P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751–800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651–700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan  and Kota Marudu P. falciparum populations . High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0.532). The genetic data from the present study highlighted the limited diversity and contrasting genetic pattern of P. falciparum populations in the malaria declining areas of Sabah.Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of  Globally, 3.3 billion people are at risk of malaria. In 2015, the number of new malaria cases reached 214 million and malaria deaths were estimated to reach 438,000 [After implementing the World Health Organization’s Global Malaria Eradication Program (1967–1981) and Malaria Control Program (1982–2010), Malaysia has managed to substantially decrease the malaria cases and enter the pre-elimination phase, with the Malaysian government now aiming to eliminate malaria by the year 2020. During the implementation of the National Malaria Elimination Plan (2011-current), indigenous human malaria cases were further decreased to only 606 cases in 2014 . Sabah, East Malaysia, contributed 93% (561 cases) of indigenous human malaria cases in 2014 . Up until November 2015, indigenous human malaria cases in Sabah have dropped to 207 cases as compared to 522 cases reported in November 2014 . On the lead up to malaria elimination, understanding the genetic diversity and population structure of the residual malaria parasite populations is crucial to guide monitoring and evaluation of malaria control strategies and antimalarial interventions.P. falciparum genetic diversity is usually performed through genotyping of polymorphic region of antigenic markers such as block 2 of the merozoite surface protein 1 (MSP-1), block 3 of the merozoite surface protein 2 (MSP-2) and RII repeat region of the glutamate rich protein (GLURP) [P. falciparum population structure and diversity such as inbreeding, epidemic population expansion, geographic isolation and gene flow or introgression of foreign parasites can be characterized. Several genetic measurements such as expected heterozygosity (He), the proportion of polymorphic loci, the number of alleles (Na), multiplicity of infection (MOI), linkage disequilibrium (LD) and F-statistics (FST) are used to estimate the factors affecting the malaria parasite population structure [The  (GLURP)  and simp (GLURP) , 6. Usintructure –8. In adtructure .P. falciparum [P. falciparum has previously been studied in a variety of transmission setting regions such as Africa, South America and Southeast Asia [P. falciparum populations with high level of He, high MOI value, low or insignificant linkage disequilibrium (LD) and low genetic differentiation (FST) [He and MOI, high or significant LD and high FST are mostly reported in the regions with low transmission setting such as Southeast Asia and South America [Effective malaria control measures have successfully reduced malaria transmission in many hyperendemic regions of sub-Saharan Africa. In future, the population structure in these regions are expected to become similar to the low transmission regions like Southeast Asia and South America. The declining malaria transmission, as a result of scaling up interventions, has been shown to affect the genetic diversity pattern and population structure of lciparum , 9–11. Tast Asia , 11–15. on (FST) . In cont America , 17.P. falciparum population structure in Malaysia could not be assessed due to the lack of genetic data and systematic genetic surveillance study. A recent genotyping based on MSP-1 and MSP-2 allelic families has demonstrated low levels of diversity in P. falciparum populations in Pahang, Peninsular Malaysia  which reflected the low transmission level in the area [P. falciparum populations in several parts of Sabah indicating that they are genetically different from each other [P. falciparum isolates collected during the malaria active case detection studies conducted in the remaining residual foci areas of Sabah, Kalabakan and Kota Marudu, by genotyping the highly polymorphic parasite antigenic markers  and 10 neutral microsatellite markers.The effect of malaria control interventions on the the area . Multiloch other . Herein,Kalabakan is located in the South of Sabah, Malaysia. Kota Marudu, is located in the North of Sabah, Malaysia . The stuThe ethical clearance was obtained from Research Review Committee of the Institute for Medical Research and the Medical Research Ethics Committee (MREC), Ministry of Health Malaysia. The recruited study subjects were otherwise healthy individuals from the selected sites in Kalabakan and Kota Marudu. The study subjects at the selected study sites were informed about the nature of the experiments and the procedure involved. Written informed consent was obtained from each participant or guardian before taking part in this study.P. falciparum-infected blood samples from Kalabakan were collected in 2008 and 2009. The P. falciparum-infected blood samples from Kota Marudu were collected in 2011 and 2014. Briefly, the blood samples were obtained by finger prick and malaria infection was diagnosed using rapid diagnostic test kits . Whenever a participant tested positive for P. falciparum, 50 μl of venous blood was spotted onto Whatman® filter paper no. 3 . The blood-spotted filter papers were allowed to dry completely, transferred into individual plastic bags, labeled and stored at room temperature in a dessicator containing silica gel until further processing. The malaria positive samples were also confirmed by Blood Film for Malaria Parasite technique (BFMP).The active case detection was performed in each study site. A total of 4257 individuals were screened for malaria infection in both study areas. The ® DNA Mini Kit , according to the manufacturer's instructions (dried blood spots protocol). The parasite DNA samples were kept frozen at -20°C until use. The Plasmodium species confirmation  was done by using a technique of Padley et al. (2003) [P. knowlesi parasite was done by using the method of Imwong et al. (2009) [Parasite genomic DNA was extracted from blood–spotted filter papers by using QIAmp. 2003)  with the03  with P. falciparum DNA samples were further analyzed by amplification of the highly polymorphic regions of MSP-1 (Block 2), MSP-2 (Block 3) and GLURP using a set of specific primers in nested and seminested PCR method as previously described [escribed . Oligonu2 free buffer, 1.5 mM of MgCl2, 200 μM of dNTPs, 0.2 mM of each primer and 2.5U of Taq polymerase enzyme. For GLURP allele detection, the PCR was carried out in a similar manner to the primary round of MSPs, except for using a MgCl2 concentration of 2.0 mM. For nested PCRs, 2 μl of primary PCR product of MSP-1 and MSP-2 and 1 μl of primary product of GLURP were used as DNA templates, which had similar reaction concentrations to the primary PCR. Cycling conditions for primary PCRs and nested PCR reactions for GLURP were as follows; the denaturation was performed at 95°C for 5 min, then followed by 25 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 45 seconds and extension at 72°C for 1.5 minutes; and a final extension was done at 72°C for 5 min. The cycling conditions for nested PCR of MSP-1 and MSP-2 started with a single step of denaturation at 95°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 45 seconds and extension at 72°C for 1.5 minute, and a final extension at 72°C for 5 minutes.All PCR reactions were performed using Eppendorf Mastercycler Gradient . For allele detection in MSP-1 and MSP-2, PCR was done in a 50 μl PCR mixture containing 50 ng genomic DNA, 1X of MgClThe amplification products were analyzed using Agilent 2100 Bioanalyzer and Agilent DNA 1000 Kit  conducted according to manufacturer’s instructions . AllelesP. falciparum DNA samples were genotyped for 10 microsatellite loci as previously described [2  or 3 mM MgCl2 , 125 μM dNTPs, 250 nM primer mix (F/R), 2 μl of DNA template and 0.4 U of Taq polymerase. The first PCR cycling conditions were as follows: Initial denaturation at 95°C for 5 minutes, 30 cycles of denaturing at 94°C for 1 minute, annealing at 52°C for 2 minutes, extension at 72°C for 2 minutes and last extension at 72°C for 5 minutes. One microliter of the first PCR product was used for the second PCR reaction (nested 2). The second PCR was performed with the reaction mixture containing 1 X PCR buffer, 2.5 mM MgCl2, 125 μM dNTPs, 250 nM primer mix (F/R), 1 μl of DNA template and 0.4 U of Taq polymerase. The second PCR cycling started with initial denaturation at 94°C for 2 minutes, followed by 5 cycles of denaturing at 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 60°C for 30 seconds, 25 cycles of denaturing at 94°C for 30 seconds, annealing at 45°C for 30 seconds, extension at 60°C for 30 seconds and final extension at 60°C for 2 minutes. Fluorescent-labelled PCR products were sent for fragment analysis  and scored using Gene Mapper 5 Software . All electropherograms or peaks generated for each isolate were visually inspected and the predominant peaks corresponding to each allele were scored. Any samples that amplified poorly for particular loci with maximum peak height less than 200 fluorescent units were discarded. Any secondary alleles that were >30% of the height of the predominant allele peaks were also scored as additional alleles which presumably were considered to be a result of polyclonal infection.escribed , 6 with P. falciparum genotypes per infected individual. The MOI was calculated as the proportion of the total number of P. falciparum genotypes for the same gene and the number of PCR positive isolates. Isolates with more than one genotype were considered as a polyclonal infection while the presence of a single allele was considered as monoclonal infection.The multiplicity of infection (MOI) was defined as the mean number of He) across loci in each population. He values were calculated using the Genetic Analysis in Excel toolkit (GenAIEx) [He) for each microsatellite loci or antigenic markers was calculated based on the allele frequencies, using the formula p represents the frequency of each different allele at a locus. He has a potential range from 0  to 1 . The genetic differentiation between populations was measured using Cockerham and Weir (1984)[To determine allele frequencies, only the predominant allele was counted for population genetic analyses. The genetic diversity in each population was assessed by