The presence of malaria parasites and histopathological changes in the placenta are associated with a reduction in birth weight, principally due to intrauterine growth restriction. The aim of this study was to examine the feasibility of studying early pregnancy placental volumes using three-dimensional (3D) ultrasound in a malaria endemic area, as a small volume in the second trimester may be an indicator of intra-uterine growth restriction and placental insufficiency.Placenta volumes were acquired using a portable ultrasound machine and a 3D ultrasound transducer and estimated using the Virtual Organ Computer-aided AnaLysis (VOCAL) image analysis software package. Intra-observer reliability and limits of agreement of the placenta volume measurements were calculated. Polynomial regression models for the mean and standard deviation as a function of gestational age for the placental volumes of uninfected women were created and tested. Based on these equations each measurement was converted into a z -score. The z-scores of the placental volumes of malaria infected and uninfected women were then compared.Plasmodium falciparum were below the 10th centile. The 95% intra-observer limits of agreement for first and second measurements were ± 37.0 mL and ± 25.4 mL at 30 degrees and 15 degrees rotation respectively.Eighty-four women  with a posterior placenta delivered congenitally normal, live born, single babies. The mean placental volumes in the uninfected women were modeled to fit 5th, 10th, 50th, 90th and 95th centiles for 14-24 weeks' gestation. Most placenta volumes in the infected women were below the 50th centile for gestational age; most of those with The new technique of 3D ultrasound volumetry of the placenta may be useful to improve our understanding of the pathophysiological constraints on foetal growth caused by malaria infection in early pregnancy. Falciparum and vivax malaria have a markedly negative impact on mothers and babies ,2. For eThe introduction of three-dimensional (3D) ultrasound has made it possible to assess intrauterine growth more accurately by allowing foetal organ and placental volumes to be measured ,15. For This relatively new technique has never been used to study placenta volumes in pregnancies complicated by malaria. The aim of this study was to examine the feasibility of studying early pregnancy placental volumes in a malaria endemic area.Plasmodium (P.) falciparum [P. vivax [The participants in the present study were attending the antenatal clinic (ANC) at Shoklo Malaria Research Unit (SMRU), which is located on the Thai-Burmese border. SMRU has focused on the epidemiology, prevention and treatment of malaria in pregnancy since 1986. The epidemiology of lciparum  and P. vP. vivax  malaria P. vivax . All womThe study was part of a larger foetal growth project , approved by the Ethics Committees of Oxford (OxTREC (14-08)) and Mahidol (TMEC 2008-028) Universities.i  with a RAB2-5-RS; 2-5 MHz/Real time 3D probe. The machine was housed in a dedicated air-conditioned room equipped with a voltage stabilizer. All scans were obtained by sonographers specifically trained in foetal growth scanning [st Project team at the University of Oxford [Ultrasound scans were performed trans-abdominally using a Voluson scanning , with ref Oxford .+0 and 13+6 weeks. The protocol and operating manual for obtaining CRL measurements were similar to those used in the International Fetal and Newborn Growth Consortium for the 21st Century (INTERGROWTH-21st) Project[Only women with singleton pregnancies were recruited. As the last menstrual period is largely unknown in this population , gestati) Project. Briefly) Project.Thereafter, women were invited to attend for a foetal growth scan every 5 weeks until delivery to take standard 2D foetal biometric measurements. If a woman did not attend a scheduled scan then she was scanned at the next available opportunity. Every woman with a malaria episode detected by peripheral smear was treated using WHO protocols  and had After standard 2D measurements were taken, a 3D sweep was obtained through the placenta. The 3D placental volume was acquired keeping the probe perpendicular to the placental plate and the size of the volume box was adapted to include the entire placenta. The sweep angle was set at 85° so as to visualize the maximum amount of placenta possible. Care was taken to minimize movement artifact. Once the scan was complete, volume data were stored on the system's hard drive for later analysis.+0 to 23+6 weeks (two sets of five weeks) were analysed. This approach was chosen because most placentas  are too large to fit in a single 3D sweep after 20 weeks. Based on published recommendations for cross-sectional studies, only one measurement per woman was included in the analysis [Only 3D ultrasound images of the placenta on the posterior uterine wall taken between 14analysis ,31; if mOne author (WEM), blinded to clinical data, performed the volume measurements using the VOCAL™ method as described by Wegrzyn et al . A sequeIntra-observer reliability and limits of agreement (mean difference ± 1.96 SD) of the placenta volume measurements were calculated as described by Bland and Altman . SystemiFrom the 15° measurements, separate polynomial regression models were fitted for the mean and standard deviation (SD), each as a function of GA , of the where K is the corresponding centile from the theoretical Gaussian distribution .Based on these equations the placental measurements of all women were converted into a z-score:The advantage of using z-scores is that it eliminates the variability of measurements by GA, which allows for direct comparability of measurements. A negative z-score denotes a placental volume that is smaller than the expected mean for the population at a given GA. The z-scores of women who were or were not infected with malaria before the scan were compared.Clinical data were entered using Microsoft Access, and analysed using SPSS version 15.0 for Windows  and STATA version 11. The student's t-test was used to compare means, the Shapiro-Wilk test-to-test normality.Between 10 February 2009 and 30 April 2010, a total of 250 women were recruited at a mean gestational age of 80 ± 10 (SD) days. Of these women, 84 (33.6%) were eligible for placental volume analysis as defined in the Methods section  mL at 30°, and 2.5 (95% CI: 0.3-4.6) mL at 15°. The 95% intra-observer limits of agreement for first and second measurements were ± 37.0 mL at 30° and ± 25.4 mL at 15°  Mean volume = - 162.83 + 3.60 × GAb) SD = - 41.55 + 0.80 × GAp = 0.36.A simple linear model with no quadratic or cubic terms for both the mean and SD provided the best fit for the model with a deviance of 717.98. The z-scores from the model did not