Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT).Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction.This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4–23.1). Microscopy-based prevalence of P. falciparum infection was 3.7%  while the prevalence was 6.9%  using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10–11.9 g/dl).The prevalence of sub-microscopic Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring  P. falciparum asymptomatically and also carry sub-microscopic asymptomatic infections below the microscopic detection threshold that can only be detected using molecular techniques. Routinely used laboratory methods appropriate for large-scale use, such as microscopy and rapid diagnostic tests (RDTs), are not sensitive enough to detect low-grade, asymptomatic infections[Plasmodium species, particularly for low parasitaemia[Individuals in malaria-endemic areas can carry microscopically detectable levels of fections. One of sitaemia. Usuallysitaemia. Since msitaemia. Polymersitaemia.The contribution of the sub-microscopic reservoir to sustaining malaria transmission depends on malaria endemicity and slide positivity rate of a given area. Studies have shown that sub-microscopic carriers are presumed to be the source of over 20% of mosquito infections in areas where slide prevalence is less than 4% while otPlasmodium species and parasitaemia[Prompt and effective malaria diagnosis not only alleviates suffering, but also decreases malaria transmission at the community level. Although microscopy remains the gold standard for malaria diagnosis, the detection threshold in Giemsa-stained thick blood film has been estimated to be 4 –20 parasites/μl. Nonethesitaemia. As malasitaemia.Plasmodium vivax than in P. falciparum infections[In Ethiopia, microscopy and RDT are commonly used for malaria diagnosis although the diagnostic performances of these tests haven’t been evaluated against PCR. A PCR- based study conducted in low-transmission settings has demonstrated that a high proportion of cases with low-density parasitaemia were not detected by microscopy or RDT,13. Studfections. This sufections. The objPlasmodium falciparum and P. vivax are the two common causes of malaria. A cross-sectional study was conducted in 12 randomly selected kebeles  to determine the prevalence of asymptomatic malaria carriages from November through December 2012. The kebeles have known population size and systematically registered households. After obtaining informed consent from parents/guardians, members of the randomly selected households who met the inclusion criteria  were requested to give finger-prick blood samples. Blood films were also collected from febrile patients attending three health centres to determine the prevalence of symptomatic malaria. Finger-prick blood was spotted on Whatman 3MM filter papers, air dried and individually kept in zip-lock plastic bag with desiccant for PCR. A total of 1,453 blood samples  were collected. Four hundred samples were randomly selected from RDT- and microscopy-negative samples and tested by PCR to determine the prevalence of sub-microscopic P. falciparum infection. All RDT- and microscopy-positive samples from both clinical patients and sub-clinical subjects were tested by PCR to determine the diagnostic performances of the two tests  without any parasite. For quantification of malaria parasites in the thick film, a total of 200 WBC were examined while simultaneously counting the number of asexual forms of malaria parasites. Plasmodial density was recorded as the number of parasites/200 WBC. This was done using a standard mean white blood cell count of 8,000 leukocytes μl-1. DensitiP. falciparum specific histidine-rich protein-II) and pLDH (Plasmodium lactate dehydrogenase) specific to P. vivax. A blood sample was considered positive for falciparum malaria if two colour bands  appeared within the result window. On the other hand, the sample was considered positive for vivax malaria if two colour bands (P. vivax test line and control line) appeared within the result window. If three colour bands  appeared within the result window, it was considered mixed infection . The presence of only one colour band (control line) was considered negative but if the control band failed to appear within the result window, it was considered invalid.SD BIOLINE Malaria Ag P.f/P.v POCT test kits  were used as per the manufacturer’s instructions. Finger-prick blood was used for the rapid assay. The kit is a one step, rapid, qualitative test intended for the detection of malaria infection in human blood samples indicating differential diagnosis between P. f HRP-II (2 square piece of blood-impregnated filter paper (~15 μl) was excised using a puncher and transferred in to a 1.5 μl tube and stored at −20°C until use. The extraction was done using the Chelex with heat extraction method[Parasites DNA was extracted from the blood spotted on Whatman 3MM filter paper samples. A 3-mmr paper ~ μl was eThis protocol was approved by the Institutional Review Boards (IRBs) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University and of the Armauer Hansen Research Institute as well as the National Research Ethics Review Committee (NRERC). Information about the objective of the study was given to the head of the household or study subjects. Verbal informed consent was sought from each eligible subject and parents of children < six years of age for blood films. Additional verbal informed assent was sought from children aged six to18 years. Illiterate people signed with their fingerprint. The privacy of participants was preserved by allocating codes/numbers to participants in the blood survey instead of names. Study subjects who tested positive for malaria by RDT were treated according to the national malaria treatment guideline in the field.P. falciparum carriage was analysed across age groups to determine age-related correlation. Statistical analysis was performed using Stata version 11. The association between haemoglobin level and sub-microscopic carriages were determined. The sensitivity and specificity of RDT and microscopy was determined taking PCR as the ‘reference’. The X2 test was used to compare the Plasmodium carriage among different age groups. Logistic regression was made to determine the association of parasite prevalence by PCR and the different independent variables. A test was considered statistically significant if the p-value was < 0.05  was 19.2% (77/400). Although sub-microscopic infections was higher in males (22.5%) than females (16.6%), there was no statistically significant difference  in sub-microscopic infections between sexes. The mean haemoglobin value observed in 172 study subjects was 16.5 g/dl (95% CI = 15.9-17.3). Anaemia (<12 g/dl) was observed in 21 (12.2%) of the study subjects . Among five individuals with body temperature ≥ 37.5°C, 20.0% (one of five) had P. falciparum infections while 19.2% (76/395) individuals with body temperature < 37.5°C had infections.After excluding 154 samples that were positive by microscopy and/or RDT, 400 negative samples from a total of 1, 299 were randomly selected and tested by PCR. The prevalence of sub-microscopic P. falciparum infection was 3.7%   and 6.9%   as diagnosed by microscopy and RDT, respectively . Moreover, the high number of false negatives by microscopy and false positives by RDT raises serious doubts about their effectiveness as the sole screening tests especially in places such as this study area. Equally important are also a high number of false negatives by RDT as well as false positives.The prevalence of  95% CI, .7-4.7  and microscopy  in positive samples. Microscopy and RDT detected on average, 38.9% (49/126) and 60.0% (80/157) of all PCR-detected P. falciparum infections, respectively.PCR consistently detected higher frequencies of n Table . The pre2 = 63; P < 0.000) and microscopy  positivity rate of microscopic P. falciparum infections, but there was no significant association between age and sub-microscopic carriages (p > 0.05). Microscopic P. falciparum prevalence ranged from as low as 1.9% (>35 years) to as high as 7.0% (two of five years) as determined by microscopy while it ranged from 1.9% (>35 years) to 12.6% (two of five years) using RDT. The prevalence of sub-microscopic P. falciparum infection ranged from as low as 10.2% (two of five years) to as high as 22.7% (26–35 years)  and multivariate analysis . In this study, the overall self-reported bed net use was 62.3% (38.2% in men and 57.4% in women). Although sub-microscopic parasite carriages were high in older age groups, the difference was not statistically significant in both univariate and multivariate analysis. Sub-microscopic carriage was not significantly different between sexes  in univariate logistic regression. Asymptomatic carriages were lowest among subjects with body temperature < 37.5°C although statistically not significant. Although sub-microscopic parasite carriage was higher among anaemic subjects than those with normal haemoglobin level, the difference was not statistically significant  all of which were determined as P. falciparum, PCR failed to detect them. They were considered false microscopy positives since PCR was considered the reference. Of the RDT positives, PCR failed to detect 20 samples, all of which were determined as P. falciparum, by RDT and hence called false RDT positives. Among many other factors, the utility of a RDT-based negative result depends on the sensitivity of the test[The PCR analyses done here helped to establish two relationships, i.e. the rate of false positives and false negatives given by microscopy and RDT, respectively. Both have implications for malaria control measures. Starting with the false positive rate and its implications, in the present study, anti-malarial drugs were prescribed for 9.3 and 20.0% microscopy- and RDT-based parasite-negative patients, respectively, as witnessed by PCR. This percentage of parasite-negative patients receiving anti-malarial drugs was comparable to countries such as Tanzania, Ugandathe test, brand[the