calculating both the mean number of alleles (A) and the mean expected heterozygosity (GenAIEx) . Brieflyir (1984) F-StatisD) between all pairwise comparisons of complete 10-locus haplotypes and the distance measure (VD) determination were done by using the program LIAN, version 3 [VD was compared with the distribution of VD in 10,000 simulated data sets in which alleles at each locus were randomly reshuffled among genotypes. Significant linkage disequilibrium (LD) was detected if the observed VD was more than 95% of the values generated in the reshuffled data sets. The standardized index of association (IAS) was used and calculated as IAS = (VD/ Ve-1)/(r-1), where Ve is the mean variance in the 10,000 reshuffled data sets, and r is the number of loci. The significant levels of the IAS values were tested using Monte Carlo method.The predominant alleles were used to construct haplotypes infection. The permutation procedure was used to test null hypothesis of random association among loci for each parasite population . The numersion 3 . To evalP. falciparum. The age of P. falciparum-infected individuals ranged from 12 to 60 years (mean age: 22.3 ± 9.3 years). The male to female sex ratio (M/F) was 1.8 (20/11). In Kota Marudu (2011 and 2014), a total of 3638 individuals from 56 sites were screened for malaria infection. Fifty four samples (1.5%) tested positive for malaria of which 29 (0.8%) were positive for P. falciparum infection. The age of P. falciparum-infected individuals ranged from 4 to 67 years (mean age: 20.3 ± 15.1 years). The male to female sex ratio (M/F) was 2.2 (20/9). The P. falciparum infected individuals were found to be blood smear-positive, asymptomatic by observation and healthy going about their routine activities. As all malaria infected individuals need to be treated in the hospital, they were advised and transported to the nearest public hospitals for malaria treatment. No clinical data was obtained in this study.A total of 4257 individuals were screened for malaria infection in both study areas. Specifically, 619 individuals from 23 sites in Kalabakan (2008 and 2009) were screened. Fifty eight (9.4%) tested positive for malaria of which 31 (5.0%) were positive for P. falciparum-infected blood samples. The frequencies of monoclonal infection were 30.4% and 4.4% for K1 and RO33, respectively  and 1 allele of each MAD20 (180 bp) and RO33 (160 bp) allelic families. K1 allelic family was predominant in 95.6% of the ectively . Sixty fectively . The MOIectively .A total of 5 different alleles were identified in MSP-2 family genotyping . Four alP. falciparum-infected blood samples ranging from 551 bp to 800 bp (50 bp bin) and coded as genotypes II-VI , 3 alleles of MAD20  and 1 allele of R033 (160 bp) allelic families. The MAD20 was predominant (57.7%) in Kota Marudu  samples . The fre samples . Only 1  samples . The MOI samples .P. falciparum-infected blood samples was 3D7 (86.4%). The frequencies of monoclonal infection were 81.8% and 13.6% for both 3D7 and FC27 allelic families, respectively  and coded as genotype I, III, IV, V and VI. Similar to Kalabakan, only 1 allele was detected in all Kota Marudu P. falciparum-infected blood samples. Frequency of genotype IV was the highest (50.0%) followed by genotypes V (21.4%), III (17.9%), I (7.1%) and VI (3.6%) (Twenty eight samples were successfully genotyped for GLURP RII repeat region . Five GLI (3.6%) . GenotypP. falciparum-infected blood samples. The allele frequencies of each microsatellite locus were shown in The Kalabakan datasets comprised of multilocus microsatellite alleles derived from 14 to 19 P. falciparum-infected samples were successfully amplified for all 10 microsatelite loci. The allele frequencies for each microsatellite marker are shown in P. falciparum-infected blood samples, there was no polyclonal infection observed in any of the samples.In Kota Marudu, twenty-four P. falciparum-infected blood samples  followed by haplotype A (37.5%). Both haplotypes were present in P. falciparum-infected blood samples collected in 2008 and 2009. In contrast, the major haplotype in Kota Marudu was haplotype C (62.5%) followed by haplotype D (8.33%), B (4.17%), E (4.17%), F (4.17%), G (4.17%), H (4.17%), I (4.17%) and J (4.17%). Except haplotype D, all haplotypes were present in P. falciparum-infected blood samples collected in year 2011. However, only haplotype C (75%) and D (25%) appeared in P. falciparum-infected blood samples collected in year 2014  values for each microsatellite locus ranged from 0 to 0.49  were 0.35 (±0.06) and 0.33 (±0.05) for Kalabakan and Kota Marudu P. falciparum populations, respectively.Low level of genetic diversity was detected for both Kalabakan and Kota Marudu  to 0.49 . The sim to 0.45 . The meaectively . The He ectively . The int of 0.70 . The lowectively . Similar of 0.69 . The aveP. falciparum parasite. Therefore, all of the P. falciparum-infected blood samples were included in linkage disequilibrium (LD) determination. To test the significance of LD, the Monte Carlo method [p<0.01) was found in both Kalabakan and Kota Marudu P. falciparum populations with IAS values of 0.495 and 0.601, respectively . The genetic relatedness between Kalabakan and Kota Marudu P. falciparum populations was illustrated in UPGMA dendogram in P. falciparum isolates were obviously grouped into 2 major clusters at 20% of similarity, cluster A and B, where each of the cluster was predominated with Kota Marudu and Kalabakan P. falciparum populations, respectively  of ≥ 0.1 to < 1.0 case per 1000 population [The P. falciparum populations with limited diversity in other areas of Sabah exist even before the implementation of National Malaria Elimination Program in 2011 (sample collection between year 1997 to 2003) [P. falciparum population structure was created by the impact of the previous malaria control interventions in the region. The genetic diversity analysis in the present study showed that P. falciparum populations in Kalabakan and Kota Marudu exhibited an overall low level of diversity (He). In addition, the occurrence of major microsatellite haplotypes in Kalabakan (2008–2009) and Kota Marudu (2011–2014) seem to be persisted in every sampling periods    suggestiventions . In otheventions , 28. Thuventions , Venezueventions , Thailanventions , 31, somventions  and Indoventions  have alsI and LD .He and MOI. It is possible that previous implementation of antimalarial drug policies in Malaysia such as the use of chloroquine (1960s) and pyrimethamine-sulfadoxine combination therapy (1970s) may have caused the selection of particular P. falciparum genotype. Indeed, the Kalabakan P. falciparum populations with chloroquine (81%) [P. falciparum has also been reported in other parts of Sabah [P. falciparum isolates carrying the chloroquine resistance genotype were also reported in low transmission area in Peninsular Malaysia like Pahang [The scale up interventions such as the usage of insecticide-treated bed nets, indoor residual spray , 36 and ne (81%) , pyrimetne (81%) . High prof Sabah . In addie Pahang . Characte Pahang .P. falciparum populations that are genetically diverse. Antigenic marker genotyping carried out in African regions like Burkina Faso, Sao Tome, Malawi, Uganda and Tanzania have identified P. falciparum populations with alleles occurring at a frequency below than 10 percent with a very high He level (0.78 to 0.99) [P. falciparum populations in African regions (except Djibouti) exhibited a high level of He (0.72 to 0.80) and low LD [P. falciparum populations showed MOI of more than 3 [High transmission regions like those occurring in many African countries are normally characterized by to 0.99)  . This followed by predomination of GLURP VI and GLURP IV genotypes in Kalabakan and Kota Marudu P. falciparum populations, respectively. Moreover, the different pattern of major microsatellite haplotypes were also observed in Kalabakan and Kota Marudu P. falciparum populations  and 10 neutral microsatellite markers with efforts to obtain a conclusive genetic diversity dataset for the areas. Although the malaria positive individuals were advised and transported to the nearest hospital for treatment, the information on clinical data could not be obtained during the study. Thus, the relationship between malaria severity or clinical symptom and genetic diversity could not be addressed in the present study. The seasonal or yearly collection of sample by passive case detection will give more information on the malaria clinical symptom and severity that may represent the population structure of P. falciparum in the study areas during the elimination process. Despite these limitations, the data from the present study has confirmed the limited genetic diversity profile and contrasting population structure of P. falciparum populations in Kalabakan and Kota Marudu, key remaining malaria residual foci in Sabah.The limitation of the present study is the small sample size collected by active case detection. As the pre-elimination phase region, the number of malaria cases in Malaysia particularly Sabah decrease drastically each year, only a small amount of P. falciparum isolates in remaining malaria residual foci areas of Kota Marudu and Kalabakan, Sabah prior to the targeted elimination of malaria by 2020. Low level of genetic diversity and predomination of clonal P. falciparum populations reflected the decline of malaria transmission in the study areas. However, the clonal characteristic of circulating P. falciparum populations will present a challenge in drug efficacy trials, as current genotyping technique may lead to false recrudescence data which could in turn have a profound impact on policy decisions.The study presented baseline genetic diversity data of S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Plasmodium falciparum infection were examined.Blood group O protects African children against severe malaria and has reached high prevalence in malarious regions. However, its role in malaria in pregnancy is ambiguous. In 839 delivering Ghanaian women, associations of ABO blood groups with Plasmodium falciparum infection was diagnosed in placental blood samples by microscopy and PCR assays. Present or past infection was defined as the detection of parasitaemia or haemozoin by microscopy, or a positive PCR result. Blood groups were inferred from genotyping rs8176719  and rs8176746/rs8176747 .P. falciparum infection was seen in 62.3% of all women. Among multiparae, the blood groups had no influence on P. falciparum