suggest any deviation from normality using the Shapiro-Wilk test, P. falciparum and 15 (79%) with P. vivax. The baseline characteristics of infected and uninfected women were similar, except for a lower mean haematocrit at the time of the volume scan, as expected following infection with malaria: 31.2 ± 2.7% versus 32.6 ± 2.8% respectively, p = 0.05 , and one woman had one P. vivax and one P. falciparum episode and was classified as P. falciparum. The median time between the last episode of malaria and the volume scan was 29  days and this was not different for the P. falciparum malaria or P. vivax malaria group. Figure +0 and 23+6 weeks obtained from the uninfected women (green dots). Most of the placental volumes of the infected women as a whole were below the 50th centile; most of those with P. falciparum were below the 10th centile  with % with P.P. falciparum infections, p = 0.003  and those who had other infections in pregnancy Table . HoweverThis pilot study demonstrates the feasibility of a) relating placental volume to gestational age between 14 and 24 weeks in a malaria endemic area, and b) comparing volumes between women with and without malaria. The findings are important because measurement of placental volume in the first or second trimester may predict which pregnancies are at high-risk of adverse outcomes ,36-38.A variety of 2D and 3D ultrasound methods to measure placental volume have been reported ,36,39,41Accuracy is also influenced by the rotation angle of the analysis method. In this study, a small error was observed between the volume measurements when comparing the two rotation methods, 30° and 15° Figure . The 30°As in studies of foetal organ volumetry , placentP. falciparum before 24 weeks' gestation appears to be associated with smaller placental volumes. In other studies, principally in developed countries, early placental volumetry has been shown to predict IUGR and adverse pregnancy outcomes [In this preliminary investigation, infection with outcomes ,48, due outcomes . All theoutcomes ,50,51.P. vivax did not seem to be related to placenta volume. The mechanisms underlying the adverse effects of P. vivax malaria in pregnancy are not fully understood [P. vivax-related growth restriction, as there is very little evidence that P. vivax sequesters in the placenta, as P. falciparum does [Infection with derstood ,52,53. SThere was a borderline difference in haematocrit at the time of the placental volume scan between the groups Table , howeverThis study, therefore, suggests that 3D placental volumetry is worthy of further investigation, in order to assess whether IUGR related to malaria is mediated via a smaller placental volume.The data presented here involve small numbers and should be interpreted cautiously. Another limitation of the study was the initial decision to include only placentas on the posterior wall to maximize the likelihood of capturing the whole target organ in the volume sweep. This enabled the inclusion of complete placental volumes up until 24 weeks' gestation, but resulted in a large group of women with an anterior placenta being excluded (n = 133, Figure Generally, ultrasound machines with 3D measurement capacity are delicate, expensive, and require a high level of technical skills and are therefore not available in most malaria endemic areas. Nevertheless, the suggestion that malaria in early pregnancy reduces placental volume in the second trimester should be confirmed by prospective studies evaluating volumes in relation to foetal growth and adverse pregnancy outcomes. Such studies should also allow for better assessment of potential confounders (such as maternal anaemia ,54, smokIn conclusion, the new technique of 3D ultrasound volumetry of the placenta may be useful to improve our understanding of the pathophysiological constraints on foetal growth caused by malaria infection in early pregnancy.The authors declare that they have no competing interests.MJR designed the study, carried out the ultrasound scanning, performed the statistical analysis and drafted the manuscript. WEM performed the volume measurements, helped with the statistical analysis and drafted the manuscript. SK carried out the ultrasound scanning and organized the study on site. GS helped with the volume measurements and organized the database. EO performed the statistical analysis and helped to draft the manuscript. JAN participated in the design and coordination of the study. SHK participated in its design and coordination of the study. RMG participated in the design and coordination of the study, helped in the statistical analysis and helped to draft the manuscript. ATP participated in the design and coordination of the study, helped in the statistical analysis and helped to draft the manuscript. FN conceived of the study and participated in the design and coordination of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.


Rapid diagnostic test (RDT) is becoming an alternative way of establishing quickly the diagnosis of malaria infections, by detecting specific malaria antigens in suspected patients’ blood between the China-Myanmar endemic borders areas, towards achieving the National Malaria Elimination programme by 2020. The objective of this study is to evaluate the performance of CareStart™ Malaria Pf/Pan RDT kit for the diagnosis of malaria infections in suspected patients. Blood examination by microscopy was taken as gold standard to evaluate CareStart™ kit’s sensitivity, specificity and predictive value and corrected with PCR assay.falciparum malaria were 88.52% and 98.26%, and for vivax malaria: 90.77% and 100%. The CareStart™ positive predictive values were 98.26%  compared to 100%  for PCR-corrected, and the negative predictive values of 89.68%  were the same in microscopy as PCR-corrected. The diagnostic accuracy of CareStart™ kit versus microscopy and PCR were 93.78%  and 94.61%  respectively. The likelihood of diagnostic of malaria positive was almost similar between microscopy and CareStart™ kit, with an entropy reduction of 60.0% compared to a weak likelihood of misdiagnosis of 0.10 , with an entropy reduction of 36.01%.Overall 126 of 241 (52.28%) malaria cases were detected by microscopy compared to 115 of 241(47.72%) CareStart™ kit and 128 of 241 (53.11%) PCR corrected assay. CareStart™ kit’s sensitivity and specificity for the diagnosis of malaria were 89.68% and 98.26% respectively, compared to standard microscopy, whereas the sensitivity and specificity for The accuracy of CareStart™ kit is comparable to gold standard microscopy in these areas, it is easy to perform and suitable for cross-border diagnosis and monitoring of local or imported malaria patterns by any local health staff in endemic remotes. P.f/P.v Combo