test, storagethe test,23.P. falciparum. The results of this study indicate that considerable numbers of P. falciparum infections were missed by both RDT and microscopy, suggesting the need for a more sensitive assay for the detection of sub-microscopic parasitaemia. There was no difference in sub-microscopic carriages in febrile and non-febrile patients . The identification of sub-microscopic P. falciparum infection in fever patients may not help to reliably confirm malaria as the cause of the fever and excludes the possibility of other diseases[P. falciparum infections may represent a significant challenge to malaria control programmes since such parasitaemic individuals may serve as a reservoir of infection and contribute to mosquito infection[P. falciparum infections in endemic areas compared to PCR[P. falciparum and mixed infections, respectively. Both RDT and microscopy underestimated the true parasite prevalence in the study area. In this study, microscopy and RDT detected 38.9 and 60% of the infection identified by PCR.For false negatives, the discussion spins around 19.2% prevalence of sub-microscopic diseases. The prenfection-26. Mored to PCR. The agrP. falciparum in Ethiopia requires further research.The limitations of commercially available RDT kits are well documented,28. The P. falciparum in areas of low transmission[P. falciparum infections while PCR detected a further 331 P. falciparum infections from microscopy-negative samples[The identification of large numbers of sub-microscopic parasitaemia in this study was supported by meta-analysis that demonstrates high proportion of sub-microscopic infection of smission, indicatsmission. In a st samples. The impP. falciparum infection decreases with age since younger age groups have the highest incidences as determined by microscopy, RDT and PCR. On the other hand, the proportion of sub-microscopic P. falciparum infections was higher in older age groups. This is because increasing age has been clearly linked to lower parasite densities[P. falciparum infection with age may explain the fact that infections will be controlled and remain asymptomatic in older age groups because clinical immunity develops overtime.The prevalence of microscopic ensities and mostensities. Childreensities. Under censities where riP. falciparum in the study area has important implications for malaria control measures in Ethiopia since such infections are important contributors to the infectious reservoir[The observation of high prevalence of PCR-based sub-microscopic eservoir-38, becaeservoir,33 and aeservoir. For assIn this study, older age was associated with increased sub-microscopic carriages. This finding is in agreement with a study by Manjurano and his colleagues where inP. falciparum infections were identified in the study area. Even though malaria transmission is seasonal and unstable in the area, significant numbers of Plasmodium-infected individuals were asymptomatic and carry sub-microscopic parasite densities below the threshold of microscopy and RDT. It is important to maximize detection of cases (symptomatic and sub-microscopic asymptomatic) presumably infectious to mosquitoes using enhanced diagnostic techniques. A molecular test (PCR) is more sensitive in detecting low levels of sub-microscopic P. falciparum infections and prevalence determination of malaria in a given population. Thus, PCR may be the best tool for measuring Plasmodium prevalence than microscopy and RDT. The presence of sub-microscopic P. falciparum infections makes control and reduction of malaria transmission easier said than done since they may not be detected by the conventional diagnostic methods, i.e. microscopy or RDTs. Sub-patent malaria infections, detectable only by molecular techniques (PCR), can be relatively common in areas of low malaria endemicity in Ethiopia and these may substantially contribute to maintaining malaria transmission in the country. The role of sub-microscopic parasite carriages in human-mosquito transmission need to be determined especially when malaria control and elimination is prioritized.Large numbers of sub-microscopic The authors declare that they have no competing interests.LG, BE, AA and GS designed the study and involved in all stages of this study. LG was responsible for sample collection and prepared the draft manuscript. BE and AA coordinated the field work. GS, NE and LG extracted the DNA and did the molecular analysis. All authors contributed to interpretation of data, writing and revising the manuscript, and have seen and approved the final version.


Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-FcγRIIA and FcγRIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding FcγRIIA and FcγRIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding FcγRIIA p.H166R and FcγRIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. FcγRIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between FcγRIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models  of inheritance. The FcγRIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p = 0.049). The FcγRIIIB-NA2*03 allotype (CTGCGA), a variant of the classical FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p = 0.0092). The present study is the first to report an association between a variant of FcγRIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical FcγRIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines. Plasmodium falciparum malaria kills nearly a million people annually and over 90% of deaths occur in children under five years of age in sub-Saharan Africa P. falciparum antigen-specific cytophilic immunoglobulin (Ig) G (particularly IgG1 and IgG3) with protection from clinical malaria in vitro systems but has so far not shown any significant correlation with protection against clinical malaria. On the other hand a correlation has been reported between neutrophil mediated antibody dependent respiratory burst (ADRB) and protection from clinical malaria in two Senegalese populations which differ in malaria transmission intensity P. falciparum clearance in Gabonese children P. falciparum infection. In the FCGR2A gene (NM_021642.3), a non-synonymous variant, c.497G>A (rs1801274) in exon 4, which specifies the ligand binding domain of the receptor, causes an arginine (R) substitution of an histidine (H) amino acid at position 166 of the polypeptide (p.H166R) FCGR3B*1, FCGR3B*2 and FCGR3B*3 alleles, respectively FCGR3B*1 and FCGR3B*2 alleles differ in five nucleotide positions; c.108C>G; c.114T>C; c.194A>G; c.244A>G and c.316A>G FCGR3B gene (NM_00570.4) which results in four amino acid changes at positions; p.S36R; p.N65S; p.N82D and p.I106V of the peptide chain with the c.114T>C being a synonymous coding SNP FCGR3B*3 is identical to the FCGR3B*2 sequence except for a SNP c.233C>A (rs5030738) encoding p.A78D resulting in the expression of the FcγRIIIB-SH allo-antigen null) the corresponding gene deletion has been described FCGR3B and FCGR2A polymorphisms and clinical malaria using data from a well characterised longitudinal cohort study.Single nucleotide polymorphisms (SNPs) which alter the affinities of both FcγRIIA and FcγRIIIB in binding IgG subclasses have been described. In the present study, we hypothesised that the implications of these polymorphisms in the described neutrophil mediated immune correlate of clinical protection from malaria (the ADRB mechanism) by Joos and colleagues p = 0.75, χ2 analysis) while the distribution of age, blood group, ethnicity and sickle cell status were significantly different between the villages  . These children were distributed across six villages as follows: Asutsuare (ASU) (169), Kewum (KEW) (138), Avakpo (AVA) (33), Mafikorpe (MAF) (36), Osuwem (OSU) (71) and Volivo (VOL) (138) . A totalnalysis) .p = 0.24, χ2 analysis) among the six villages (p(LR test) = 0.013) . These individuals were considered susceptible to clinical malaria while those who never had clinical malaria were considered protected. The protected group was sub-categorized into two: (1) any individual with no malaria episode and (2) only individuals with no malaria episodes but with parasites detectable by microscopy at any time point during the follow up period. The overall number of susceptible and protected individuals did not differ significantly (villages . Logisti= 0.013) .p = 0.690, χ2 analysis). Nucleotide sequencing of exon 3 of FCGR3B identified the six polymorphisms , defining the FcγRIIIB-NA1/NA2/SH allotypes. The previously described c.197T>G, and c.297G>T polymorphisms were also found in 87 (14.9%) and 46 (7.9%) individuals respectively. A new SNP, c.232G>A was identified in five (0.85%) of the study participants. All the SNPs were in HWE, p>0.05  and FCGR3B-c.194A>G  SNPs (The FcγRIIA-p.H166R (c.497A>G) genotypes were determined by gene specific polymerase chain reaction amplification followed by allele specific restriction enzyme digestion (PCR-ASRED). The minor allele frequency  was 41.0% and there was no deviation from HWE  SNPs .FCGR2A-c.497A>G (FcγRIIA-p.H166R) genotype frequencies among the susceptible and protected groups did not differ significantly in a multivariate analysis and had no influence on the outcome of P. falciparum infection (p(LR test) = 0.80) (FCGR3B-c.233C>A (FcγRIIIB-p.A78D) polymorphism showed a statistically significant association with the outcome of P. falciparum infection (p(LR test) = 0.009)  analysed using a control group that included all individuals with no clinical malaria during follow up .The distribution of the  = 0.80) . The FCG= 0.009) . The A-aollow up . The conollow up  under th2 values between these SNPs (0.08≤r2≤0.86) from linkage analysis revealed a significant linkage between most of the SNPs (p = 0.0092) while the FcγRIIIB-SH allotype showed a borderline significant association with protection   . When onFCGR3B was sequenced in 132 native Danish blood donors and genotype distributions compared to the Ghanaian population to investigate differences and possible selection. Sequence analysis found all the six SNPs defining the FcγRIIIB-NA1/NA2/SH system. All SNPs were in HWE (p>0.05). One person carried synonymous variant (c.201C>T). The c.233C>A genotype frequencies were significantly different  between the Ghanaian and Danish populations  and Danish  populations. Comparisons with data from the 1000 Genomes Project database showed that the A-allele frequency for the African (AFR) (17.9%) super population was similar to that in the Ghanaians (21.4%) in this study. Similarly, the A-allele frequency for the Ad Mixed American (AMR) (3.3%) and European (EUR) (0.8%) super populations were comparable to the Danes (3.8) . The AFR and the Ghanaian population shared a pairwise FST value of 0.004 while all comparisons between the malaria endemic (Ghanaians and AFR) and the malaria non-endemic  populations yielded consistently higher FST values (0.098≤FST≤0.194) (Exon 3 of ulations . Althouges (3.8) . Thus, tT≤0.194) .FCGR2A and FCGR3B respectively, the association between SNPs which alter the affinity of these receptors for IgG subclasses and clinical malaria were studied in a cohort of Ghanaian children. There was no association between the FCGR2A-c.497A>G (FcγRIIA-p.H166R) polymorphism and clinical malaria while the A-allele of FCGR3B-c.233C>A polymorphism (rs5030738) was strongly associated with protection from clinical malaria. Haplotype analysis identified the FcγRIIIB-SH allotype (CTGAAA) containing the c.233A-allele (in bold) to be associated with protection while a variant of FcγRIIIB-NA2, the FcγRIIIB-NA2*03 allotype (CTGCGA) was associated with susceptibility to clinical malaria. Individuals of the Ewe ethnic group showed a reduced risk to clinical malaria compared to other ethnic groups, however, the distribution of genotypes was not different between the ethnic groups except for FCGR2A-497A>G and FCGR3B-194A>G. These genotypes had no association with clinical malaria. Also, while the ethnic group distribution was different between the villages, the number of susceptible and protected individuals was not different between the villages. Thus, the genetic associations with protection and susceptibility observed here could not be due to a bias caused by ethnicity. Heterogeneity in malaria exposure is an important confounder in association studies P. falciparum malaria. The Tajima's D estimates show that FCGR3B exon 3 is under strong selection pressure in the Ghanaian populations and also to a lesser extent in the Danish population.Using PCR-ASRED and exon 3 nucleotide sequencing data for in vitro studies P. falciparum infection. This notion is supported by both in vitro studies of ROS toxicity on intra-erythrocytic malaria parasites P. falciparum clearance Both sero-epidemiological FCGR3B which was associated with protection from clinical malaria in the present study causes a replacement of the hydrophobic amino acid alanine (A) with the negatively charged aspartic acid (D) at position 78 in the NA2 protein. This substitution results in the SH allotype which also showed association with protection. The functional implications of this p.A78D substitution in receptor-ligand (antibody) interactions have not been conclusively shown. Koene and colleagues The c.233A-allele of FCGR2A and FCGR3B have found individuals carrying the NA2 allotype in combination with FcγRIIA-166H have an increased risk of developing cerebral malaria FCGR3B-exon 3 nucleotide sequencing data, we find a previously reported variant of FcγRIIIB-NA2, FcγRIIIB-NA2*03  data of FCGR3B to be under selection pressure in both the Ghanaian and the Danish populations; however, it is not clear what may be driving the selection in these different populations. Malaria has been considered the strongest known selective pressure in the recent history of the human genome ST indices (0.098≤FST≤0.194) between the malaria versus non-malaria endemic populations. These observations support the notion that malaria may be a significant contributor to the selection pressure, at least in the Ghanaian and African (AFR) populations. However, other possible factors that could explain the selection in both the malaria and non-malaria endemic populations may include inflammatory diseases such as rheumatoid arthritis FCGR3B. It is however, worth noting that the present data does not clearly show which modes of natural selection may be at play in these two populations. Further studies are needed to clearly define the forces of selection on FCGR3B-exon 3 in these populations.The Tajima's D analysis showed exon 3 of FCGR3B-c.233C>A (rs5030738) and the FcγRIIIB-SH (CTGAAA) haplotype to be associated with protection from clinical malaria. The FcγRIIIB-NA2*03 (CTGCGA) variant of FcγRIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria in the Ghanaian population. The study provides the justification for a more detailed functional characterisation of the FcγRIIIB-SH and FcγRIIIB-NA2*03 haplotypes in relation to neutrophil functionality especially in respiratory burst activity.In conclusion, the present study has identified the c.233A allele of Ethical approval for the study was given by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research (NMIMR) of the University of Ghana, Accra, Ghana. Written informed consent was given by the parents and guardians of children before they were enrolled into the study. Ethical approval for Danish blood donor samples was given by the Scientific Ethics Committee of Copenhagen and Frederiksberg, Denmark. DNA samples from a total of 132 anonymous Danish blood, obtained for control purposes from Copenhagen University Hospital, were analysed in order to allow population comparisons of genotype distributions. These individuals are resident of central Copenhagen and provided written consent to have a small portion of their blood stored, anonymously, and used for research purposes. Blood donors in Denmark must be between the ages of 18 and 60. All data were analysed anonymously.P. falciparum constitutes 98% of all infection with the remaining 2% due to P. malariae and P. ovaleThe study was conducted in Asutsuare (ASU) (about 120 km north-east of Accra) and five neighbouring villages: Kewum-Atrobinya (KEW), Avakpo (AVA), Mafikorpe (MAF), Osuwem (OSU) and Volivo (VOL) of the Damgbe West District of the Greater-Accra Region of Ghana. The villages are only about 2-5 km apart. Like many other parts of Ghana, the climate of the area is characterised by two major seasons: a dry or the harmattan season (December to March) and a wet or the rainy season (June to August), however, there are also some few rains in November and early December, just before the onset of the harmattan season. Malaria transmission occurs throughout the year but peaks during and after the rains (September and January). Altogether, 798 children (aged 1 to 12 years old) were enrolled and were followed up actively and passively for malaria case detection in a 42 week longitudinal cohort study. Genomic DNA for analysis was available for 585 of the 669 children who successfully completed the follow up without missing at most three successive weekly visits. Of these 585 children, there were 316 singletons, 91 families with two children enrolled, 17 families with 3 children enrolled and 9 families with 4 children enrolled.. At baseline (enrolment), 5 ml EDTA-anticoagulated venous blood and thick and thin film blood slides were obtained from all individuals prior to the malaria transmission season (May 2008) for baseline immunological and parasitological determinations. Blood group and sickle cell status of each individual were determined by a commercial blood grouping kit  and the sodium metabisulphite test respectively and haemoglobin (Hb) level was measured using the Hemocue-Hb 201 . Blood was centrifuged to separate plasma and peripheral blood mononuclear cells (PBMCs) and stored at −80°C and in liquid nitrogen respectively. The thick and thin blood film slides were stained with Giemsa and examined for baseline asymptomatic parasitaemia. A slide was negative if no parasite was visualised in 200 oil fields of a thick film. For slides that were positive, parasites were counted per 200 white blood cells (WBCs) and parasite densities calculated by assuming 8,000 WBCs/µl blood. A standardised questionnaire was used to obtain epidemiological, anthropometrical and clinical data of all study participants during the enrolment.DNA samples from a total of 132 anonymous Danish blood donors, obtained for control purposes, were analysed in order to allow population comparisons of genotype distributions. These individuals are resident of central Copenhagen and provided consent to have a small portion of their blood stored, anonymously, and used for research purposes. Blood donors in Denmark must be between the ages of 18 and 60 P. falciparum parasitaemia with at least one other sign of malaria such as vomiting, diarrhoea, or malaise. Malaria was treated with artesunate-amodiaquine combined dose therapy which was the recommended standard treatment for malaria in Ghana. At the end of the study, on the basis of the clinical and parasitological data obtained, the study population was divided into three groups: (1) those susceptible, in which parasitaemia was associated with febrile disease, and (2) those apparently protected against clinical manifestation despite parasitaemia and (3) those apparently protected against clinical manifestation without detectable parasitaemia by microscopy.Parasitological surveillance for malaria infection was carried out monthly for each study participant during the follow-up period. This involved obtaining thick and thin blood film slides from finger pricks by trained medical personnel. In addition, about 500 µl of blood was collected during the monthly finger pricking to obtain plasma for immunological analyses. The remaining packed cells were stored at −80°C for DNA purification for genetic analyses. The active case detection surveillance comprised weekly visits to each participant's home, where a morbidity questionnaire (investigating symptoms occurring in the preceding week) was administered by trained Field Assistants. The presence or absence of fever  was ascertained. Study participants complaining