infection. In contrast, primiparae with blood group O had significantly less present or past infection than women with non-O blood groups . In multivariate analysis, the odds of present or past placental P. falciparum infection were reduced by 45% in blood group O primiparae .The majority of women had blood group O (55.4%); present or past in vitro results. The data underline the relevance of host genetic protection among primiparae, i.e. the high-risk group for malaria in pregnancy, and contribute to the understanding of high O allele frequencies in Africa.The present study shows a clear protective effect of blood group O against malaria in primiparae. This accords with findings in severe malaria and  Plasmodium falciparum malaria has long been suggested to influence the global distribution of the ABO blood groups[in vitro observations and functional hypotheses support a protective role of blood group O .P. falciparum infection and malaria. In areas of high transmission, malaria in pregnancy is frequently asymptomatic but consequences involve anaemia, abortion, stillbirth, low birth weight (LBW), preterm delivery (PTD), and, annually, up to 200,000 infant deaths[P. falciparum erythrocyte membrane protein-1 (PfEMP1) mediate adhesion to the placental syncytiotrophoblast  and thereby placental sequestration of infected erythrocytes, deposition of haemozoin , and, commonly, the local accumulation of inflammatory cells[P. falciparum infection among pregnant women in hyper- to holo-endemic Ghana using PCR for blood group genotyping.In contrast, findings are notably ambiguous with respect to the impact of the ABO system on malaria in pregnancy. Pregnant women – particularly primiparae – are a high risk group for t deaths. In pregry cells. In highry cells-12. In Try cells,14. In Sry cells. In contry cells, and in ry cells. Againstper 100 high-power fields and 500 white blood cells, respectively. Placental parasite densities were expressed as parasites/100 fields and peripheral ones as parasites/μL assuming a mean white blood cell count of 8,000/μL. Leukocyte-associated haemozoin in placental samples was recorded. Following DNA extraction , nested P. falciparum-specific PCR assays were performed[P. falciparum infection was defined as the presence of placental parasitaemia or haemozoin by microscopy, or a positive P. falciparum PCR result on placental samples. Plasma concentrations of pyrimethamine, at that time recommended for malaria chemoprophylaxis in pregnancy, and of chloroquine were measured by ELISA assays[Eight-hundred and thirty-nine women with live singleton delivery were recruited from January 2000 through January 2001 at the Presbyterian Mission Hospital in Agogo, Ghana. The study protocol was approved by the Committee on Human Research Publications and Ethics, School of Medical Sciences, University of Science and Technology, Kumasi, and informed written consent was obtained from all women. Agogo is a community of some 30,000 inhabitants located in the forested hills of Asante Akim North District. Subsistence farming, trade and mining are the main income sources in that region, and malaria is hyper- to holo-endemic. Study performed. PresentA assays.ABO gene, namely rs8176719, rs8176746, and rs8176747 were typed by melting curve analysis employing the LightCycler 480 device  and using commercially available primers and probes . ABO blood groups were inferred from rs8176719  and from rs8176746/rs8176747 [n = 38), PCR-generated fragments containing the polymorphisms of interest were subjected to sequencing .Three loci on the  allele). In caset-test, analysis of variance, Mann–Whitney U-test, and Kruskal-Wallis test as applicable. Associations of blood groups with P. falciparum infection, anaemia, LBW, and PTD were identified by χ2-test, and odds ratios (ORs) and 95% confidence intervals (95% CIs) calculated. Adjusted ORs (aORs) were derived from logistic regression models with stepwise backward removal of factors not associated in multivariate analysis (P > 0.05). For placental malaria, previously identified associated factors were a priori included, i.e. delivery in rainy season, age and plasma pyrimethamine concentrations[P-value of <0.05 was considered statistically significant.Continuous variables were compared between groups by trations. A P-valP = 0.43; chloroquine plasma concentrations, 24.3% (110/453) vs. 20.2% (72/357), P = 0.16).ABO genotyping was successful in 827 of 839 (98.6%) women with a live singleton delivery. The majority of women (55.4%) had blood group O. Blood groups B, A, and AB were present in 22.6, 18.0, and 4.0%, respectively. Blood groups were in Hardy-Weinberg equilibrium; genotypes are detailed in Table P. falciparum infection in placental samples  was seen in 62.3% (515/827) of all women. This figure was significantly lower in women with blood group O  than in women with non-O blood groups . Notably, this difference was pronounced among primiparae  but insignificant among multiparae  vs. 102 (51–204) parasites/100 high-power fields, P = 0.63). In contrast, the reduced infection prevalence among blood group O primiparae was obvious taking into account PCR results, and even more so when considering placental haemozoin . Correspondingly, the O allele was significantly less frequent in primiparae with present or past placental infection than in those without .Small numbers prevented a meaningful attribution of infection risks to individual genotypes. Nevertheless, present or past placental infection was observed in 88.9% (16/18) of primiparae with homozygous non-O genotypes, in 74.1% (80/108) of O-heterozygous women, and in 61.5% (107/174) of first-time delivering women with blood group O (cvs 69.2% (54/78), P = 0.65).In multiparae, none of the above malaria-protective features of blood group O were discernible; blood group A even tended to come along with lower infection prevalence Table . ParticuP = 0.006) whereas the difference was small and not significant among primiparae with evidence of present or past infection . Likewise, the proportions of malaria-associated LBW or preterm delivery did not differ significantly between O and non-O blood groups, neither in primiparae nor in multiparae.Blood group O was furthermore associated with reduced odds of maternal anaemia among all women, and among primiparae in particular Table . This waP. falciparum infection. In this highly endemic setting, blood group O was associated with protection against placental malaria among primiparae but not in multiparae, and particularly against late or chronic infections, i.e., infections characterized by the presence of pigment. This finding accords with the clearly protective role of blood group O in severe malaria[P. falciparum. In pregnant women, P. falciparum-infected erythrocytes sequester in the intervillous space by adhering to ligands on the syncytiotrophoblast, followed by the local accumulation of haemozoin and inflammatory cells. Specific antibodies capable of blocking parasite adhesion prevent this placental malaria only after successive pregnancies exposed to P. falciparum infection[P. falciparum infection due to blood group O in the present study was limited to the relatively immune-naïve group of primiparae. At higher parity, the effects of adaptive immunity may override the protection afforded by blood group O.In the present study from Ghana, almost two in three women had evidence of present or past placental  malaria-8 and th malaria,4,9). Se malaria-12, but nfection,24,25. TPlasmodium vivax. Therefore, it is questionable whether these findings from Thailand can be compared with the African studies. Blood group O in the present study was also associated with reduced odds of anaemia, particularly in primiparae. Protection from malaria-related anaemia in individuals with blood group O has been observed before[Notably, however, two studies from The Gambia and Malawi have reported results contrasting the present finding, i.e. an increased risk of active placental malaria in blood group O primiparae and a reduced risk in multiparae,14. The d before but, remAs a limitation of the present study, protection by blood group O was statistically significant only when considering placental haemozoin or when combining results of all diagnostic means . Statistically significant findings were observed in the relatively small group of 300 primiparae of whom 174 had blood group O. The study was sufficiently powered to detect an influence of blood group O on present or past infection but power declined with declining diagnostic sensitivity, i.e. prevalence of infection. Limited sample sizes in subgroups may thus have interfered with the analysis of infection risks in individual genotypes and of potential modification of malaria-related outcomes, e.g., LBW. Also, data on some potentially interfering factors were not available, e.g., bed net use or transfusions. This should be kept in mind when interpreting the data.Anopheles vectors, shared ABO antigens with P. falciparum, impaired merozoite penetration as well as reduced cyto-adherence  to uninfected erythrocytes [P. falciparum-infected RBCs form smaller and less firm rosettes than in non-O RBCs[P. falciparum rosetting[P. falciparum isolates[P. falciparum strains bind, i.e. chondroitin sulphate A (CSA) and hyaluronic acid[P. falciparum-infected O-RBCs has been shown[The hypotheses on why blood group O protects from malaria, and from severe malaria in particular, include aspects of differential attractiveness to iewed by,4,9). Thiewed by and recemalaria). Bindingmalaria). In bloomalaria), and P. n-O RBCs. In factosetting. Howeverisolates and the isolates,31,32. Nnic acid,34, are nic acid. Thrombonic acid. Recentlen shown. Consideen shown, ABO-depP. falciparum infection in primiparae living in an area of high malaria endemicity. Considering the consequences of placental malaria in terms of infant mortality, the influence of the ABO polymorphism on malaria in pregnancy may contribute to the high frequencies of the O allele in sub-Saharan Africa.The present study demonstrates a protective influence of blood group O against The authors declare that they have no competing interests.GBA, KT, and FPM designed the study. GBA, TAE and FPM were responsible for patient recruitment, clinical and laboratory examinations. PPG and SM did the PCR analyses, TAE and KT contributed to data interpretation, and FPM did the statistical analyses. GBA and FPM wrote the paper with major contributions of the other authors. All authors read and approved the final manuscript.