province, Southeast China. Within Yunnan, malaria occurs mainly at the border areas, in the drainage areas of the Yuanjiang River and amongst the high population of motile ethnic minority groups at risk living in forests and on forest fringes for economic reasons, on the China-Myanmar borders [Malaria remains unevenly endemic across Yunnan The CareStart™  borders . While p borders .Although malaria cases and deaths have dropped substantially over the past decade, efforts must be intensified in the malaria-endemic communities to abolish the public threat of malaria by prompt diagnosis and effective treatment . HoweverPlasmodium vivax and Plasmodium falciparum, whilst monitoring the indicators for malaria elimination [P. falciparum-specific tests generally have greater sensitivity (over 90%) than the pLDH-based tests when compared with microscopy in clinical cases, whilst sensitivity of pLDH tests for non-P. falciparum species was low [P.f/P.v combo test (detecting histidine-rich protein (pHRP-2) and P. vivax-specific lactate dehydrogenase (pLDH) has been evaluated in a few field settings, including higher sensitivities for P. falciparum (99.4%) reported in Ethiopia with P. falciparum samples with parasite densities above 100/μl, [P. falciparum low values at parasite densities <100/μl (60.0%) and increasing sensitivity at higher parasite densities (100% at >500/μl) reported in Madagascar [P. vivax were significantly higher than those found in Ethiopia with pLDH (pan) (91.0%), but similar results for the CareStart™ malaria pLDH (pan/Pf) reported in Myanmar [P. falciparum ranging from 87.5-99.0% [P. vivax, RDTs detecting pan-pLDH showed sensitivities ranging from 62.0-95.0%, [P. falciparum) and 500/μl (P. vivax) [Technical support for malaria prevention and control in China is provided at national level by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (CDC), based in Shanghai. The strategies of the National Malaria Elimination programme 2010–2020 include greater access to early diagnosis and prompt treatment coverage in remote areas, free distribution of long-lasting, insecticide-treated bed nets (LLINs), strengthening quality and regulatory mechanisms for anti-malarial drugs, strengthening malaria surveillance and information systems, epidemic preparedness, increasing the awareness of malaria, involvement and empowerment, basic and applied research on mination . Moreovemination  The use mination . There amination . The var was low ,7. Speci was low -10. The  100/μl, , sensitidagascar , and sen Myanmar . Similar <100/μl 0.0% and .5-99.0%  and for 0-95.0%,  compared0-95.0%, . The inc <100/μl 0.0% and . vivax) ,17,18.The aim of the study was to evaluate the performance of CareStart™ malaria Pf/pan test kit in early diagnosis for prompt effective case management of malaria.The CareStart™ malaria HRP-2/pLDH (Pf/pan) combo test was evaluated in suspected patients attending TengChong CDC, China and Health Unlimited clinic in Laiza City, Kachin State, Myanmar, near the China-Myanmar border. Standard microscopy method was done for all thick smears corrected by polymerase chain reaction (PCR) assay. The assessment of the diagnostic performance was performed in comparison the CareStart™ (CS) kit with gold standard microscopic results, corrected by PCR assay.The study was conducted in July to December, 2011 in TengChong CDC, China and Health Unlimited clinic in Laiza City, Kachin State, Myanmar, near the China-Myanmar border on all suspected patients. Individual biodata and malaria history in the previous one year were documented from each suspected case as well as an informed consent. All patients had venous blood samples collection for microscopy and CS kit evaluation and also samples were collected in EDTA and stored at −20°C condition for PCR analysis.The study was reviewed and approved by the Ethical Committee of Yunnan Institute of Parasitic Diseases, Yunnan province and Health Ministry of Kachin State of Myanmar. All patients provided informed consent before admission into the study.Duplicate thin and thick slides were made from all blood, stained using 5% Giemsa stain and dyed for 30 minutes, read under x100 electron microscopy. The malaria microscopic examination was performed by two independent experienced microscopists, for identification of the malaria parasite species. The number of parasites was counted against 200 leucocytes and quantification of parasite density was estimated by assuming 8,000 leucocytes/μl blood. Samples were considered negative when no parasite was detected after examining 100 microscopic fields . In ordePlasmodium species  and the other line (test line 1) consists of a monoclonal antibody specific to histidine-rich protein 2 (HRP2) of the P. falciparum species. The conjugate pad is dispensed with monoclonal antibodies, which are pan-specific to pLDH and P. falciparum specific to HRP2. The CS RDT kit is designed for the differential diagnosis between P. falciparum and other pan-specific species. The CS RDT kit was supplied by the China-England Advanced Communication Project and is produced by American Access Bio Company; batch number C201R, period of validity to February 2012. The test reproducibility was evaluated by testing 15 samples representing all species at variable parasite densities on three consecutive occasions in Teng Chong CDC.The CS RDT kit contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line 2) is pan-specific to lactate dehydrogenase (pLDH) of the Tests were performed according to manufacturer’s instructions. Readings were carried out at daylight, assisted by a standard electric bulb, by three health worker-observers performing and interpreting the test results. The first observer performed readings at 20 minutes, reading time recommended by the manufacturer, followed by observers 2 and 3 within the next 10 minutes. The observers were blinded to each other’s readings and to the results of microscopy and PCR assay.In the case when no control line appeared, the test was considered invalid and was repeated. To score line intensities, a scoring system of five categories was used as defined previously : none  had P. falciparum, and 4.76% (six of 126) mixed infections of P. vivax and P. falciparum. There were only two cases of missed diagnosis observed by microscopy.A total of 241 blood samples from suspected patients were analysed for malaria parasites by the CS RDT kit and the results were compared with results from gold standard microscopy, corrected by PCR assay. The male: female ratio was 3.54:1. The mean age was 29.62 ± 11.21 years (range 3–58 years old) of which the majority 89.21% (215) were older than 18 years and only four patients (1.66%) were under five years. The microscopic results indicated that 52.28% (126 of 