of symptoms suggestive of malaria were referred for treatment at the respective health centres. In the passive case detection surveillance, visits by participants to the health centres without prior referral from a field assistant were documented. In both the active and passive case detection, Hb level was measured and thick and thin blood films were obtained from study participants with febrile temperature (>37.5°C) or reported febrile temperature prior to treatment. Clinical malaria was defined as slide positive for any asexual stUI restriction digestion products were visualized as 343 bp  and 322 bp  bands on 3% agarose  with ethidium bromide  staining. Both fragments were present for heterozygous individuals.Genomic DNA was purified from packed cell samples using the Maxwell®16 system  following manufacturer's guidelines. Genotyping of FcγRIIA-p.H166R was done by the gene specific polymerase chain reaction (PCR) amplification followed by allele specific restriction enzyme digestion (ASRED) method FCGR3B gene from genomic DNA. First, an approximately 4.3-kb fragment of the FCGR3B gene using the sense primer (5′-CTCCATTGCGAGACTTCAGAT-3′) placed in exon 1 and the antisense primer (5′-CGTGGTTTCTAAGGTGTCACAGG-3′), positioned within intron 3. A 30-cycle amplification process consisting of denaturation at 95°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 5 mins was performed using PfuUltra® high-fidelity DNA polymerase . The product was gel purified using E.Z.N.A® Gel Extraction Kit  and used as template in a nested PCR to amplify exon 3 of FCGR3B with the M13 tagged (in lower case) sense (5′-tgtaaaacgacggccagtGTCAGCTTCATGGTCTTGGATTG-3′) and antisense (5′-caggaaacagctatgaccACACATTCACATTGTATGCACTCCA-3′) primers. The 38 cycle amplification consisted of denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 45 sec using TEMPase hotstart DNA polymerase . The nested PCR product was then sequenced with M13 primers. The low affinity FCGR locus contains regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. However, previous FCGR3B CNV data determined by the SALSA® multiplex ligation probe amplification (MLPA®) kit P110-B1/P111-B1 FCGR  (MRC Holland) on the same samples in the present study found no association with and clinical malaria .We designed a protocol to specifically amplify and sequence exon 3 of the 2-test). Since the risk of clinical malaria is known to be high in children aged 5 years and below, age was modelled as a categorical variable with two levels . Logistic regression analyses and likelihood ratio tests were carried out to evaluate the association of the variables, age group, sex, sickle cell status, blood group, use of bed net and ethnic group with clinical malaria. The genotype distribution of the SNPs was compared across ethnic groups (χ2-test). To test Hardy Weinberg equilibrium (HWE), we used the data of n = 267 children who had diploid copies of FCGR3B, were the first sibling in a family and were not susceptible to clinical malaria in the observation period. The exact test (HWE.exact) implemented in the R-package ‘genetics v.1.3.6’ (http://CRAN.R project.org/package = genetics) was used. For each of the SNPs, the minor allele (single marker) association with clinical malaria was calculated under the three genetic models of inheritance  using logistic regression adjusting for age group, sex, sickle cell status, blood group, use of bed net and ethnic group. Two different control groups were defined: (1) all individuals with no malaria episodes but with definitive exposure as indicated by parasites detectable by microscopy during the follow up, plus all individuals with no malaria episodes but without parasites detectable by microscopy and (2) only individuals with no malaria episodes but with definitive exposure as indicated by parasites detectable by microscopy during the follow up. The second analyses involving control group (2) was to account for any possible confounding due to heterogeneity in exposure. This was because, the group comprising individuals apparently protected but without detectable parasitaemia by microscopy may also include individuals who did not have malaria due to lack of exposure to the parasite during the follow up period. Additional sensitivity SNP association analyses (logistic regression) were performed with a generalized estimating equation (GEE) approach to correct all confidence limits and p-values for the family structure in the data. Linkage disequilibrium (LD) in the FCGR3B SNPs and visualisation of pairwise r2 LD values were evaluated using Haploview v. 4.2 FCGR3B haplotypes with clinical malaria was analysed using the Hapassoc package v. 1.2–4 in R 2 visualization, all statistical analyses were performed using R v. 2.13.2 (http://www.R-project.org). DnaSP v. 5.10 (http://www.ub.edu/dnasp/) was used to estimate Tajima's D for exon 3 of FCGR3B the Ghanaian and Danish populations. The window length for the Tajima's sliding window analysis was 50 with step size of 10 nucleotides. Genotyping data for rs5030738 was retrieved from the 1000 Genomes Project database (http://browser.1000genomes.org/Homo_sapiens/Variation/Population?db=coreg=ENSG00000162747r=1:161592986-161601753t=ENST00000531221v=rs5030738vdb=variationvf=8673417); for the African (AFR); Ad Mixed American (AMR) and European (EUR) super populations and allele frequencies compared among all 5 populations. Pairwise FST distances were calculated for all 5 populations using the POPTREE2 software Demographic and clinical characteristics of the study population were compared across village of residence Click here for additional data file.Table S2Univariate analyses of FCGR2A and FCGR3B genotypes association with clinical malaria.a encodes the FcγRIIA-166H/R polymorphism. b adjusted for age groups, sex, sickle cell status, blood group, bed net use and ethnic group(DOC)Click here for additional data file.Table S3Single marker association of FCGR3B alleles with clinical malaria using sub-set of controls. Odds ratio (OR) and 95% confidence intervals (CI) were determined using multivariate logistic regression controlling for age, gender, ethnicity, sickle-cell status, FCGR3B copy number, blood group, family structure and use of bed net. MAF: minor allele frequency. ¥All individuals who never had malaria despite parasitaemia at any time point during the study(DOC)Click here for additional data file.


Plasmodium falciparum incidence rate in children up to 3 years of age who were cleared of parasites and monitored for 8 months was 163/100 person-years under UPS and 63 under ITPS . In Tobanda incidence was 157/100 person-years under UPS and 134 under ITPS . Protective efficacy was 61% under fully lined ITPS and 15% under roof lined ITPS. Anemia rates improved under ITPS in both camps. This novel tool proved to be a convenient, safe, and long-lasting method of malaria control when used as a full shelter lining in an emergency setting.A double-blind phase III malaria prevention trial was conducted in two refugee camps using pre-manufactured insecticide-treated plastic sheeting (ITPS) or untreated polyethylene sheeting (UPS) randomly deployed to defined sectors of each camp. In Largo camp the ITPS or UPS was attached to inner walls and ceilings of shelters, whereas in Tobanda the ITPS or UPS was used to line only the ceiling and roof. In Largo the  It has been estimated that up to one in three malaria-related deaths occurs in countries affected by conflict or natural disaster.Plasmodium falciparum in children < 7 years of age in this area of Sierra Leone.Anopheles gambiae s.l. and Anopheles funestus s.l.The study was conducted in Largo  and Tobanda  refugee camps situated in southeast Sierra Leone, West Africa . The twoThe United Nations High Commission for Refugees (UNHCR) negotiated the establishment of the refugee camps and provided basic shelter. Both camps were established to accommodate ∼8,000 refugees each. The camps were populated solely by Liberian refugees fleeing conflict in bordering provinces. The populations were represented by various ethnic groups from Lofa and Grand Cape Mount counties of Liberia. It was anticipated that refugees were likely to remain for at least 1–2 years, which was sufficient time to complete the study.P. falciparum malaria incidence was monitored in children 4–36 months of age, the age group most at risk from malaria.19Taking into account the anticipated lifespan of the camps, the 2-year funding commitment from the Humanitarian Aid department of the European Commission (ECHO), and the time required for construction, refugee sensitization, and baseline monitoring, we elected to run a prospective cohort study of 8 months duration in which Each refugee camp was divided into four sections in which intervention (ITPS) and control untreated polyethylene sheeting (UPS) arms were randomly allocated to the outer sectors of each camp . The siz2 (4 × 6 m) in which walls were rough casted from earth and a roof with eaves was covered with thatch. The shelter kits provided in both camps and within each sector were identical. The quantity of polyethylene sheeting distributed to families differed between the two camps. In the Largo camp all interior walls and ceilings were lined with either ITPS or UPS (full coverage). This design simulated the acute phase of a humanitarian crisis in which rudimentary shelters are constructed from polyethylene sheeting. The ITPS was attached to the roofing struts to create a plastic ceiling that met the tops of the four walls. The open eaves of the hut were set above the plastic ceiling layer thereby restricting mosquito entry into the living space via the eaves. By contrast, in the Tobanda camp, only enough ITPS or UPS was given out to form a roof and ceiling that was attached to the roofing mainframe. The ceiling-only coverage in Tobanda simulated usage of polyethylene sheeting in more established camps during the transition from acute to chronic phase emergency when families construct mud walled huts. The ITPS was identical to UPS in all respects apart from the pre-treatment with deltamethrin. Installation