Background. South eastern parts of Iran remain endemic for malaria infection. There is some concern that malaria infection may spread into Bushehr, which is located in the south western part bordering the Persian Gulf and at the periphery of the declared endemic region Hormozgan province due to frequency of visitors from eastern endemic areas and from neighboring malaria endemic countries. We investigated malaria prevalence in Bushehr. Methods and Results. Attempts were made to identify malaria active infection in blood smears and malaria specific antibody and antigens in serum samples. Traditional blood smears prepared from 1955 blood specimens yielded no definitive malaria positive case by microscopic technique. A total of 270 (13.8%) serum samples were positive for malaria antibodies. Using specific ELISA kits, presence of histidine rich proteins and lactate dehydrogenase antigens were investigated in serum samples. No histidine rich proteins specific for P. falciparum were detected amongst 270 antibody positive samples. However, six samples representing 0.3% of total population, were found to be positive for plasmodium pan specific lactate dehydrogenase antigens. This suggested the possibility of low level exposure to malaria in Bushehr community. Conclusions. Out of a total of 1955 samples tested, 270 (13.8%) were positive for malaria antibodies and six (0.3%) of these were positive for plasmodium-specific lactate dehydrogenase antigen suggesting a low level exposure to malaria in a hypoendemic region based on immunological testing. Since none of the 270 antibody samples were positive for histidine rich protein antigens, there is scope for further testing of blood samples by molecular methods such as polymerase chain reactions to confirm the plasmodium species and provide information valuable for future investigations. Our testing strategy for hypoemdemic malaria can be used as a template for investing malaria in 32 eliminating countries for testing ongoing transmission. This approach may be useful as a method in epidemiological studies. Several publications show importance of malaria control and elimination programs for economic progress . In IranInformed consent was obtained from all participants and this project was approved by The Persian Gulf Tropical Medicine Research Center, Bushehr University of Medical Sciences. Blood samples used in this study were provided by voluntary individuals who willingly participated in this study of malaria investigation. The health status of voluntary blood donors met the required condition as stipulated by the Ethical committee for using their blood samples in this investigation. Of the sample donors, none was suffering from any symptoms suggestive of malaria, none had ever been hospitalised with malaria. The region where work was undertaken is marked on the map . Region  P. falciparum and P. vivax parasites were used as reference during malaria microscopy. For P. malariae and P. ovale, slides were not available, therefore published pictures were used as a reference. Cross-checking of slides to confirm diagnosis was also performed between technicians.Thick and thin blood smears were prepared based on the traditional methods described in the Medical Parasitology manual and other publications , 8. The Plasmodium falciparum, P. vivax, P. malariae, and P. ovale). This kit has been independently validated against other diagnostic methods with (95.5% sensitivity and 92.2% specificity) [F-test and Pearson coefficient tests were used to assess any significance of antibody positivity between 10 centers of blood collection.Malaria specific antibodies present in the serum samples were detected by using a commercially available ELISA kit . The kit is based on the indirect immunosorbent assay (ELISA) principle and designed for detection of malaria specific IgG antibody response in serum against four human malaria species  [. Serum samples diluted 1/2 in PBS/T were tested in the ELISA wells coated with capture monoclonal antibodies. It is known that detectable levels of P. falciparum specific HRP antigens are present in serum [ P. falciparum parasites) and negative (media blank) controls. Any readings above the cutoff level OD > 0.12 (mean + 3SD of negative samples) were classified as positive.Histidine rich proteins specific forstralia) , 11. SerA second line of testing was performed for circulating plasmodium genus-specific lactate dehydrogenase (Pan-pLDH-ELISA) antigens in serum samples . Presenc P. falciparum and P. vivax blood smear positive slides and published blood stage photographs of P. malariae and P. ovale were used as reference while examining the stained smears prepared from a total of 1955 blood samples. No active malaria cases were identified based on blood smear microscopy. Then we tested the serum samples in a validated malaria antibody detection ELISA identifying sero-positive individuals and malaria antigen ELISAs were used for identifying individuals carrying malaria. Results of microscopy, antibody, and antigen levels that were found in 10 different centers are documented in F-test and Pearson coefficient test).The flow chart  details Of the 1955 individuals enrolled for the study, a total of 1085 (55.5%) were men and 870 (44.5%) were women. The mean age was 32.82 ± 14.61 years. There were 1422 local residents and 533 travelers. About 5 mL blood samples were collected from each volunteer by venipuncture, and the whole blood was stored at 4C in the refrigerator until use in the assays. Serum samples were frozen at −20C until use. Travelers were also included in this study for testing the possibility of them bringing active malaria infection from endemic into nonendemic regions. All samples from local residents as well as from travelers were tested under identical condition for prevailing malaria antibodies as well as for malaria antigens.Pan Malaria IgG antibody test was employed. The kit positive control reacted fairly strongly (mean OD = 2.581) which is 44x higher than the OD reading of the negative control. This showed that Pan Malaria antibody kit possesses a high level (>40x) of discrimination between negative and positive samples. A battery of in-house negative samples plus kit negative control samples were initially tested and a cutoff (mean + 3SD) (OD = 0.150) was determined. This cutoff served as the basis for differentiating positive samples in malaria antibody assay. In a similar manner we also developed a cutoff level for malaria antigen ELISAs. Data in Out of a total of 1955 samples tested in Pan Malaria IgG antibody CELISA ; 6 antibody positive samples (PS)  reacted positively in pLDH antigen assay . When weThe national malaria control programs initiated in Iran during the 1950's resulted by 1977 –4 in the P. falciparum active infection amongst the exposed group of 270 samples, with sensitive and well validated histidine rich proteins ELISA tests that have been used with field malaria samples [We subsequently focused our investigation towards identifying those individuals exposed to malaria. It is known that antibodies prevail in the circulation for many years after exposure to malaria infection . It is a samples , 11. Uti samples –12. P. falciparum specific histidine rich proteins ELISA results were consistently negative, it is reasonable to assume that these six individuals may have been exposed to non-falciparum malaria species.We further tested those malaria antibody positives with the pLDH CELISA kit to detect plasmodium specific lactate dehydrogenase enzyme. Six individuals were found to be positive at a very low level of pLDH. Because theA point of explanation is needed on our results. Here our observation has shown only the “exposure” situation and not as any degree of “infection.” It is assumed that Bushehr community is naive to malaria. It is postulated that malaria is introduced into Bushehr by a traveller from an adjacent malaria endemic area, and that results in local transmission. Our findings substantiate this situation very well as Giemsa-based blood smear microscopy was consistently negative and no active malaria case was observed. Results can be attributed only as “exposure” situation but not as “infection” situation in Bushehr. If we see overall malaria antigen results, it is clearly apparent that absence of histidine proteins shows there is no active infection prevailing. However in absence of histidine proteins, occurrence of pLDH in some of the individuals may show that they may carry some sub-microscopic stages of P. vivax invariably in the form of dormant hypnozoites , 17. HypBecause we restricted antigen testing only to antibody positive samples, a question may be raised on the possibility of antibody negative samples showing some level of antigens. This criticism can be answered by stating that, if malaria is endemic in the community, then such antigen positive individuals should have tested positive for malaria antibodies, because malaria antibodies develop soon after infection. It is unlikely that antibody negative individuals would be positive for malaria antigens. We, therefore, conclude that our approach of only testing for malaria antigen in antibody positive individuals is appropriate. P. vivax and P. falciparum have been reported from central Iran, aside from known records of infection from southeastern endemic regions [P. vivax in 94% cases and P. falciparum in 6% cases) in Iran. Despite a recent report of a malaria slide positivity rate of 1.19% in Iran [Recently regions , 19. TheOur work has shown the usefulness of immunoassays in identifying individuals exposed to malaria and also in detecting individuals carrying potentially active infections albeit at a possibly low level parasitaemia that are not detectable by Giemsa microscopy. Asymptomatic malaria in hypoendemic areas is very important , 21. Thi