241) of patients were infected with malaria. Among the malaria-positive patients, 51.58% (65 of 126) harboured The geometric mean parasite density was 5,990, ranged  positive cases initially misdiagnosed by microscopy were identified by PCR assays and CS RDT kit. There was no significant difference between the sensitivity of PCR and standard microscopy, however the specificity of PCR assay was 100% against 98.26% for microscopy. The diagnostic accuracies of both diagnostic methods were comparable. The likelihood of ratio of a positive test and negative test were 51.57 (19.32-137.7) and 0.11  respectively. The negative entropy after a positive microscopic test was 60.44%, whereas the microscopy and PCR entropy reduction after a negative test were 36.1% and 35.96% respectively. There was no significant difference between the Cohen’s kappa values of microscopy compared to PCR assay (see Table P. vivax represented 51.30% (59/115), P. falciparum or P. falciparum mixed other species was present 48.70% (56/115). Two cases microscopically negative were CS kit P. falciparum positive whereas 13 cases  originally detected by microscopy were not detected by CS RDT kit. The summary results of microscopy and CS kit results are showed in Table The CS kit results indicated that 47.72% (115/241) of the patients were infected with malaria. Among the positive samples, malaria species identified showed that P.f/P.v Combo test sensitivity ranged between 88.55 - 90.77% and specificity ranged 98.26 - 100% (95%CI) when compared to gold standard microscopic blood films for detection of malaria. The mean CS malaria P.v/P.f rapid kit sensitivity was 89.68%  and mean specificity was 98.26% . The positive predictive value was 98.26%  and negative predictive value of 89.68%  when compared to traditional blood films for falciparum and vivax malaria detection. The diagnostic accuracy of CS compared to microscopy was 93.78% (89.99-96.19%)  were falciparum against the predominant 50.72% (65/128) vivax malaria in the areas. In contrast, CS diagnosis had a lower specificity to P. vivax of 98.26%, but same specificity for P. falciparum when compared with corrected PCR assay results, as shown in Table Real-time PCR for diagnosis of malaria, and identification of parasite species showed a difference in  49.22% 6/128 wereP. falciparum was 98.3% and 99.3% in samples with parasite densities above 100/μl and 1,000/μl respectively whereas the sensitivity for P. vivax was 97.6% for parasite densities above 500/μl. No positive result occurred among the Plasmodium-negative samples, which is consistent with the recent study of CS kit in Ethiopia [P. vivax-specific pLDH CS kit based on specific monoclonal antibody, detected histidine-rich protein 2 and four kinds of lactic acid dehydrogenase of Plasmodium in human blood  in a card detection device, aseptic and single packing. The comparison of the sensitivity and specificity of the CS-RDTs versus microscopy corrected by PCR assay  13/126 documented in the study can be explained only partly by the CS threshold level of >100 parasites/μl and probably the CS storage conditions and duration in the field resulted in loss of sensitivity, since the quality assurance and control testing was 100%.As shown in Tables P.f/P.v Combo (detecting HRP-2 and P. vivax-specific pLDH) test has been evaluated in Ethiopia [P. falciparum (99.4%). No clear reason could be given for increasing sensitivity and possibly included exclusively P. falciparum samples with parasite densities above 100/μl, which is above the detection threshold of most RDTs [P. falciparum low values at parasite densities <100/μl (60.0%) and increasing sensitivity at higher parasite densities (100% at >500/μl) [The CareStart™ Ethiopia -25 compaost RDTs . Also, C>500/μl) .P. vivax were significantly higher than those found in Ethiopia with pLDH (pan) (91.0%), but results were similar for the CareStart™ malaria pLDH (pan/Pf) [The two products  combo test) and CareStart™ brand ), evaluated in Myanmar, reported sensitivities for the detection of (pan/Pf) -25.P. falciparum by the RDT, but negative by microscopy [A study in The Philippines compared the results of the ICT malaria P.f/P.v RDT with microscopy and found a high discordant rate involving cases positive for croscopy .P. falciparum that are comparable to the present study, but there were only nine P. vivax samples included, making comparison difficult [The CareStart™ malaria pLDH (Pf/pan) combo test field study in Madagascar reported sensitivities for ifficult . Moreoveifficult .P. vivax and P. falciparum positive and negative predictive values of CS were 100% and 94.96% compared to 100% and 94.17% respectively. In addition, P. vivax diagnostic accuracy of CS was 96.67% compared to 94.89% for P. falciparum. The differential explanation could be the lack of differentiation of other species of Plasmodium species as reported in Thailand [When PCR was the reference method, the microscopic method showed a low specificity (88.2%). The P. vivax were significantly higher than those found in the present study in case of the CareStart™ malaria pLDH (Pf/pan) (88.52%), but for the CareStart™ malaria pLDH (Pv/pan) they were in line with the previous findings (90.77%) [This study recorded two misdiagnosed microscopic cases, corrected using PCR assay, whereas 13 cases were not detected by CS kit. The reported sensitivities for the detection of (90.77%) ,11.P. falciparum where they ranged from 87.5-99.0%, with one exception of 76.2%, whereas for P. vivax, RDTs detecting pan-pLDH showed sensitivities of 33.5% and 62.0%-95.0% [Studies evaluating other RDTs in non-endemic countries report similar sensitivities as those found for the CareStart™ malaria HRP-2/pLDH (Pf/pan) combo test in this study for 0%-95.0% .The specificity and sensitivity of CS kit were 89.68% and 98.26%, a difference not statistically significant than previous CS results in southwest Ethiopia  combo test performs satisfactory well for the detection of Plasmodium-specific parasite lactate dehydrogenase; PCR: Polymerase chain reaction; CI: Confidence interval; FIND: Foundation for innovative new diagnostics; Pf-pLDH:Plasmodium falciparum-specific parasite lactate dehydrogenase; pLDH: Plasmodium-specific parasite lactate dehydrogenase; Pv-pLDH: Plasmodium vivax-specific parasite lactate dehydrogenase; RDTs: Rapid diagnostic test; WHO: World health organization.CS: CareStart™; HRP-2: Histidine-rich protein 2; pan-pLDH: Pan The authors declare that they have no competing interests.ET, SX and XZ participated in the design of the study, execution, statistical analysis and drafted the manuscript. WC, CZ, ET and DY performed the PCR assays. WJ, WJ collected the blood sample. All authors read and approved the manuscript.