teams and refugee groups were blinded as to which polyethylene sheeting was treated or untreated. Both ITPS and UPS were produced by Vestergaard Frandsen, Denmark, under strict quality control that ensured batch separation between ITPS and UPS from manufacture to field delivery. Community latrines in the camps were also covered with UPS/ITPS in accordance with the respective sheeting type given to shelter coverage in each section of the camp. The time interval by which the majority of family plots had completed construction, and the earliest time in which monitoring of study cohorts could commence was 5–9 months in Largo and 3–5 months in Tobanda. Throughout the trial any UPS/ITPS that became storm damaged was replaced.Refugees were allocated individual family plots and provided with shelter kits by UNHCR. Shelter kits provided refugee families with the appropriate components to construct a wooden framed structure of 24 mA tiered sensitization program ran concurrently with camp construction and the distribution of polyethylene sheeting. The purpose of the trial was explained to refugee leaders through camp meetings. Here, the reason for the randomized allocation of sheeting type was explained further and consensus to proceed with the trial was sought. Later tiers went into more detail about informed consent, the risks and benefits of participation, responsible use of polyethylene sheeting, and possible adverse events.The primary outcome was the malaria incidence rate between children in each study arm. Children 4–36 months of age, taken equally from control and intervention arms were monitored as per the study profile . With anP. falciparum HRP-2 antigen detecting rapid diagnostic tests (RDTs), on a random sample of the whole population in ITPS and UPS arms in each camp in November 2003. This determined whether gradual exposure to ITPS during camp establishment and construction had affected parasite prevalence since arrival. Longitudinal monitoring of the child cohort commenced in December 2003 in both camps and terminated in July 2004.A baseline cross-sectional prevalence survey was conducted using Visitect  Seven refugee communities in “full coverage” Largo and 12 in “ceiling/roof coverage” Tobanda were randomly selected for epidemiological monitoring. Location of the communities was stratified on the basis of distance from surrounding water-bodies  and the positioning of ITPS/UPS covered latrines .In December 2003, all children 4–36 months of age within the clusters received directly observed 3-day combination therapy, with a daily dose of amodiaquine (10 mg/kg) and artesunate (4 mg/kg) from Co-treatment Blister Packs  to provide clearance of any malaria parasites.Inclusion criteria for the recruited children were residence in the camps, 4–36 months of age, and guardian informed consent. Children who had a serious illness other than malaria or who had experienced adverse reactions to amodiaquine or artesunate on a previous occasion were excluded from monitoring. Families were excluded if they anticipated moving out of the camp during the upcoming 12 months.Between December 2003 and July 2004, daily monitoring of children in both camps was conducted from health screening points staffed by nurses and laboratory technicians who rotated between each screening point over the course of the study. Any child presenting with fever or reported fever in the last 24 hours was administered a clinical questionnaire based on the Integrated Management of Childhood Illness (IMCI), after which a RDT  was taken to confirm malaria positivity. Unwell children were referred to the camp health facility for further examination to identify any other potential causes of illness prior to treatment. If a child became ill outside of normal screening hours, they could attend the health facility and study nurses were notified the following day.A malaria episode was defined as an individual with fever (≥ 37.5°C) or reported history of fever plus patent parasitemia confirmed by RDT. In addition to RDTs, a reference slide (thick and thin blood smears) were collected for confirmation of parasitemia. Unfortunately some of these deteriorated and were unreadable. Confirmation of malaria was therefore based on the RDT results.®, Ängelholm, Sweden) that was calibrated daily when used.In the study cohort hemoglobin (Hb) levels were monitored at 3 monthly intervals using a HemoCue photometer . These symptoms or conditions were classified as potential adverse related events whether recorded on the IMCI assessment form or recorded directly as an adverse related event. This prevented the loss of any adverse events that were recorded as another child associated condition. A symptom listed repeatedly within a 7-day period for each child was considered to be the same adverse event as was any child having more than one of the symptoms present on a single day.The baseline prevalence of malaria between control and intervention arms in each refugee camp was compared using a random effects multivariable logistic regression, adjusting for the potential intra-cluster correlation at the community level.Malaria incidence rate was estimated as the total number of malaria episodes per person year over the course of the trial. This was analyzed using a random effects multivariable Poisson regression model to account for intra-cluster correlation among individuals from the same intervention clusters. The analysis was per protocol; any child who repatriated or became lost to follow-up was only included up to the date of loss or repatriation. The incidence rate ratios (IRR) between ITPS and UPS arms were estimated after adjusting for age and gender. Protective efficacy (PE) was calculated as (1 – IRR) × 100.The time to first detected infection (symptomatic or asymptomatic) for children in UPS and ITPS arms was estimated using Kaplan-Meier survival analysis and log rank tests.The effect of the intervention on anemia (Hb levels) was calculated by running a random effects linear regression, adjusting for repeated observations on the same children.2 test with Phi symmetric measure being used to measure the strength of association.Differences in potential adverse events between treatment groups were analyzed using Pearson's χAll analysis was performed using STATA 9 .Approval for the study was obtained from the Ethics Committee for Research of the Sierra Leone Ministry of Health and Sanitation. The UNHCR Field Office granted permission for the study to be conducted in Largo and Tobanda refugee camps. The trial is registered on clincaltrials.gov with Identifier: NCT01456858. Refugees were fully informed about the trial and the risks/benefits before giving consent. Granting of informed consent was a two stage process. Heads of families gave informed consent to receiving at random ITPS or UPS. Heads of families who had children 4–36 months of age gave informed consent to longitudinal clinical and parasitological monitoring. Participation was voluntary. Refugee names were not recorded and locations were marked by only unique identification codes. Verbal information was given in English  and in the refugees' local language.N = 827) and Tobanda (N = 839). Although more females than males were sampled  the proportions were consistent in control and intervention arms (data not shown). Parasite prevalence was 54% (446 of 831) for Largo and 45% (355 of 779) for Tobanda. Having lived 5–9 months under polyethylene sheeting before the prevalence survey, the population in Largo under ITPS showed a decrease of 9.3%  in prevalence of infections compared with those living under UPS  or Tobanda (P = 0.9). Because of voluntary repatriation, 60% of the overall study cohort did not complete longitudinal monitoring (P = 0.252) and Tobanda (P = 0.49) and there was no significant difference in the age distribution of UPS and ITPS groups among those that completed the trial.Baseline characteristics of the study cohorts are shown in nitoring . The losP < 0.001). In Tobanda incidence was 157 and 134 per 100 child-years in the control and intervention arms, respectively, giving an adjusted incidence RR of 0.85 and a PE of 15% (P = 0.008). Adjustment for age and gender as potential confounding variables had a limited effect on the IRR point estimates.P = 0.0032) and the risk of malaria did not change over the course of the intervention. In Tobanda there was no significant difference between intervention and control arms .P = 0.002), adjusted for survey and repeated sampling. Mean Hb was higher in the ITPS arm during the first survey, possibly indicating protection from ITPS during the camp construction period before the survey. There was a significant increase of 0.6 g/dL of mean Hb under ITPS at 3 months  and 0.7 g/dL at 6 months  compared with the baseline. The UPS also showed a similar level of increase in mean Hb over this interval (P < 0.001).The mean Hb concentrations by survey and cohort groups are summarized in P = 0.002) under ITPS compared with baseline, adjusted for survey and repeated sampling, but not in the UPS group.By contrast, there was no difference in mean Hb concentration at baseline between control and intervention groups in Tobanda (ceiling/roof only coverage camp). However over the 6 months of repeated surveys there was a significant increase in Hb of 0.7 g/dL  offers an effective and safe tool for malaria control in displaced populations. The ITPS supplied as only roofing treatments are not recommended on their own as any effect would be insufficient to control malaria. The use of ITPS is a viable option in acute phase emergencies. As the situation stabilizes and refugee population's switch from polyethylene shelters to construction of habitations from local materials, LLINs should be used for protection. Distribution of ITPS is easier to organize than IRS or LLINs at the outset of a humanitarian crisis and may provide efficiencies in program costs and speed of delivery compared with separate shelter and malaria control interventions. Consideration should be given to the routine supply of ITPS in emergencies in malaria-endemic areas.