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.Volunteers (n = 426) from four malaria endemic areas in Colombia  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.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.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.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  Glucose 6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme involved in prevention of cellular oxidative damage by stimulation of detoxification of free radicals. It catalyzes the production of nicotinamide adenine dinucleotide phosphate (NADPH), which is necessary for maintenance of reduced levels of glutathione (GSH) important to protect erythrocytes from oxidative damage and to reduce susceptibility to haemolysis , 2. HoweG6PD locus is located on chromosome X (Xq28) and displays a great polymorphism with ~190 mutations identified coding for ~400 biochemical or allelic enzyme variants [The variants –7, some ome X Xq2 and dispvariants . This trDifferent prevalence of G6PDd has been reported in Africa 20 %), the Mediterranean (4–30 %) and Southeast Asia (10–20 %) . In Lati0 %, the Plasmodium vivax clinical relapses [Plasmodium falciparum infections to prevent gametocyte development and further parasite transmission to mosquitoes [P. vivax and treated with PQ. According to the Colombian guidelines for P. vivax malaria treatment, PQ must be administered for 14 days at a daily dose of 15 mg/day in combination with chloroquine (CQ) at a total dose of 25 mg/kg; no PQ is currently used for treatment of P. falciparum infections [P. vivax malaria is highly prevalent in endemic communities, many of which are of African descent. This is the first study including genotyping to evaluate locally important G6PDd variants in a large number of individuals from rural malaria endemic areas in the northwest of Colombia. Preliminary evaluation of both G6PD enzyme activity along with variant identification was important to study correlations between genotype and phenotype in these populations.Primaquine (PQ), an 8-aminoquinoline, is currently considered the most effective drug to prevent relapses , as wellsquitoes . Howeversquitoes , 20 and squitoes –25. Howefections . This stHuman blood samples were collected as part of a malaria epidemiology study in the context of an International Center of Excellence for Malaria Research (ICEMR) programme. The protocol was reviewed and approved by the IRB of Malaria Vaccine and Drug Development Center-MVDC  (Code 004-2010). The screening process was considered to be of minimal risk according to resolution 8430 of 1993 of the Colombian Ministry of Health (MoH) . ResearcThis study was performed in four municipalities: Buenaventura , Quibdo (Choco), Tierralta (department of Cordoba) and Tumaco (Nariño). These municipalities were selected based on the source of the malaria cases reported in the Colombian National Surveillance System (SIVIGILA).P. vivax) were reported by SIVIGILA in 2014 [Buenaventura is a municipality located to the West of Colombia on the Pacific coast at 7 m above sea level (m a s l). It has a population of ~392,000 people  . A total in 2014 , placingP. vivax, contributing 65 % of all malaria cases in Colombia according to SIVIGILA [Quibdo is located to the northwest of Colombia at 45 m a s l. It has ~160,000 inhabitants and most of them are Afrocolombian (~95 %). This municipality shows ~47 % of malaria cases caused by SIVIGILA .P. vivax) [Tierralta is located to the northwest of Colombia, 51 m a s l and ~50 km from the Colombian Atlantic coastline with a mean annual temperature of 27.3 °C . Its pop. vivax)  and is cP. falciparum is by far the most prevalent malaria pathogen. It reported 1309 malaria cases  in 2014 [Tumaco is a municipality located to the southwest of Colombia on the Pacific coast at ~40 km from the border with Ecuador. It has a population of ~195,000 inhabitants . It diff in 2014 , which pBlood samples used here G6PD studies were randomly selected from a larger group of samples collected of healthy male and female donors of any age, randomly selected in cross sectional studies to determine malaria prevalence in sentinel sites of Buenaventura (n = 118), Quibdo (n = 100), Tierralta (n = 119) and Tumaco (n = 89) . Venous The G6PD activity was measured in all 426 volunteers. Initially, the haemoglobin (Hb) level was determined in all samples using 500 µL of whole blood and an automated haematology analyzer . The G6PD activity was measured in 10 µL of whole blood samples using the quantitative G6PD kit from Trinity according to the manufacturer’s instructions  and was expressed in terms of Hb concentration. Normal, intermediate and deficient controls supplied by manufacturers were included in each determination. Enzyme activity was determined using a spectrophotometer  at 340 nm and it was reported as international units per gram of Hb(U/gHb). The reference values for enzyme activity were: normal 4.6–13.5 U/gHb, intermediate deficiency 2.1–4.5 U/gHb, and severe deficiency <2.1 U/gHb.G6PD gene to characterize the genotypic variants A−, A+ and Mediterranean [® Colorless Master Mix according to manufacturer’s recommendations (Promega), 2 μL of DNA and specific primers for each region. The PCR conditions used to amplify the three fragments were: 94 °C/5 min, 35 cycles of 95 °C/30 s, 62 °C/30 s, and 72 °C/30 s, and final extension of 72 °C/5 min for regions including mutations 376 A>G (90 bp product) and 202 G>A (109 bp product). For the region including the mutation 563C-T (263 bp product) the PCR conditions for amplification were similar except for annealing and extension time which were 45 s. Finally, the PCR products were detected in 3 % agarose gels. The PCR products were used in RFLP with specific restriction enzymes. All restriction reactions were performed according to manufacturer’s recommendations. The mutation 376 A>G (G6PD A+ variant) was identified using the enzyme Fok I (New England Biolabs). The 202 G>A transition (G6PD A− variant) was detected by restriction with NIaIII (New England Biolabs). Finally, MobII (New England Biolabs) was used to detect the 563 C>T mutation associated with the Mediterranean variant. The digested products were analysed through electrophoresis on 3 % agarose gels.According to a previous estimation of 10 % prevalence of G6PDd in Colombia , 21, a serranean , 32. DNADescriptive measures  and a Chi squared test were performed to describe and to test the relationship of variables such as age, gender, previous malaria infection, and number of previous malaria episodes, ethnicity and provenance with the presence of G6PDd Tables , 2. A diA total of 426 volunteers, 251 females and 175 males, were screened for G6PD activity: 118 from Buenaventura, 100 from Quibdo, 119 from Tierralta and 89 from Tumaco (Table None of the patients with G6PDd had severe anaemia (Hb <7 g/dL) Table . Five inAnalysis of G6PD allelic variants by PCR–RFLP was successful for 151 samples, 77 (50.99 %) female and 74 (49.01 %) male individuals, including 123 patients with normal G6PD activity, 22 with intermediate deficiency and six that showed severe deficiency. A total of 23 individuals (15.23 %) showed genotypes associated with G6PD deficiency , which is in agreement with a previous study showing that A− was the predominant G6PDd allelic variant in some countries in Latin America and the Caribbean . These sP. vivax malaria infections. The lowest proportion of individuals with G6PDd variants was found in Quibdo.Tumaco (26.66 %) and Buenaventura 24.39 %) showed the highest allelic G6PDd frequencies . In Honduras, a greater proportion of females with G6PDd allelic variants were reported compared to hemizygous males .In some individuals it was founded G6PDd probably associated with the presence of A + and A− genotypes. A significant association between deficient enzyme activity and the presence of G6PDd genetic variants was found (p = 0.04098). Eight individuals presented functional G6PDd and displayed the corresponding genetic variants, however, these variants were not detected in the remaining 20 individuals that showed reduced G6PD enzyme activity. A plausible explanation is that G6PDd in these individuals is probably associated with other variants not evaluated in this study, which have not been characterized for the Colombian population and could be identified in the future by DNA sequencing of all exons.P. vivax malaria on the Colombian Pacific coast. Additionally, this study shows an important association between genotype and phenotype in these regions. Previous studies did not report allelic variants in individuals with reduced G6PD activity [This is the first report regarding frequency of G6PDd allelic variants in regions with endemic activity . Here thP. falciparum  and P. vivax  PQ is important for elimination of liver hypnozoites in P. vivax malaria. The PQ regimen for P. vivax malaria consists of a much higher dose than the currently recommended WHO single low dose (0.25 mg base/kg) to block P. falciparum malaria transmission which is associated with a considerably lower risk of haemolytic toxicity [P. vivax malaria [P. vivax is the main parasite causing malaria, it is recommended that PQ administration requires prior determination of G6PD status , However, this recommendation is not followed in most endemic countries of the continent, and it is uncertain what proportion of malaria cases, mainly P. vivax that are treated with therapeutic protocols including PQ, develop haemolysis. Substantial reduction of malaria by implementation of mass drug administration (MDA) using PQ has been observed in regions with high prevalence of P. vivax and varying prevalence of G6PDd [Used for treatment of both toxicity . Acute h malaria , 34. Theof G6PDd . The parof G6PDd  must be of G6PDd , includiG6PD alleles. Further mapping of G6PDd prevalence and variants will help design appropriate malaria elimination strategies including MDA with PQ. A prospective study with a larger sample size including a close follow up to detect potential development of haemolysis and haemolytic anaemia in P. vivax patients under PQ treatment was recently initiated in collaboration with the MoH of Colombia. The MoH is currently preparing pilot activities towards malaria elimination which include the introduction of PQ treatment for P. falciparum cases, and more intensive search and treatment of asymptomatic cases, which are mostly P. vivax cases demanding 2 week PQ regimens.This study offered estimates of G6PDd prevalence in malaria endemic areas with considerable populations of African descent in a relatively small sample, a limitation of our study. Importantly, genotyping for the first time in these regions showed a high proportion of the G6PD A− variant in these populations. However, more information is needed about G6PDd prevalence in malaria endemic areas in Colombia, and other endemic countries of the Latin American region, including additional variants not evaluated in this study that may be associated with G6PDd in 20 individuals with no identified P. vivax infections and are treated for 14 days with PQ, no official reports of PQ-induced haemolytic crises are available. These results underscore the need to more carefully assess G6PDd in malaria endemic populations, certainly in a larger population, including other variants and sequencing as well as clinical follow up of PQ response. This study emphasizes the need to implement appropriate G6PDd diagnostic methods, particularly in view of possible MDA with PQ for both P. vivax and P. falciparum elimination.A high frequency of G6PD A− genotype, followed by A+ genotype was found in the analysed population, along with a moderately high proportion (6.56 %) of G6PDd based on enzymatic activity. Although 70 % of national malaria cases correspond to