Malaria, which frequently occurs in pregnant women in the tropics, is a leading cause of maternal anaemia and low birth weight (LBW) in infants. Few data exist concerning malaria infections that are present at submicroscopic levels during pregnancy and their LBW delivery in babies.A case–control study (87 in each group) was conducted at the Medani Hospital, Central Sudan. Cases were women who had LBW deliveries where the infants weighed < 2,500 g. Controls were parturient women without having LBW babies. Obstetrical and medical characteristics were gathered from both groups through structured questionnaires. Both cases and controls were investigated for malaria using microscopic blood film analysis, placental histology and polymerase chain reaction (PCR). Microscopic and PCR analyses were conducted on maternal peripheral blood, placenta, and umbilical cord samples. Infant weights were recorded immediately after birth.Plasmodium falciparum-positive blood films were not obtained from any of the women (cases or controls). Twenty-seven (31.0%) versus 22 (25.3%) (P = 0.500) of the cases and controls, respectively, had placental malaria infections as determined by histological examination. In comparison to the controls, the submicroscopic malaria infection prevalence rates were significantly higher in the cases; 24 (27.6%) vs six (7.0%), P < 0.001. Multivariate analysis showed that while malaria infection of the placenta (based on histology) was not associated with LBW, submicroscopic P. falciparum infection , or a combination of histologically determined and submicroscopic infections , were significantly associated with LBW.In Central Sudan, pregnant women were at a higher risk of having an LBW delivery if they had submicroscopic infections rather than a histological diagnosis of placental malaria. Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) variant surface antigen occurs, there was no significant difference in the body mass index [23.8 (3.3) vs 24.3 (2.2) kg/m2] in the LBW delivery vs the control women, respectively. The bed net coverage was low in both groups, but there was no statistically significant difference  were of LBW babies. Eighty-seven of these women with LBW deliveries fulfilled the inclusion criteria and had complete data, including placental histology and PCR diagnoses, and were, therefore, included in the final analyses. Such data were compared with an equal number of controls with complete data. There were no significant differences between the two groups (case or control) in their level of education, residence, antenatal care attendance and other socio-demographic characteristics  vs 22 (25.3%) (P = 0.500) of the cases vs controls had placental malaria infections on histological examination. Three (3.4%), one (1.1%) and 23 (26.4%) vs two (2.3%), two (2.3%) and 18 (20.7%) of the placentae showed acute, chronic and past infection on histopathology examination in the two groups (case–control), respectively, while 60 (69.0.4%) vs 65 (74.7%) of them showed no signs of infection; P = 0.500, ; P < 0.001. None (0%), 19 (21.8%) and six (7.0%) vs four (4.6%), two (2.3%) and none (0%) were maternal, placental or cord submicroscopic malaria infections in the two study groups, respectively. One case had a placental and umbilical cord submicroscopic malaria infection, Table In comparison to the controls, the prevalence of submicroscopic malaria infection was highly significant in the cases; 24 (27.6%) vs 26 (30.0%), P = 0.002. Out of these malaria infections, six  had histologically positive placental malaria infections as well as submicroscopic malaria infections ; 46 (53.0%)  Figure .vs 3,319.2 (358) g, P < 0.001 in the cases and controls, respectively. While there was no significant difference in the mean (SD) of the birth weight between those who had placental malaria infections based on histology  and those who did not [2843.0 (601.0) vs 2834.7 (533.0) g], the mean (SD) of the birth weights was significantly lower in those who had submicroscopic malaria infection  than in those who had no evidence of a submicroscopic malaria infection [2551.7 (497.0) vs 2896.9 (545.0) g]  of birth weight was 2,387.2 (152) P. falciparum infection (by histology) was not associated with LBW, submicroscopic infections with this parasite  and all P. falciparum infections  were significantly associated with LBW ,  between the two groups, significantly higher numbers of the cases had submicroscopic malaria infections than the controls . While placental malaria infections that were positive by histology were not associated with LBW, submicroscopic malaria infections were, and this was a statistically significant finding. In fact, the impetus for this study arose out of the knowledge that two cross-sectional studies in Eastern Sudan failed to show significant associations between LBW delivery and placental malaria infection, as diagnosed by histology[The main findings of the current study were as follows. While there was no significant difference in the prevalence of placental malaria by histological examination [Interestingly, in the current study only six of the malaria infections (28.0%) had both placental malaria infections (histology) and submicroscopic malaria infections. The performance of PCR l Sudan). The lowl Sudan).P. falciparum infections had a 13-fold higher risk of LBW delivery compared with non-infected pregnant women[In the current study, while women with placental malaria infections (confirmed by histology) were not at risk of having an LBW delivery, women who had submicroscopic malaria infections had a seven-fold higher risk of such an event. In addition, birth weights were significantly lower in women who had submicroscopic malaria infections. Interestingly, it has been shown that pregnant women in Burkina Faso with submicroscopic malaria infections delivered infants that weighed significantly less than those delivered by women with no placental infections. Yet in the same study, delivery of LBW babies was not more common among women with submicroscopic placental malaria parasitaemia than those women without malaria. In Gabont women. Remarkant women.P. falciparum infection and LBW was observed.Conversely, the current study identified an association between submicroscopic malaria and LBW; this contrasts with previous reports from Malawi,31 and GP. falciparum malaria on pregnancy outcomes such as anaemia. This is because it was designed to investigate LBW through a case–control study; another study is needed if the effects of submicroscopic P. falciparum malaria on haemoglobin are to be investigated. The second limitation is the sample size, where the current study failed to yield enough maternal, placental and umbilical cord submicroscopic P. falciparum malaria infections to enable parasite genotyping to be conducted. Therefore, a larger cross-sectional study is needed to address the effect on haemoglobin and parasite genotyping.One of the limitations of the current study was the inability, by its design, to investigate the other effects of submicroscopic In Central Sudan, pregnant women were at higher risk of having an LBW delivery if they had submicroscopic infections rather than a diagnosis of placental malaria based on histology.The authors declare that they have no competing interests.AHM and IA coordinated and carried out the study, and participated in the statistical analysis and procedures. EME and BBE participated in the clinical work and statistical analysis. MMS, AAM and SEE conducted the laboratory work. All the authors have read and approved the final version of this manuscript.