Malaria and Soil Transmitted Helminthiasis (STH) are co-endemic and major public health problems in Ethiopia. The aim of the study was to assess the prevalence of malaria and STHs co-infection and to determine the association risk factors.A cross-sectional community based study was conducted on 388 pregnant women living in three districts around Gilgel Gibe Dam area, southwestern Ethiopia. Socio-demographic and socio-economic data, single stool sample and blood sample were collected from each participant.Ascaris lumbricoides (A. lumbricoides) 58 (15%) and Trichuris trichiura (T. trichiura) 13 (3.4%). Habit of eating soil (Adjusted Odds Ratio (AOR) = 4.64, 95% CI: 1.50-14.36, P=0.008), presence of stagnant water near study participants’ house  and habit of using human feces as a fertilizer  were found to be significantly associated with malaria and STH co-infection among the pregnant women. Hookworm parasitic load was positively correlated with malaria parasitic load  while A. lumbricoides parasitic load was negatively correlated with malaria parasitic load .The prevalence of STH and malaria was 159 (41%) and 45 (11.6%), respectively and the prevalence of STHs/malaria co-infection was 30 (7.7%). Hookworm was the most prevalent 114 (29.4%) soil transmitted helminthiasis infection followed by Intestinal parasite and/or malaria co-infection is a health problem among pregnant women living around Gilgel Gibe dam area. Therefore, intervention including improving sanitation, removing stagnant water, and health education to the pregnant women should be given. Parasitic diseases pose major obstacles to health growth and socio-economic development in developing countries. Parasitic diseases such as malaria are life threatening as well as the leading cause of mortality in endemic countries; more severe to some risky groups, like pregnant women . In pregImmunological factors are expected to influence rates of co-infection because helminths modulate host immune responses both to themselves, and to concurrent infections. That is why pregnant women, immunologically compromised, are highly susceptible for parasitic infections such as malaria and Soil transmitted helminthiasis . ActuallIt was estimated that over a third of the world's population, mainly in the tropics and sub-tropics, is infected with parasitic helminthes and Plasmodium species  and an eIn Ethiopia, the study done in Alaba Kulito Health Center, southern Ethiopia, reviled that only few cases of helminth and malaria co-infected patients showed signs and symptoms of severe malaria as compared with severe manifestation in patients infected with plasmodium parasite alone . This iGenerally, there is poor understanding of the association between malaria and helminths infection and the basic factors for their co-occurrence especially in pregnant women. There is no satisfactory published literature on the prevalence and associated risk factors for co-infection of malaria and intestinal helminths among pregnant women in Ethiopia in general and in Gilgel Gibe dam area in particular. Therefore, this study was intended to investigate the prevalence of malaria and soil transmitted helminthes co-infection among pregnant women in Gilgel Gibe dam area and to assess the possible risk factors for these co-infections.A cross-sectional community based study was conducted from August to September, 2011 in Gilge Gibe Dam area, 260 km south-west of Addis Ababa, near Gilgel-Gibe hydroelectric dam which started operating in 2004. The total population was 55,000. The study area lies between latitudes 7°42'50"N and 07°53'50"N and between longitudes 37°11'22"E and 37°20'36"E, at an altitude of 1,672-1,864 m above sea level. The area has a sub-humid, warm to hot climate, receives between 1,300 and 1,800 mm of rain annually and has a mean annual temperature of 19°C. The rainfall pattern of the area is similar to other parts of Ethiopia with the long rainy season starting in June and extending up to September, while the short rainy season begins in March and extends to April/May. Participants from six rural and six urban kebeles were assessed through house to house visit. The study participants were then randomly selected using systematic random sampling from the sampling frame which was constructed after identifying the pregnant women.After getting their written consent, stool samples were collected from participants. The study participants were provided with labeled screw caped stool cap and informed on how to collect about 5 gram stool sample. The collected stool samples were soon transported to Jimma university clinical laboratory where it was processed following the standard procedure of McMaster concentration technique. The type of parasite and parasitic load were recorded in the prepared laboratory format.Within the same day when the participants donate their stool sample, capillary blood sample was also collected from all the 388 pregnant women following aseptic technique. Then, the finger was cleansed with alcohol-moistened swab, dried with a piece of dry cotton, punctured with a disposable blood lancet. Through wiping off the first drop of blood, thick and thin films were made on the same slide. After being air-dried in a horizontal position, the slides were placed in a slide box and carefully transported to Asendabo Health Center where parasitological test was carried by experienced laboratory technicians and technologists.Before staining the blood films, the thin blood films were fixed in methanol for 30 sec. Then smears were stained with 10% Giemsa solution for 10 minutes. The staining techniques and blood film examination was conducted according to WHO guideline. Microscopic examination of thick films using high power magnification for the presence of parasites and parasite species identification using thin films under 100x oil immersion objective were done by experienced laboratory technicians and technologists. In addition to the qualitative examination, parasitic load was determined following WHO guide line.A semi structured questionnaire was developed and administered to identify possible risk factors of malaria/STH co-infection in the study area. The questionnaires address the individuals’ socio-demographic information, use of malaria and STH preventive measures, housing conditions, their knowledge and attitude concerning malaria and soil transmitted helminthes transmission and other related issues to assess some possible risk factors for the co-infection.X2) test Odds ratios (OR) with a 95% confidence interval were computed to compare the strength of association between explanatory and outcome variable. Multivariable logistic regressions were also employed for those variables that had significant association with disease outcome to determine the main predictors of infection. P-value ≤ 0.05 was considered significant during the analysis.Data from both the laboratory and survey were checked and cleaned for completeness and consistency. Data were then analyzed using SPSS version 16.0 software package. For the analysis of demographic data descriptive statistics was employed. Point estimation of prevalence and intensity of STH calculated based on the stool and blood sample results. The comparison of prevalence of STH and malaria co-infection between different explanatory variables was done using chi-squire  live in rural areas and the rest 193 (49.7%) live in urban areas. The minimum and maximum ages were 16 and 40, respectively with mean age of 25 years. During the study 88 (22.7%), 157(40.5%), 143 (36.8%) were in their first, second and third trimester, respectively. The proportion of primigravida (pregnant for the first time) and multigravida were 125(32.2%) and 263(67.8%), respectively. Majority of the participants 372 (95.9%) were Oromo in ethnicity and more than 92% of the respondents were Muslims (Table Hookworm 114 (29.4%), A.lumbricoides 58 (14.9%), T.trichuria 13 (3.4%), H.nana 8 (2.1%), E.vermicularies 5(1.3%). The prevalence of STH and malaria co- infection among pregnant women was 30 (7.7%)  and 45 (11.6%), respectively. Parasitic infection was detected in 65 (33.9%) and 97 (48%) urban and rural residents pregnant women, respectively. STHs detected in the stools of the pregnant women were worm 114 9.4%, A.lSocio demographic and others variables were analyzed in relation to Malaria and STH co-infection by bivariate and multivariate logistic regression analyses model. In the bivariate logistic regression analysis, Malaria and STH co-infection was associated significantly with thatched roof, presence of stagnant water in the residence area, use of human faeces as fertilizer, and habit of soil eating Table .Habit of eating soil , Presence of stagnant water near study participants’ house  and habit of using human feces as a fertilizer  were found to be significantly associated with malaria and STH co-infection among the pregnant women on multivariate logestic regression model  and T.trichuira  were found to increase malaria prevalence. There was statistically significant association between number of helminthes parasite species and malaria infection rate (P<0.001) and it was found that the rate of malaria infection was higher in those pregnant women infected with more than one helminth species .Pregnant women infected with STH were likely to be three times more infected with malaria compared to these pregnant women with no STH infection . Infections with hook worm , Most of the time malaria and STH infections share endemicity in Ethiopia. The prevalence of malaria/STH co- infection among the pregnant women in our study was 7.7% with rate of co-infection higher in rural (9.7%) than urban (5.7%) pregnant women. Though it is not statistically significant, co-infection was more prevalent in those within the age group range of 26–30 year which was different from the previous report from Nigeria where pregnant women less than 20 were the most co-infected group . The preA. lumbricoids parasitic load was negatively co-related to malaria parasitic load; therefore as the parasitic load of A. lumbricoids increases the severity and parasitic load of malaria decreases. This finding was comparable with the studies which showed the protective effect of A. lumbricoids to reduce the severity of malaria [There was strong association between malaria and STH infection in which those pregnant women infected with STH were 3.3 times likely to be infected with malaria which is almost similar to a study from Ghana where women with intestinal helminth infection (s) were 4.8 times more likely to have malaria infection , was lower than the findings from Bushulo health center, Southern Ethiopia 58.2%  but was l report  as well l report . This dil report  and Kenyl report  but lowel report .P.falciparum and P.vivax proportion of 71.1% and 15.6%, respectively; whereas mixed infection with both species and maximum parasitic load were 13.3% and 36,480 parasites/μL, respectively. Malaria prevalence found in this study was by far lower than the results of similar studies done in Nigeria [P.vivax was reported to be more prevalent than P.falciparum[Malaria is the most serious public health problem in Ethiopia where it is highly distributed throughout the country. In our study malaria prevalence in Gilgel-Gibe dam area was 11.6% with  Nigeria , but alm Nigeria . This dialciparum.In this study presence of stagnant water, habit of eating soil and habit of using human feces as a fertilizer were associated risk factors for malaria and STH co-infection among pregnant women in Gilgel Gibe dam area in which pregnant woman who lives in house which is near stagnant water was three times more likely to be co-infected. Study done in Thailand, Ghana and Nigeria however identified parity, low income, being young age and marital status (being single) as possible factors associated with malaria STH co-infection ; Yatich Intestinal parasite and/or malaria co-infection is a health problem among pregnant women living around Gilgel Gibe dam area. Therefore, intervention including improving sanitation, removing stagnant water, and health education to the pregnant women should be given.The authors declare that they have no competing interests.MG conceived the study, designed, participated in data collection, conducted data analysis, drafted and finalized the manuscript for publication. DY, AZ and KT assisted in data collection and reviewed the initial and final drafts of the manuscript. MG, DY, AZ and KT interpreted the results, and reviewed the initial and final drafts of the manuscript. All authors read and approved the final manuscript.