Plasmodium falciparum, the most pathogenic of the malaria parasite species. We conducted a cross-sectional study in Cruzeiro do Sul, Acre state, Brazil, an area of low transmission and where both P. vivax and P. falciparum circulate. We collected peripheral and placental blood and placental biopsies, at delivery from 137 primigravid women and measured levels of the angiogenic factors angiopoietin (Ang)-1, Ang-2, their receptor Tie-2, and several cytokines and chemokines. We measured 4 placental parameters  and associated these with the levels of angiogenic factors and cytokines. In this study, MiP was not associated with severe outcomes. An increased ratio of peripheral Tie-2:Ang-1 was associated with the occurrence of MiP. Both Ang-1 and Ang-2 had similar magnitudes but inverse associations with placental barrier thickness. Malaria in pregnancy is an effect modifier of the association between Ang-1 and placental barrier thickness.Malaria in pregnancy remains a substantial public health problem in malaria-endemic areas with detrimental outcomes for both the mother and the foetus. The placental changes that lead to some of these detrimental outcomes have been studied, but the mechanisms that lead to these changes are still not fully elucidated. There is some indication that imbalances in cytokine cascades, complement activation and angiogenic dysregulation might be involved in the placental changes observed. Nevertheless, the majority of studies on malaria in pregnancy (MiP) have come from areas where malaria transmission is high and usually restricted to  Plasmodium infections during pregnancy represent a substantial health burden for mothers and their neonates leading to underweight babies, preterm deliveries, abortions, stillbirths, and even maternal mortality. The placenta, in its role as a bridge between mother and foetus, will either suffer or mediate the injuries which eventually lead to these adverse outcomes. Plasmodium infections during pregnancy can affect the metabolism of the placenta, its functions and even its architecture. These changes are well described in Sub-Saharan Africa, where P. falciparum infections predominate, but little is known in areas where P. vivax is predominant. The state of Acre in Brazil is one such area. We have recently shown that malaria in pregnancy is associated with placental pathology in this region. In the present study we sought to find out the association between the placental and peripheral levels of angiogenic factors, which are known and important players in the formation and function of the placenta, and the occurrence of placental pathology in Plasmodium-infected women. We found that, lower levels of Angiopoietin-1 in women who experienced malaria during pregnancy were associated with specific modifications occurring in the placenta. This provides a possible pathway through which these pathological changes occur. Plasmodium spp., malaria in pregnancy (MiP) remains an important health burden that can result in maternal anaemia, small-for-gestational-age babies, low birth weight, and even miscarriages and stillbirths : 19.7 (4.2) vs 18.5 (2.7), p = 0.064, p<0.001, p = 0.001, A total of 92 out of the 137 women were defined as uninfecteds after microscopic, histologic or molecular examination revealed no evidence of P. vivax-only infections during pregnancy. Twenty-one percent of those women presented with infections at delivery  of the single species infections detected during pregnancy, with 5 women showing infections at delivery  in those women who underwent a caesarean (Levels of cytokines and chemokines (with the exception of IFN-γ and IL-10) were found to be higher in the placenta than in the periphery, although this result should be taken with caution due to the small number of samples evaluated . Similaraesarean , possiblPlasmodium spp. infection during pregnancy and those without : Uninfected (n = 88): 12.67 , vivax (n = 4): 9.19 , p = 0.975, falciparum (n = 5): 8.89 , p = 0.098; ratioTie2:Ang-1: Uninfected (n = 88): 0.89 , vivax (n = 4): 0.76 , p = 0.766, falciparum (n = 5): 1.42 , p = 0.096).Placental and peripheral levels of both cytokines and chemokines did not generally vary between women with  without . Similar without . However without  Figs. Ad without . Though p-value) 0.30 , p = 0.004 and Ang-1–0.30 , p = 0.045. Increased Ang-1 was also associated with a decrease in placental weight -36.34 , p = 0.033. Rather then segregating the women into MiP positive or negative, thus reducing the power of our analysis, we investigated whether these associations were modified by the presence or absence of malaria during pregnancy by adding Plasmodium spp. infection as an interaction term in the model. Interestingly, the association between Ang-2 and placental barrier thickness was not modified by the presence of Plasmodium infection; however, there was some evidence that malaria during pregnancy altered the effect of Ang-1 on placental barrier thickness   revealedhickness .In this study, we were able to measure and compare cytokine and angiogenic factors levels at delivery between women who had malaria during pregnancy and those who did not. Additionally, we associated the levels of these molecules with the occurrence of histopathological changes in the placenta.P. falciparum, the placental events and mechanisms that contribute to adverse obstetric outcomes have been extensively studied and include adhesion of the parasite to the placenta [P. vivax is also present, both P. falciparum and P. vivax infections are able to significantly impact placental development and foetal outcomes [P. vivax and P. falciparum seem to have a similar magnitude of effects on the placenta in areas of low malaria transmission and where treatment is readily available [Plasmodium spp. infections together for evaluation.Malaria during pregnancy in this region of Brazil has previously been associated with detrimental outcomes for both mother and child ,29–31. Tplacenta , accumulplacenta , histoloplacenta , complemplacenta –9, disruplacenta ,11. In aoutcomes –36, but outcomes . Becausevailable ,26, we gPlasmodium-infected and uninfected women had very similar epidemiological characteristics. Additionally, apart from an increase in the axillary temperature in those who were infected, no other clinical features were significantly different between the groups of women, including haemoglobin levels, prevalence of anaemia, newborn birth weight and proportions of prematurity and low birth weight. These results may be a positive reflection of the active and prompt diagnosis and treatment policies in place in Acre state, where our study site is located and where women are often diagnosed and treated under supervision within 48 h [In this study, the hin 48 h .Placental parameters between infected and uninfected women differed when the number of infections during pregnancy was taken into account. Consistent with our previous report , the womThe observation that placental levels of both cytokines and angiogenic factors were significantly higher in the placenta than in the periphery, independently of malaria infection status, may reflect the different time-points of collection of samples  but is also a clear indication of the distinct nature of both these compartments. When studying placental parameters, one should focus on the placental milieu and not rely on that of the periphery.P. falciparum infections. Neither the level of placental Ang-1 nor the placental ratio Tie-2:Ang1 were different between women with and without infection. This may be a reflection of the low grade placental infections or, most likely, it may reflect different regulatory mechanisms in the peripheral and the placental compartments regarding the levels of these factors. Peripheral levels of all cytokines and chemokines (with the exception of IL-6) were increased in the women who had malaria at delivery, and this appears to be true for those women with P. vivax infection only, but the low numbers from whom these levels were available do not allow for a more substantial analysis. In the placenta, levels of IL-10 were significantly increased in the women who had malaria at delivery compared to the uninfected women. Once more, low numbers dictated the analysis but women with P. vivax only also appeared to have higher levels of placental IL-10. This finding substantiates the role of IL-10 not only as a marker of MiP but of malaria infection in general [Similar to research findings described in other populations ,38, we o general .Plasmodium infection. In uninfected women, levels of Ang-1 did not alter placental barrier thickness while there is a negative association between levels of Ang-1 and placental barrier thickness in women who experienced malaria during pregnancy. A study with a larger number of samples from infected women is necessary in order to verify this observation.Increased levels of Ang-1 were associated with decreases in placental weight and with a decrease in placental barrier thickness in the entire cohort of patients. An analysis of the effect of malaria during pregnancy on this association revealed that the burden of the interaction occurred in the women who had a P. falciparum transmission and where the detrimental effects of MiP are substantial [Plasmodium-infected women.Decreased levels of peripheral and placental Ang-1 have previously been associated with the occurrence of malaria in pregnancy in African women, in an area of high stantial ,38,40. AS1 FigA) and the percentage of mononuclear cells (B) are plotted according to the number of P. vivax-only infections detected during pregnancy. Uninfected (n = 84), one infection by P. vivax (n = 11) and more one infection by P. vivax (n = 8).The thickness of the placental barrier ((TIF)Click here for additional data file.S2 FigP. vivax infection status during pregnancy. Uninfected (n = 12), past infection (n = 11) and current infection (n = 4).The Ln-transformed levels of cytokines and chemokines (in pg/mL) are plotted against the (TIF)Click here for additional data file.S3 FigP. vivax infection status during pregnancy. Uninfected (n = 78), past infection (n = 15) and current infection (n = 4).The Ln-transformed levels of cytokines and chemokines (in pg/mL) are plotted against the (TIF)Click here for additional data file.S1 TableMultivariate linear models were used to evaluate the association between angiogenic factors or cytokines with the various placental parameters of interest. A different model was run for each ln-transformed independent variable (angiogenic factors/cytokines) and the residuals tested for normality. Coefficients represent the increase or decrease in placental parameter associated with a two-fold increase of the independent variable. Boldface type represents statistical significance below the 0.05 level. All models were controlled for delivery method and gestational age at delivery.(DOCX)Click here for additional data file.S1 Checklist(DOCX)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.