Plasmodium falciparum, has gradually expanded in last three decades [Plasmodium falciparum exhibits resistance to their drug. Resistance to the combination of sulfadoxine-pyrimethamine was also already emerged [Plasmodium falciparum were investigated experimentally. The oxygen distribution and the morphological shape of farnesyltransferase inhibitor-treated mitochondria were examined under in vitro condition. From this study, we found farnesyltransferase inhibitor is very important to understand the mitochondrial function of Plasmodium falciparum as an antimalarial drug.Malaria is one of the world’s public health problems in terms of medical emergency with a high risk of mortality. The protozoan malaria parasites are transmitted by infected female mosquitoes . The mos decades . Choloro emerged . Farnesy emerged ,4. There emerged . In addiCulture of malaria parasites.Plasmodium falciparum.Synchronization of Determination of oxygen gradients in malaria.Plasmodium falciparum. Farnesyltransferase inhibitor has noticeable effects on mitochondrial function of malaria parasites, compared to the control case for non-infected RBCs.In this study, farnesyltransferase inhibitor was treated to RBCs (Red blood cells) uninfected by in vitro condition. The farnesyltransferase inhibitor was observed to be very important to understand the mitochondrial function of malaria parasite as an effective antimalarial drug.Oxygen distribution and morphological shape of farnesyltransferase inhibitor-treated mitochondria were investigated under


Plasmodium vivax has been considered rarely threatening to life, but recent studies challenge this notion. This study documented the frequency and character of severe illness in paediatric patients admitted to a hospital in south-eastern Pakistan with a laboratory-confirmed diagnosis of vivax malaria.Infection by P. vivax; 48 Plasmodium falciparum; and four mixed infections of these species. Patients were classified as having severe illness with any of the following indicators: Glascow coma scale <11; ≥2 convulsions; haemoglobin <5g/dL; thrombocytes <50,000/mL; blood glucose <45mg%; >70 breaths/min; or intravenous anti-malarial therapy. Additionally, 64 patients with a diagnosis of vivax malaria were treated during 2009, and the 21 of these having severe illness were included in analyses of the frequency and character of severe illness with that diagnosis.An observational study of all 180 paediatric patients admitted with any diagnosis of malaria during 2010 was conducted: 128 P. vivax or P. falciparum were classified as having severe disease. Including the 2009 records of 64 patients having vivax malaria, a total of 60 (31%) patients with severe illness and a diagnosis of P. vivax were available. Altered mental status  dominated at 54% of the 83 indicators of severe illness manifest among the patients with vivax malaria, as was true among the 37 children with a diagnosis of falciparum malaria and being severely ill; 58% of the 72 indicators of severe disease documented among them. No statistically significant difference appeared in frequencies of any other severe disease indicators between patients diagnosed with vivax or falciparum malaria. Despite such similarities, a diagnosis of falciparum malaria nonetheless came with 3.8-fold (95% CI = 1.8-8.1) higher risk of presenting with severe illness, and 8.0-fold (95% CI = 2.1-31) greater likelihood of presenting with three or more severe disease indicators. Two patients did not survive hospitalization, one each with a diagnosis of falciparum or vivax malaria.During 2010, 39 (31%) or 37 (77%) patients with a diagnosis of Vivax malaria caused a substantial burden of potentially life-threatening morbidity on a paediatric ward in a hospital in south-eastern Pakistan. As a general rule vivax infections exhibit relatively benign characteristics…instances of death  due to this parasite alone must indeed be rare.” This remained the prevailing view of malariologists over the ensuing decades. Only recently has evidence emerged suggesting that, on the contrary, life-threatening illness with a diagnosis of vivax malaria may often be encountered in many endemic settings [Plasmodium vivax represents a conspicuously important global health issue.In Russell’s 1949 text of malariology [settings -6. Kitchsettings ,8, the fP. vivax have long been noted [Plasmodium falciparum in the same population.Endemic falciparum and vivax malaria occur in Pakistan . At a hoen noted . PerhapsLiaquat University Hospital (LUH), affiliated with Liaquat University of Medical and Health Sciences, is a 1,600-bed private teaching hospital in Hyderabad, Sindh Province, Pakistan. The LUH is the primary referral hospital for the city of Hyderabad and surrounding rural communities. In 2009 the hospital admitted 2,956 adult patients (at least 17 years of age) and 6,395 paediatric patients. Malaria accounted for 433 (15%) and 148 (2.3%) of adult and paediatric admissions, respectively. Most adults had falciparum malaria (72%), whereas children had approximately equal shares of falciparum (57%) and vivax (43%) malaria.P. vivax mono-infection and classified as having severe illness ; and 2) observational study of the records of 176 patients diagnosed with either P. falciparum or P. vivax mono-infections and treated in the paediatric department (April to December 2010). All data were gleaned from records of routine patient care. A protocol describing these studies was reviewed and approved by the ethics review board of Liaquat University of Medical and Health Sciences.Two paediatric patient populations composed distinct analytical approaches to the studies undertaken: 1) a retrospective case series of 21 patients diagnosed with P. vivax, along with 10 others diagnosed as having severe illness and P. falciparum, were evaluated by expert microscopists and by standard nested PCR [P. vivax (July to October 2009) were evaluated by expert microscopy at the Eijkman-Oxford Clinical Research Unit in Indonesia. Two certified  <11; ≥2 episodes of convulsions; haemoglobin level <5 g/dL; respiratory rate >70 breaths/min; thrombocytes <50,000/mL; blood glucose level <45mg%; or the administration of anti-malarials intravenously. Patients exhibiting none of these features were classified as having uncomplicated malaria. Excepting intravenous antimalarial therapy, these criteria were extracted from the WHO criteria for classifying patients with a diagnosis of falciparum malaria as having severe, complicated, and life-threatening disease . We applP. vivax was confirmed by PCR in 12 of these children, and review by two expert microscopists among all 21. Twelve of those children suffered at least two convulsions (57%) and five had GCS <11 (24%), three of those patients having both. Eight patients (38%) had severe anaemia (<5g/dL), with two of those also having both ≥2 convulsions and GCS < 11. None of the patients exhibited respiratory distress , only one of 12 evaluated had severe thrombocytopaenia , another had hypoglycaemia (22mg%), and just one showed a bleed time >8min. All of these children recovered after treatment by either chloroquine syrup or quinine infusion.During 2009 the paediatric ward admitted 64 children with a diagnosis of vivax malaria Figure . AdditioP. falciparum and P. vivax; these patients were excluded from subsequent statistical analyses. Blood films from all of the 39 patients classified as having vivax malaria and severe illness yielded the same diagnosis from the two blinded reads by expert microscopists in Indonesia. Vivax malaria accounted for 71% of malaria cases evaluated, 51% of