Pentoxifylline (PTX) affects many processes that may contribute to the pathogenesis of severe malaria and it has been shown to reduce the duration of coma in children with cerebral malaria. This pilot study was performed to assess pharmacokinetics, safety and efficacy of PTX in African children with cerebral malaria.Ten children admitted to the high dependency unit of the Kilifi District Hospital in Kenya with cerebral malaria (Blantyre coma score of 2 or less) received quinine plus a continuous infusion of 10 mg/kg/24 hours PTX for 72 hours. Five children were recruited as controls and received normal saline instead of PTX. Plasma samples were taken for PTX and tumour necrosis factor (TNF) levels. Blantyre Coma Score, parasitemia, hematology and vital signs were assessed 4 hourly.One child (20%) in the control group died, compared to four children (40%) in the PTX group. This difference was not significant (p = 0.60). Laboratory parameters and clinical data were comparable between groups. TNF levels were lower in children receiving PTX.The small sample size does not permit definitive conclusions, but the mortality rate was unexpectedly high in the PTX group. The mortality of severe falciparum malaria in children admitted to African hospitals has not decreased significantly in recent decades despite the introduction of new anti-malarial drugs, including the rapidly acting artemisinins. Disruption of the pathogenic mechanisms may improve the outcome.The pathogenesis of severe malaria is unclear and several different processes may be responsible. Enhanced production of the cytokine tumour necrosis factor (TNF), induction of nitric oxide, cytoadherence and sequestration, rosetting (the aggregation of parasitized and non-parasitized red blood cells), and decreased red blood cell (RBC) deformability are thought to contribute to the pathogenesis of severe malaria ,2.Pentoxifylline (PTX), a methylxanthine, which acts as a phosphodiesterase inhibitor, has effects on many of these processes. It was initially developed to increase RBC deformability and enhance blood flow in the microvasculature. It leads to an overall improvement in haemorheological characteristics such as RBC deformability, blood viscosity, platelet aggregation, and plasma fibrinogen concentration . It may Pentoxifylline as an adjunctive therapy for severe malaria was first proposed in the early 1990 s and to date four clinical trials have investigated the efficacy of the drug in severe malaria. Trials in adults have shown conflicting results; a trial from India showed a significant reduction in duration of coma and mortality , but triThe pharmacokinetic properties of PTX are well described in healthy adults , but litThe study was approved by the Institutional Review Boards at the Michigan State University, USA, the International Center for Infectious Diseases Research protocol review committee within the National Institute of Allergy and Infectious Diseases, U.S. National Institutes of Health, USA and the National Ethics Committee of Kenya. The trial was registered at ClinicalTrials.gov, with the identifier NCT00133393.P. falciparum; inability to localize a painful stimulus  in patients who present with hypoglycaemia and 30 minutes after cessation of convulsive activity in patients who are convulsing on admission); and informed consent given by parents or guardians. Exclusion criteria were: mean blood pressure (BP) < 60 mmHg (mean BP = 2/3 diastolic BP + 1/3 systolic BP); platelet count < 50/nL; spontaneous bleeding; haematocrit < 20% or haematocrit between 20-25% with parasitaemia > 10%; neck stiffness; or clinical evidence of lower respiratory infections.The study was conducted between January 2002 and September 2004, in the coastal region of Kenya, an area of moderate to high malaria transmission . RecruitThe study was planned as a step-wise dose-escalation controlled study of ten subjects in each group, starting with 10 mg/kg, increasing by 10 mg/kg with 40 mg/kg as the maximum dose. The first four patients were assigned to the control group and two controls were to be added between each dosage group. However, after preliminary analyses, the study was stopped after 15 patients due to patient safety concerns raised by the National Ethics Committee of Kenya and the Data and Safety and Monitoring Board (DSMB).Children were admitted to the high-dependency research ward of the Kilifi District Hospital and were treated with a loading dose of 15 mg/kg quinine and a maintenance dosage of 10 mg/kg quinine every 8 hours thereafter . MannitoOut of the 15 children recruited, 10 children received a continuous infusion of 10 mg/kg/24 hours PTX in 0.9% saline (5 mg PTX/mL) for 72 hours. Five children (the first four and the last one recruited) acted as controls and received continuous infusion of normal saline only.On admission, demographic details of the patient, history of disease, and clinical findings   were recDuring the study, parasitaemia, glucose, and haematocrit were assessed 4-hourly until three consecutive slides were negative for parasites. Vital signs were assessed quarter-hourly during the first 4 hours and 4-hourly thereafter. Blantyre coma score was assessed every 4 hours. A full blood count and differential was performed at 12, 24, 48, and 72 hours. Lactate was measured at 4, 8, 12, and 24 hours. Creatinine and AST were measured after 72 hours. Blood for determination of TNF, PTX and metabolite (1-(5'-hydroxyhexyl)-3,7-dimethylxanthine) concentrations was taken on admission and at 1, 2, 4, 8, 12, 24, 48, and 72 hours after PTX administration. Blood was collected in heparinized tubes, centrifuged to separate plasma, and the plasma was stored frozen (-20°C) until assayed to PTX and its major metabolite.Follow-up visits were conducted at one and three months after admission. During these visits, neurological status was assessed and the examinations performed on admission were repeated.Plasma PTX and metabolite concentrations were determined using a validated High Performance Liquid Chromatographic method developed in our laboratory. Peak drug/metabolite concentrations and the corresponding times were the experimentally observed values, while the area under the plasma drug/metabolite concentration-time curve was determined using the trapezoidal method . HoweverTNF levels were measured from plasma samples by standard sandwich enzyme-linked immunosorbent assay using pairs of cytokine specific monoclonal antibodies according to the manufacturer's instructions  with a detection limit of 11 pg/mL. Each plate included a standard curve of recombinant human cytokine. All specimens were measured in duplicate and the mean value was used in all analyses. The optical density of each well was measured at 450 nm by a dual-wavelength plate reader . The correlation between optical densities ranging from 1 to 1000 pg/ml was strong in all cases.Outcome variables included the number of children who died, the number who survived with neurological sequelae , and resolution time of coma, parasitaemia, and fever. Parasite clearance time was defined as the time  when the first of two consecutive 4-hourly peripheral blood films was negative. Coma resolution time was defined as the time since start of PTX when a Blantyre Coma Score of 5 was first noted. Fever clearance time was defined as the time since start of PTX when the first of five consecutive 4-hourly rectal temperatures was below 37.5°C. Laboratory values were compared between intervention groups by flagging values outside of normal ranges. Serious adverse events were defined as any untoward medical occurrence that is life-threatening, results in death, or in prolongation of hospitalization. Fisher's exact tests were used to compare categorical variables and Wilcoxon rank sum tests and Student's t-tests were used to compare continuous variables.Admission characteristics of the children allocated to the PTX and control groups were similar Table . With thOutcome variables are shown in Table Salmonella sepsis on admission, required two blood transfusions and was subsequently found to be HIV positive. Two children in the PTX group needed transfusion, compared to none in the control group. The blood culture was positive in two subjects in the PTX group , and no positives were found in the control group.Two children experienced a serious adverse event and the PTX infusion was stopped as a consequence. One child developed hypotension (mean blood pressure below 60 mmHg), and the other demonstrated frequent vomiting (seven episodes over a four-hour time period) and gastrointestinal bleeding. The latter patient had Twelve children (80%) had haemoglobin below 8 g/dL on at least one occasion, 2 (13%) had aspartate transaminase above 150 U/L at least once, and 3 (20%) had creatinine above 100 μmol/L at least once. There was no statistically significant difference in the number of children with laboratory values outside of the normal range between the control and intervention groups for any laboratory parameter.TNF values were consistently and significantly reduced in the PTX group starting 2-4 hours after start of administration Figure . There wPentoxifylline has often been proposed as an adjunct therapy in cerebral malaria -22. ThisA study conducted in 56 Burundian children showed that PTX (10 mg/kg/day) caused a significant reduction in median coma resolution time, which was six hours in the PTX group compared to 46 hours in the control group. The reduction in mortality (17% vs. 0%) was not statistically significant, but the sample size may have been too small to detect such a difference. However this was not a double blind randomized trial, and it is unclear if the dose used was the optimal dose.In adults, the studies of PTX in cerebral malaria are contradictory. In a study of 52 Indian adults with cerebral malaria, there was a significant reduction in duration of coma, but not mortality in the PTX group . In a stThe rationale for the present study was the need to describe pharmacokinetics in children with cerebral malaria in order to decide on a dosage for large-scale studies. However, we found that the efficacy of the drug was associated with more side effects than the Burundian study, despite using the same dose. The study was not designed as an efficacy study and as a consequence there was no randomization of the intervention and the number of subjects was small, due to the early termination of the study. The difference between the Burundian and this study remains unexplained, since the entry criteria and regimens appear to be similar.The study shows that PTX was effective in reducing TNF levels, even at low PTX dosages. In the neonatal sepsis, PTX was given at a dosage of 30 mg/kg per day, which led to a reduction of TNF and other cytokines compared to placebo . In ThaiThe pharmacokinetic properties of PTX administered orally are well described in adults . ConsideThe use of PTX in children with cerebral malaria remains unresolved. It reduces the TNF levels, but whether this effect reduces mortality can only be examined by further studies in moderate malaria and a large-scale multi-center trial.The authors declare that they have no competing interests.BL: participated in the data and sample collection, data analysis and drafted the manuscript. CK: participated in the data and sample collection. BW: participated in the data and sample collection. CHOO: carried out the data management and participated in the data analysis. EK: participated in the data and sample collection. GK: carried out the analysis and interpretation of pharmacokinetic data. DW: participated in the design of the study, the data management and data analysis. SM: participated in the design of the study, and in the data and sample collection. TET: participated in the design of the study, and the writing of the study protocol. PGK: participated in the design of the study, and the writing of the study protocol. CRJCN: participated in the design of the study, the writing of the study protocol and carried out the study coordination. All authors read and approved the final manuscript.


Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) concentrations, a measure of parasite biomass, have been correlated with malaria severity in adults, but not yet in children. We measured plasma PfHRP-2 in Tanzanian children with uncomplicated (n = 61) and cerebral malaria . Median plasma PfHRP-2 concentrations were higher in cerebral malaria (1008 [IQR 342–2572] ng/mL) than in uncomplicated malaria . In cerebral malaria, natural log plasma PfHRP-2 was associated with coma depth  and mortality . In this relatively small cohort study in a mesoendemic transmission area of Africa, plasma PfHRP-2 was associated with pediatric malaria severity and mortality. Further studies among children in areas of Africa with higher malaria transmission and among children with different clinical manifestations of severe malaria will help determine the wider utility of quantitative PfHRP-2 as a measure of parasite biomass and prognosis in sub-Saharan Africa.Plasma  Plasmodium falciparum, and results from the ability of parasitized erythrocytes to cytoadhere to endothelial cells in post-capillary venules Plasmodium falciparum parasites in the deep tissue circulation of the brain compared to peripheral blood Sequestration of parasitized red blood cells within the deep organ microvasculature is thought to explain the pathogenicity of P. falciparum that facilitates heme polymerization to hemozoin Histidine-rich protein-2 (PfHRP-2) is a protein synthesized by Although there is a clear association with disease severity in Asia-Pacific adults, the greatest burden of severe malaria morbidity and mortality is in African children We conducted a prospective observational study in Dar Es Salaam, Tanzania, a city (population ∼3.2 million) with mesoendemic transmission of falciparum malaria P. falciparum parasitemia on peripheral blood smear; unarousable coma (Blantyre Coma Score ≤2) not attributable to hypoglycemia (blood glucose level <40 mg/dL) and persisting more than 60 minutes after any convulsion; no other identifiable cause of coma P. falciparum parasitemia >10,000 parasites/µL on Giemsa-stained blood film and positive P. falciparum rapid diagnostic test (Paracheck-Pf [Omega Diagnostics]); and no other cause of fever identified.Children were aged from 6 months to 6 years. Inclusion criteria for cerebral malaria were defined by WHO: any level of Plasmodium species; bacterial co-infection as evidenced by a positive blood, urine or cerebrospinal fluid (CSF) culture; CSF analysis indicative of bacterial meningitis; quinine or artemesinin combination therapy initiated >18 hours prior to enrollment; and/or hemoglobin <5 mg/dL. Erythrocyte transfusions were not readily available at our study sites. Children were also excluded from enrollment as uncomplicated malaria if they exhibited WHO warning signs suggestive of severe disease: inability to suckle, eat and/or drink; excessive vomiting; abnormal respiratory rate or respiratory distress as evidenced by accessory muscle use, suprasternal retractions, or intercostal retractions; recent history of convulsions; altered mental status; or inability to sit unaided.Exclusion criteria for both groups with malaria were any of the following: microscopic evidence of mixed infection with any other At presentation, demographic information, clinical history, and examination were documented using standardized case report forms. Capillary blood samples were obtained for Giemsa-stained thick and thin blood smears as well as on-site rapid diagnostic testing (Paracheck-Pf [Omega Diagnostics]). Venous samples for routine laboratory analysis included complete blood count , sodium, potassium, chloride, bicarbonate, blood urea nitrogen, creatinine, and venous blood gas (i-STAT® 1 [Abbott Laboratories]). Blood cultures were obtained in all children with uncomplicated and cerebral malaria to rule out concomitant bacteremia. Lumbar puncture with opening pressure was performed in all patients with coma to evaluate for meningitis. Cerebrospinal fluid analysis included the following: glucose and protein levels; cell count with differential by trained microscopists; and bacterial culture.P. falciparum HRP-2 . Concentrations were derived from standard curves utilizing purified PfHRP-2 kindly provided by D Sullivan . Samples with ODs outside the linear part of the curve were repeated at an appropriate dilution until an accurate concentration was determined. The lower limit of detection was 1.5 ng/mL.Plasma PfHRP-2 was measured by ELISA as previously described Children with uncomplicated malaria and cerebral malaria received anti-malarial therapy and other supportive care as per standard Tanzanian national protocols . Treatment was initiated as soon as the diagnosis of malaria was suspected. Children with cerebral malaria were re-examined daily until death or hospital discharge.Statistical analysis was performed using SAS (version 9.2). Results are presented as mean with 95% confidence intervals for normally distributed continuous variables or median with interquartile range for variables with non-parametric distribution. Non-parametric tests and natural log transformation of variables were used when the assumption of normality was violated. Differences between PfHRP-2 values in uncomplicated malaria and cerebral malaria cases were compared using Wilcoxon rank-sum scores two-sample test. Further comparisons of PfHRP-2 values between uncomplicated malaria, cerebral malaria survivors, and cerebral malaria fatal cases were performed using a Kruskal-Wallis test as well as the Wilcoxon rank-sum tests with the Holm’s Step Down Procedure for partitioning alpha. Differences between normally distributed continuous variables were compared using Student’s t-test. Correlation between variables was evaluated using Spearman’s correlation coefficients. In the cerebral malaria group, logistic regression was used to determine the association between death and plasma PfHRP-2 concentrations and clinical variables previously shown to be associated with mortality (coma score and plasma bicarbonate) We screened 227 febrile children for uncomplicated malaria between November 2007 and January 2011. Of these, 67 were consented, six were excluded because they did not meet inclusion/exclusion criteria, leaving 61 enrolled participants . We screened 153 febrile children with central nervous system conditions (mostly stupor or coma) for cerebral malaria. Fifty-one met inclusion criteria for cerebral malaria and were consented. Of these, six were excluded, leaving 45 enrollees . Baseline characteristics comparing the two groups are shown in The median concentration of plasma PfHRP-2 in cerebral malaria was significantly higher than in uncomplicated malaria  . One patOn univariate analysis, natural log plasma PfHRP-2 concentration correlated with lower Blantyre Coma Score  and with hemoglobin concentration , but not with plasma creatinine . Natural log plasma PfHRP-2 concentration correlated with the natural log peripheral parasitemia in uncomplicated malaria , but did not correlate with natural log peripheral parasitemia in cerebral malaria  or in an aggregate analysis of all patients enrolled  .Seven children died, all of whom had cerebral malaria. Median plasma PfHRP-2 concentrations in those with fatal outcomes (4000 ng/mL [IQR 1194.5–5287.3 ng/mL]) were higher than in those who survived (926.1 ng/mL [IQR 330.7–2028.3]) . A comparison of plasma PfHRP-2 values between uncomplicated malaria cases, cerebral malaria survivors, cerebral malaria fatal cases found a significant difference among the three groups . In patients with cerebral malaria, increased natural log plasma PfHRP-2 was associated with an increased risk of death on univariate analysis . Coma score and plasma bicarbonate were not significantly associated with death. In multivariate models natural log PfHRP-2 remained significantly associated with death (p = 0.04) while natural log peripheral parasitemia and plasma bicarbonate did not .Plasma concentration of PfHRP-2, a measure of total parasite biomass, was associated with malaria severity, depth of coma and mortality in Tanzanian children living in an area with mesoendemic malaria transmission. This supports similar findings in adults from areas of relatively unstable transmission in Asia and Indonesian Papua Two previous studies have measured plasma PfHRP-2 in children with severe malaria. A small Kenyan study did not relate PfHRP-2 with disease severity or outcome P. falciparum parasitemia, higher levels of host neutralizing antibody to PfHRP-2, or a dysregulated host inflammatory response despite low burden of infection. Another limitation of our study is the relatively small sample size, which could lead to a type I error (i.e. rejecting the null hypothesis when in fact the null hypothesis is true).Strengths of our study include rigorous exclusion of bacteremia and meningitis that may mimic severe malaria. A limitation lies in the reliability of the WHO clinical case definition for cerebral malaria. An autopsy study (without blood culture exclusion of bacteremia) found that the clinical definition has a specificity of only 77% We conclude that plasma PfHRP-2 is associated with malaria severity and mortality in Tanzanian children from a mesoendemic area of transmission. Although further studies are needed in other areas of Africa with higher malaria transmission and in children with different clinical manifestations of severe malaria, our findings support the utility of plasma PfHRP-2 in clinical research to estimate the total parasite biomass in falciparum malaria, in children as well as adults.