Plasmodium falciparum mono-infected malaria patients (age ≥ 10 years) seeking medication in Halaba Kulito and Jimma Health Centers. In addition, incidences of severe malaria symptoms were assessed. The data were analyzed using SPSS (version 20) software. Prevalence of current khat chewer malaria patients was 57.38% (95%CI =53-61.56%). Malaria symptoms such as hyperpyrexia, prostration and hyperparasitemia were significantly lower (P<0.05) among khat chewer malaria patients. However, relative risk to jaundice and renal failure were significantly higher (P<0.05) in khat chewers than in non-khat chewer malaria patients. Longer duration of khat use was positively associated with incidence of anemia. IgM and IgG antibody titers were significantly higher (P<0.05) among khat chewer malaria patients than among malaria positive non-chewers. Although levels of IgG subclasses in malaria patients did not show significant differences (P>0.05), IgG3 antibody was significantly higher (P<0.001) among khat chewer malaria patients. Moreover, IgM, IgG, IgG1and IgG3 antibodies had significant negative association (P<0.001) with parasite burden and clinical manifestations of severe malaria symptoms, but not with severe anemia and hypoglycemia. Additionally, a significant increment (P<0.05) in CD4+ T-lymphocyte population was observed among khat users. Khat might be an important risk factor for incidence of some severe malaria complications. Nevertheless, it can enhance induction of humoral immune response and CD4+ T-lymphocyte population during malaria infection. This calls for further investigation on the effect of khat on parasite or antigen-specifc protective malaria immunity and analysis of cytokines released upon malaria infection among khat chewers.Although more emphasis has been given to the genetic and environmental factors that determine host vulnerability to malaria, other factors that might have a crucial role in burdening the disease have not been evaluated yet. Therefore, this study was designed to assess the effect of khat chewing on the incidence of severe malaria syndromes and immune responses during malaria infection in an area where the two problems co-exist. Clinical, physical, demographic, hematological, biochemical and immunological data were collected from  Plasmodium parasites , and mono-infected with Prior information on socio-demographic characteristics, and frequency and duration of khat use among patients were assessed using mini-structured interview questions. Moreover, clinical data on malaria syndromes (complicated and uncomplicated) were assessed by health professionals working in the selected health centers. Nutritional status of each patient was checked by measuring his/her body mass index (BMI) using their height and weight values. Classification of patients based on BMI was made following WHO classification criteria . AccordiAfter written consents were obtained from the study participants or guardians of the patients with complicated severe malaria and children <18 years, blood samples were collected. Thin and thick blood smears were prepared in duplicate per patient for microscopic examination. Having fixed only the thin smear in methanol, the whole smears were stained with 10% Giemsa (pH = 7.2) for 10 min. Based on the morphological appearance of the parasite in infected red blood cells (iRBCs), parasite identification was carried out under a microscope . Parasite load was calculated by counting the number of asexual stage parasites per 200 white blood cells (WBCs), assuming mean human WBC is 8,000/μL . Each blAbout 5mL of venous blood samples were collected from each study participant by vein puncture of the antecubital vein using a 21 gauge hypodermic sterile needle and syringe for different analyses. The blood samples were then transferred into clean sterile EDTA pre-coated centrifuge tubes. Few drops of blood samples (~10μL) were taken for measurement of blood glucose level using Hemocue instrument . After completing the sampling and data collection, participants with non-complicated severe malaria, hence not admitted to the health centers, were treated with artemether-lumefantrine (20mg base/kg for three days twice a day); but admitted patients were treated with IM quinine (10mg base/kg every 8 hours for five days) as per the recommendations of the National Malaria Treatment Guideline .rd Generation).From the collected blood samples, small quantity (~10μL) were used for quantification of total WBCs, lymphocytes, RBCs, hematocrit (HCT), hemoglobin (Hb) and platelets using CBC machine [Automated complete blood cells (CBC) Analyzer: Sysmex KX-21]. Some of the venous blood samples (~1mL) were centrifuged at 10,000 rpm for 10 minutes at room temperature; the sera were transferred into new eppendorf tubes and stored at -80°C until processed. Then, the sera were used for determination of liver biomarkers: serum glutamic oxaloacetate transaminase (sGOT), serum glutamic pyruvic transaminase (sGPT), total bilirubine and albumin levels. In addition, kidney function tests such as measurement of creatinine and urea and inflammation biomarkers such as uric acid (UA) and C-reactive protein (CRP) were quantified using automated immunochemical analyzer , (ii) parasitologically confirmed P. falciparum positive with clinical manifestation of malaria infection, self-reported khat chewers and aged ≥10 years (n = 120), (iii) neither malaria infected nor khat chewers and aged ≥10 years (n = 60), (iv) khat chewer and aged ≥10 years, negative for malaria infection under microscope and rapid diagnostic test (RDT) (n = 60). About 2mL of the venous blood samples were centrifuged at 10,000 rpm for 10 minutes at room temperature; the sera were transferred into new eppendorf tubes and stored at -80°C until processed. Finally, the sera were used for quantification of serum immunoglobulins (Ig): IgG, IgM, IgG1, IgG2, IgG3, and IgG4 using nephelometric assay  and between IgG subclasses . Although IgG4 was negatively associated with most Igs, significant correlation was not observed (P>0.05).The link interconnecting each of the antibodies assessed in malaria positive (khat chewer and non-khat chewer) patients were investigated using the Spearman’s correlation rank test. As shown in below, multiple positive correlations were identified between antibodies:  from non-chewer malaria positive patients. Likewise, although there was slightly increasing pattern in count of CD4+ among khat chewers, significant difference was not (P>0.05) observed from neirher chewer nor infected or healthy controls. On the other hand, cytotoxic-T lymphocytes or CD8+ count did not show significant differences (P>0.05) among different groups of the study participants  and in vivo study on animals model showed that khat has anti-inflammatory role on the pro-inflammatory cytokines responsible for induction of inflammation that`leads to fever during malaria infection by inhibiting their production and enhancing secretion of anti-inflammatory cytokines [Most of the hyperpyrexia and its associated signs and vomiting symptoms have been largely attributed to production of various cytokines such as TNF-alpha produced in response to the parasite and toxin products released during rupture of infected RBCs . Also, hytokines , 38.The parasite load observed among khat users was also lower than the one among non-khat chewers. Even though anti-plasmodial activity of khat is not known yet, there are its reports on its antimicrobial activity against some human pathogens , 40, andP. falciparum are the commonest form of malaria pathology [P. falciparum parasite.There is a well established fact that changes that occur in liver function biomarkers induced by athology . It coulathology . Althougathology . In thisathology  and repoathology , 46. Alsathology . AccordiPlasmodium parasite [The renal impairment caused by khat could have further effects on the incidence of severe malaria complications through production of increased level of inflammation biomarker (uric acid). The significantly high level of CRP observed among khat chewer malaria patients is an indication for presence of excess uric acid (UA) in the bloods of the chewers. Source of this UA could be from the influence of khat on proper functioning of kidney, supposed to excrete it , and theparasite , 50. Excparasite , 52. Theparasite .Although significant differences were not observed on some hematological tests among the two groups, as frequency (daily) and duration (chronic) of khat use increase, it is evidenced by the possibility of developing anemia and leucopenia among khat chewer malaria patients. Although there was no standard data available or generated on the status of patients’ renal failure, there were irregular complaints on urine retention problems among the khat chewer malaria patients.P. vivax and P. falciparum [Thrombocytopenia, an early sign of malaria infection, is among the most common malaria-associated hematological complications, with the incidence ranging from 40.5–85% in lciparum . Its meclciparum , 56, thulciparum –59. A siIt is generally believed that khat chewing causes loss of appetite , and accAccording to different literature and a very recent assessment made in the site of this study, people traditionally believe that khat has medicinal value for treatment of malaria –21. Howe+ T-lymphocyte population during malaria infection. Different studies indicated that khat enhances in vitro secretion of anti-inflammatory cytokines and had anti-inflammatory role in vivo in rats, while suppressing secretion of pro-inflammatory cytokines [Khat use was positively associated with significant induction of antibodies secretion and CD4ytokines , 38. Theytokines . Then, Tytokines , 69. ThuAlthough both cellular and humeral immune responses are crucial for protection against malaria parasite, the antibody-dependent