admissions for malaria, and 51% of cases classified as severely ill. Additional file P. vivax. Among the 13 of these managed as outpatients, 12 had severe thrombocytopaenia only, and the other had severe anaemia alone as consistent with the classification as severely ill. Three inpatients also had only severe thrombocytopaenia, but, in contrast to the relatively brief bleed times of those managed as outpatients, they all bled in excess of nine minutes. That observation prompted admission to hospital.Figure P. vivax- and P. falciparum-infected patients, no statistically significant differences emerged (P = 0.171-0.802). GCS < 11 and ≥2 convulsions dominated among severe disease syndromes. The proportion of patients classified with either or both of these syndromes was not significantly higher with a diagnosis of P. falciparum vs P. vivax .Figure P. vivax or P. falciparum. Vivax malaria came without severe disease much more frequently than falciparum malaria . Likewise, a diagnosis of P. falciparum more often came with three or more severe disease syndromes than with P. vivax . Expressed differently, a diagnosis of falciparum malaria was 3.8 times (95%CI = 1.8-8.1) more likely to be classified as severe (≥1 syndrome), and 8.0 times (95%CI = 2.1-31) more likely to involve more than two indicators of severe illness.Figure P. falciparum and one with P. vivax. The death with falciparum malaria occurred in a three month-old female admitted with GCS of 6, convulsive, respiratory distress (70b/min), and severe thrombocytopaenia . She died three days after admission. Death with diagnosis of vivax malaria occurred in a 12 year-old girl admitted with GCS of 10, convulsive, and hypoglycaemic (34mg%). She died seven days after admission.Two deaths attributed to malaria occurred during the study period, one with a diagnosis of Other studies in the South Asia region report somewhat similar findings in hospitalized paediatric populations. Among 103 children hospitalized with a diagnosis of vivax malaria at Bikaner, India, 65 (63%) were classified as having severe disease . A similThe observational studies described in this report have important limitations. A number of other endemic infections may have affected the patients in this study, but the laboratory capacities required to systemically assess these were lacking. Underlying disease, malnutrition for example, may represent important determinants of exacerbated vivax malaria but none were systematically assessed in these studies. Nonetheless, almost all of the patients in these studies recovered with anti-malarial therapies (few received presumptive antibiotics) and association between the confirmed diagnosis and clinical disease may be considered at least probable. Further prospective studies in endemic zones like south-eastern Pakistan supported by wider arrays of diagnostics for infections and other disease states are needed.P. vivax show little propensity for such adhesion [The means of classifying severe illness in this study represents another limitation with these studies. The standard WHO thresholds for disease severity derive from thorough statistical assessments of clinical and laboratory indicators associated with risk of fatal outcome with a diagnosis of falciparum malaria . No suchadhesion . NonetheP. falciparum carried significantly higher risks of more often severe and more complicated illness, a diagnosis of vivax malaria nonetheless accounted for 51% of the burden of severe illness attributed to malaria. The range and frequency of syndromes – dominated by altered mental status, severe thrombocytopaenia, and severe anaemia – were indistinguishable between patients with either diagnosis. The expressed caveat of Kitchen on the improbability of threatening disease with vivax malaria, i.e., “……”, may prove historically important as better understanding of vivax malaria as it occurs in endemic zones emerges, especially in children. Although relative risks varied between falciparum and vivax malaria, both of these diagnoses came with the same range of potentially life-threatening syndromes at very substantial rates with respect to overall burdens. A diagnosis of infection by P. vivax, regardless of what co-factors or determinants may come into play, often came with clinically threatening conditions in the patients evaluated in this study. The long-held view of vivax malaria as a benign infection may threaten patients by inviting inappropriately casual attention to its clinical management, research, and control. The commitment of just 3.1% of global funding for malaria research to P. vivax between 2007 and 2009 [Although a diagnosis of malaria caused by and 2009  suggest LUH: Liaquat University Hospital; NAMRU-2: U.S. Naval Medical Research Unit No.2; GCS: Glascow coma scale; WHO: World Health Organization.The authors declare that they have no competing interests.SS organized and supervised the collection of data from patient files. HM, AS, IA, and BI identified, collected, entered, and quality-assured the data from patient files. JKB advised on the collection, analysis, and reporting of the data. All authors contributed to writing the manuscript. All authors read and approved the final manuscript.Table S1. Case series summary of 21 patients classified as having severe disease with a diagnosis of vivax malaria during 2009.Click here for fileTable S2. Case series summary of 39 patients classified as having severe disease with a diagnosis of vivax malaria during 2010.Click here for file


The epidemiology of malaria in the Senegal River Gorgol valley, southern Mauritania, requires particular attention in the face of ongoing and predicted environmental and climate changes. While “malaria cases” are reported in health facilities throughout the year, past and current climatic and ecological conditions do not favour transmission in the dry season . Moreover, entomological investigations in neighbouring regions point to an absence of malaria transmission in mosquito vectors in the dry season. Because the clinical signs of malaria are non-specific and overlap with those of other diseases (e.g. acute respiratory infections and diarrhoea), new research is needed to better understand malaria transmission patterns in this region to improve adaptive, preventive and curative measures.Plasmodium spp. parasite rates were tested among children aged 6–59 months who were recruited from a random selection of households using a rapid diagnostic test and microscopic examination of Giemsa-stained thick and thin blood films. Acute respiratory infection and diarrhoea were the two other diseases investigated, administering a parental questionnaire to determine the reported prevalence among participating children.We conducted a multipurpose cross-sectional survey in the city of Kaédi in April 2011 (dry season), assessing three major disease patterns, including malaria. Plasmodium infection was found in any of the 371 surveyed preschool-aged children using two different diagnostic methods. Acute respiratory infections and diarrhoea were reported in 43.4% and 35.0% of the participants, respectively. About two thirds of the children with acute respiratory infections and diarrhoea required medical follow-up by a health worker.No Malaria was absent in the present dry season survey in the capital of the Gorgol valley of Mauritania, while acute respiratory infections and diarrhea were highly prevalent. Surveys should be repeated towards the end of rainy season, which will enhance our understanding of the potential changes in malaria transmission in a region known as ‘hot spot’ of predicted