud-Est departments were screened using a highly sensitive enzyme-linked immunosorbent assay (ELISA).Malaria transmission continues to occur in Haiti, with 25,423 confirmed cases of 19). The classification of previous exposure was established by using a threshold value that fell three standard deviations above the mean absorbance for suspected seronegative population members . The observed seroprevalence values were used to fit a modified reverse catalytic model to yield estimates of seroconversion rates.Between February and May 2013, samples were collected from four different sites including a rural community, two schools, and a clinic located in the Ouest and Sud-Est departments of Haiti. A total of 815 serum samples were screened for malaria antibodies using an indirect ELISA coated with vaccine candidates apical membrane antigen (AMA-1) and merozoite surface protein-1  were positive for either AMA-1 or MSP-1; indicating rates of previous infections between 21.1% and 30.3%. Not surprisingly, age was highly associated with the likelihood of previous infection . After stratification by age, the estimated seroconversion rate indicated that the annual malaria transmission in the Ouest and Sud-Est department is approximately 2.5% .Of the samples screened, 172 of 815 (21.1%) were AMA-1 positive, 179 of 759 (23.6%) were MSP-1These findings suggest that despite the absence of sustained malaria control efforts in Haiti, transmission has remained relatively low over multiple decades. Elimination in Haiti appears to be feasible; however, surveillance must continue to be strengthened in order to respond to areas with high transmission and measure the impact of future interventions. Plasmodium falciparum, and the principal mosquito responsible for malaria transmission, Anopheles albimanus, is primarily zoophilic making it a poor vector of disease . When seroreversion was included in the model, the probability at age x was modeled using the equation P(x) = λ/(λ + ρ) [1 ‒ exp(-(λ + ρ)x)]. The functions were optimized (using R) to give estimated seroconversion/reversion rates The observed cross-sectional seroprevalence was used to estimate the seroconversion rate using a method similar to those previously described , 18. BriCharacteristics of the resulting study population can be found in Table Using the average absorbance from each serum sample and a threshold of three standard deviations from the sample mean to indicate the presence of AMA-1 or MSP-1 antibodies, 172 of 815 (21.1%) had the presence of AMA-1 antibodies, 179 of 759 23.6%) had the presence of MSP-1 antibodies, and 247 of 815 (30.3%) had the presence of either AMA-1 or MSP-1 antibodies. Using thresholds from three to five standard deviations gave similar estimates of seroprevalence that ranged from 16.3% to 21.1% for AMA-1, 17.4% to 23.6% for MSP-1 and 24.0% to 30.3% for either AMA-1 or MSP-1. The seroprevalence by age group (ranging from 2 to 80 years) is presented in Table .6% had tAs expected, the prevalence of seropositive participants increases with participant age. The prevalence of previous infections determine by AMA-1 response was not significantly different  between age classes under 20 years of age. However compared to participants younger than 20 years of age, the likelihood of previous infection in those over 20 years of age was 6.0 times higher . For MSP-1 responses, differences in prevalence compared to the youngest age class were significant  beginning at the third age class (9 to 13 years of age), with those over 20 years of age having 3.7 times the likelihood of previous infection . Of participants who were tested with both AMA-1 and MSP-1 (n = 759), 104 were classified as seropositive using both antigens (13.7%), 75 were MSP-1 positive and AMA-1 negative (9.9%), 57 were AMA-1 positive and MSP-1 negative (7.5%), and 523 were negative for both AMA-1 and MSP-1 (68.9%). The distribution of positive ELISA responses for AMA-1 or MSP-1 antigens appears graphically in Figure The prevalence and the estimated probability of previous infection as determined by AMA-1 and MSP-1 antigen response are presented in Figure P. falciparum transmission, which could hold valuable information on PQ tolerance and malaria elimination strategies abroad. Findings suggest that these regions have experience a relatively low and constant state of P. falciparum transmission, given the stable increase in seroprevalence by age observed in this study. In samples that had positive responses to either AMA-1 or MSP-1, the estimated SCR of 2.5% , was added to the malaria national treatment policy for Haiti in 2010 [ in 2012 . Howevera priori exclusion of a seroreversion rate from the final model was justified in this circumstance. When comparing the estimated seroconversion rates from study participants under 20 years of age, the continuity in the age-specific seroprevalence curve could indicate that over multiple decades, a relatively constant state of low malaria transmission has occurred in these regions, even in the absence of sustained malaria control efforts. Entomological studies investigating the vector competency of A. albimanus mosquito, may better explain this phenomenon, of stable low transmission.The inclusion of a seroreversion rate in the model also slightly increased the estimated seroconversion rates. Due to the cross-sectional nature of this study and the long duration of antibody detection for AMA-1 and MSP-1, it was appropriate to set the seroreversion rate to zero. When seroreversion was included in the model, the seroreversion rates were -0.006 , however, this potential sampling bias was adequately addressed by excluding all malaria RDT positive individuals 5/815) from final analysis. This study also only screened for previous exposure to /815 from3, howeveAs reported cases of malaria in the Dominican Republic have reached a 15-year low of 952 cases [Future studies should expand seroprevalence methodologies to other departments in order to establish countrywide trends. Research examining barriers to access, protective host characteristics, the extent of heterogeneous malaria transmission in other departments, and vector proficiency could also enhance elimination models in Haiti. Elimination in Haiti appears to be feasible; however, surveillance must continue to be strengthened in order to respond to areas with high transmission, while measuring the impact of future interventions.