mechanisms play an important role in reduction of parasitaemia and suppression of clinical symptoms in humans . In agreP. falciparum patients was elevated and strongly associated with lower level of parasitemia and less incidence of severe clinical malaria pathologies.Different studies suggested that among the four subclasses of IgG, IgG1 and IgG3 are the predominant subclasses produced in response to merozoite antigens –73. IgG1+ cells are able to confer suppression of parasite growth and provide protection [+ regulates the immune response against malaria by proliferation and production of cytokines, and activation of B cells to produce parasite specific antibodies [et al [+ T-lymphocyte during malaria infection, as significantly higher count of this T- lymphocyte population was observed. Thus, besides direct involvement of CD4+ cells in parasite growth suppression and provide protection among khat users, it might support B lymphocytes activities for production of plasma antibodies.T cells play a major role in the gaining and maintenance of protective immune response to malaria infection. Different studies from human and animals suggested that CD4otection . Helper tibodies , 78. In s [et al , chronicEven though some common malaria symptoms were not frequently observed among khat chewer malaria patients, khat might be an important risk factor for incidence of some severe malaria complications such as jaundice and renal impairment. Also, khat use was positively associated with high levels of antibodies titer and T- helper lymphocytes population. Moreover, the elevated levels of these antibodies were negatively correlated with parasite burden and manifestation of severe malaria pathologies.S1 File(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Plasmodium falciparum.This case report describes a case of presumed acute myocardial infarction in a returned traveler who was later diagnosed to have severe malaria. Emergency coronary angiography was normal and subsequent peripheral blood film was positive for  Plasmodium falciparum infection among returned travellers can easily be missed by treating physicians, in the event a detailed travel history is not taken at presentation. The many ways a severe malaria can present further complicates the issue as is illustrated in the case below whereby fever was not a predominant component in the presenting complaint but rather an organ-specific complication that prompted the patient seeking medical attention at emergency department (ED) with severe chest pain. This quickly escalated into a prompt cardiology referral and subsequent invasive angiography following an abnormal electrocardiogram that strongly suggested a case of myocardial infarction. Fortunately, the sole sample sent from the ED for malaria blood film examination proved invaluable in this case, which allowed the targeted treatment for malaria to be instituted 24 hours following presentation.Severe -pack/year history of smoking. On examination, he was afebrile and his blood pressure and pulse rate were 94/53 mmHg and 125/min, respectively. He was conscious and alert, had mild pallor, icterus, tea-coloured urine and hepatomegaly of three finger-breadths under right hypocondrium with dull Traube’s space. There were no remarkable findings on respiratory and cardiovascular examination.In this case, a 51-year old local man presented to the emergency department (ED) with acute onset of central chest pain. The worst intensity was experienced four hours before arrival and was self-graded as 6/10. There was associated profuse sweating. His only cardiovascular disease risk factor was 409/mm3 and platelet 20 × 109/mm3, respectively). Of note, a blood film was sent for malaria parasite detection from the ED as the travel history might have prompted this, as this is not routinely carried out in all the febrile cases presenting to our ED unit.The electrocardiogram (ECG) revealed greater than 2 mm ST segment elevation in leads V1 to V3 suggestive of acute anteroseptal myocardial infarction (AMI)  rapid diagnostic test and confirmed to be sed test . The parUnfortunately, a few hours after ICU admission he developed progressive hypoxemia due to acute respiratory distress syndrome (ARDS) which necessitated invasive ventilation. He underwent continuous veno-venous haemodialysis (CVVHD) due to oliguric acute kidney injury with a peak urea of 14.3 mmol/L and creatinine of 450 micromol/L with noradrenaline support. Intravenous ceftriaxone was added to cover for possible super-added bacterial infection. Normoglycaemia was maintained throughout his stay in ICU with intravenous 5% dextrose solution. After six hours of commencement of the first dose of IV artesunate, the parasite count rapidly declined to 0.07%. Following anti-malarial therapy, the inotrope requirement was gradually reduced. The parasite count was undetectable after 48 hours of IV artesunate.He was extubated after six days in ICU. His renal function and urine output continued to improve and he eventually did not need renal replacement therapy. On discharge, his urea and creatinine were stable at 7.6 mmol/L and 200 umol/L, respectively. The antibiotics were ceased after ten days as baseline and subsequent blood cultures did not grow any bacteria. He received in total ten days of weight-adjusted doses of IV artesunate, followed by four doses of fixed combination pills of artemisinin and lumefantrine and 14 days of oral doxyxycline. A month later on follow up, his urea and creatinine normalized to 3.5 mmol/L and 85 umol/L, respectively.P. falciparum infections, 23 patients (14.3%) had either non-specific T-segment changes or abnormal conduction delay [Malaria is a serious and potentially life-threatening disease, especially so in travellers due to: 1) lack of protective immunity to the parasite and, 2) due to delay in recognizing and treating the infection upon return. The clinical presentation of malaria is non-specific and is often misdiagnosed . In a statients 1.3% had ePlasmodium falciparum ability to produce distinctive virulence factors that increase the risk for the human host to develop end organ complications. It increases microvascular sequestration via the expression of several molecules on the infected erythrocyte such as Ag332 [P. falciparum erythrocyte membrane protein [Transient myocardial ischemia manifested by ST segment changes may not cause elevation in the cardiac biomarkers. This might be due to diffuse distal vessel epicardial spasm and microvascular spasm . This isas Ag332 , modifieas Ag332  and P. f protein . This maOf note, hypokalaemia and hypercalcaemia which can mimic the ST-T changes of myocardial infarction was not present in this patient on admission. .This case report illustrates that diagnosis of malaria based on clinical features alone is difficult and requires a high index of suspicion among clinicians. The key to diagnosing malaria is to elicit detailed travel history and request a specific malaria test in anyone who feels unwell and has recently been to an endemic country. The diagnosis of acute coronary syndrome due to severe malaria should be considered in those who present with features suggestive of myocardial ischaemia.All travelers to malarious areas should be made aware of the risk of contracting malaria and the seriousness of this infection. They should be advised on preventive measures, which include complying with anti-malarial chemoprophylaxis and avoiding mosquito bites. Furthermore, patients need to be informed about the importance of seeking medical treatment immediately should they become ill during travel or up to one year after returning home.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for the review by the editor of this journal.


Plasmodium falciparum which is initiated through protein interactions of pathogen with the host proteins. It is essential to analyse the protein-protein interactions among the host and pathogen for better understanding of the process and characterizing specific molecular mechanisms involved in pathogen persistence and survival. In this study, a complete protein-protein interaction network of human host and Plasmodium falciparum has been generated by integration of the experimental data and computationally predicting interactions using the interolog method. The interacting proteins were filtered according to their biological significance and functional roles. α-tubulin was identified as a potential protein target and inhibitors were designed against it by modification of amiprophos methyl. Docking and binding affinity analysis showed two modified inhibitors exhibiting better docking scores of −10.5 kcal/mol and −10.43 kcal/mol and an improved binding affinity of −83.80 kJ/mol and −98.16 kJ/mol with the target. These inhibitors can further be tested and validated in vivo for their properties as an antimalarial drug.Malaria, a life-threatening blood disease, has been a major concern in the field of healthcare. One of the severe forms of malaria is caused by the parasite Plasmodium falciparum. It takes away millions of lives with the rate increasing each growing year. According to WHO's Factsheet on the World Malaria Report 2013, 1.2 billion people out of a total of an estimated 3.4 billion are at a high risk of malaria. Malaria is highly prevalent in sub-Saharan Africa where 90% of all malaria deaths occur (WHO 2013). A lot of research has been going on in the field of malarial therapeutics. Nowadays, there are a wide variety of antimalarial drugs, such as chloroquine and artemisinin, and strategies available for the control and treatment of malaria phosphoryl} oxy)-2-(hydroxymethyl)-6-(trifluoromethyl)phenol and 5-({ethoxy[(piperidin-1-yl)amino]phosphoryl} oxy)-4-(hydroxymethyl)-2-(trifluoromethyl)benzene-1,3-diol were the two best molecules which can be considered as drug molecules for