climate change. Plasmodium falciparum[Malaria continues to be a major health challenge globally with an estimated 655,000 to 1.2 million deaths in 2010 [alciparum. The natalciparum, and alsalciparum,4. Sincealciparum,5. ConveAnopheles arabiensis as the predominant vector species in the region, while An. pharoensis and An. funestus were reported in close proximity to irrigation schemes [P. falciparum parasite rates (PfPR) are scarce and conflicting [Recent entomological investigations have identified  schemes . However schemes ,4, furth schemes  and repoflicting ,9. WhethPlasmodium parasitaemia rates in the human population in the dry season, comparisons can be made with entomological studies and laboratory data that could contribute to a better understanding of malaria transmission in the Gorgol valley. Of note, most published studies in the region on malaria are dated, they were primarily based on hospital data, and only few attempts have been made to characterize malaria infection in the preschool-aged population in the dry season. To contribute to filling in these gaps, we conducted a multipurpose study, including a determination of malaria infection among a random sample of children aged 6–59 months who were recruited during a cross-sectional household survey in the dry season of 2011.By studying Approval for this study was obtained from the ethics committee in Basel, Switzerland  and the national ethics committee of the Ministry of Health in Mauritania (reference no. 00158). Local government and health administrative authorities were involved in the planning, approval and announcement of the objectives and procedures of the study to the local residents. Parents  of participating children signed a written informed consent sheet before finger prick blood samples were taken and questionnaires administered.Our study was carried out in March 2011 in the city of Kaédi, southern Mauritania. The design was cross-sectional, and we aimed at recruiting approximately 350 children aged 6–59 months from various communes in Kaédi. We randomly selected 246 households, following a method described elsewhere . In brieA finger prick blood sample was collected and a malaria rapid diagnostic test (RDT) was administered according to the manufacturer’s instructions . Two additional drops of blood were used for thick and thin film preparations on microscope slides. The RDT results were read out on site, while the slides were transferred to a nearby laboratory, stained with 10% Giemsa and later analyzed under a microscope by experienced technicians. Each slide was examined by two technicians, both of whom were blinded to the report of the other before final results were entered. Provisions were made for treatment of any positive cases using national guidelines with an artemisinin-based combination therapy (ACT).For each child recruited during the survey, a questionnaire was administered to the mother or legal guardian of the child, enquiring about the occurrence of symptoms of acute respiratory infections and diarrhoea in the preceding 2 weeks, using definitions put forth by the World Health Organization (WHO) ,12.P. falciparum infection among the 371 preschool-aged children.As shown in Figure It is important to note that clinical signs and symptoms of malaria are non-specific and fever is a common presentation that overlaps with other clinical entities such as respiratory tract infections and gastrointestinal illnesses . We addifPR concurs with recent findings obtained in entomological investigations in neighbouring regions in the dry season of 2009, as reported by Ndiath and colleagues [Anopheles specimens during a 16-month period with an overall infection rate of 1.1%. No infected mosquitoes were observed between January and June 2009. However, some transmission was observed in January of 2010, albeit at low levels. Malaria transmission may occur between November and January because, in the absence of rainfall, the annual flood recession from the Gorgol River and the Senegal River during these months can provide residual breeding sites for mosquitoes. Our observations are also consistent with older studies in the Gorgol region conducted shortly after the filling of the Foum Glieta dam, as Baudon and colleagues reported the absence of malaria transmission in March during the dry season [versus 568 mosquitoes). In 2009, Dia and co-workers [Plasmodium infection rate when they examined 394 An. arabiensis mosquitoes with only one specimen being infected with P. falciparum, owing to an infection rate of 0.25%. Furthermore, there was a considerable difference in infection rates of mosquitoes in these same months (0% compared to 1.06%), suggesting that transmission appears to occur only during the rainy season [Our observation of a nil Plleagues . Indeed,y season  also not-workers  found a Laboratory data from the Kaédi regional hospital report 4% slide positivity rate in April 2009 (unpublished observations), while Cortes and colleagues recorded a dry season prevalence of 16.6% in April-May 2003 ,14. FurtPlasmodium parasitaemia among preschool-aged children in our multi-purpose cross-sectional survey conducted in Kaédi, southern Mauritania points, once more, to very little or no malaria transmission in the dry season. However, the possibility of highly focal transmission zones cannot be discounted because local transmission may occur during the annual flood recession in the months immediately following the end of the rainy season (November to January). Between February and June when the swamps have dried up and ambient temperatures are very high, acute respiratory infections and diarrhoea appear to be the underlying aetiology in most febrile illnesses among preschool-aged children. The implications are that public health efforts to control malaria should be strengthened in the rainy season, and the months immediately following the cessation of the rains. Nevertheless the predicted climate change patterns in this region require careful surveillance of malaria transmission. The case management of febrile children under the age of 5 years should adopt a broader consideration of differential diagnosis, which should include acute respiratory infection and diarrhoea , as more likely aetiologies in the dry season. Importantly, malaria treatment with ACT should be strictly contingent on a positive malaria test (RDT or microscopy), which has been shown to be a safe and effective strategy to reduce unnecessary malarial treatment of febrile cases [The absence of le cases ,16. MoreThe authors declare that they have no competing interests.ST, HB, MSW, JU and GC conceived the study. ST, HB, OB, MK and CBOAS conducted field and laboratory work. DT and IS co-coordinated the geographic survey, while MSW processed the geo-referenced data and produced the map. ST performed statistical analysis, interpreted the data and drafted the manuscript. HB, MSW, JU and GC revised the manuscript. All authors read and approved the final submitted version of the manuscript.


Plasmodium falciparum malaria in pregnancy, but the roles of the various underlying genes and mechanisms predisposing to the disease are poorly understood.Innate immunity plays a crucial role in the host defense against malaria including 98 single-nucletoide polymorphisms were genotyped in a set of 17 functionally related genes of the complement system in 145 primiparous Ghanaian women with placental malaria, defined by placental parasitaemia or malaria pigment, and as a control, in 124 non-affected primiparae.MBL2, MASP2, FCN2 and in properdin. In particular, the main African mannose-binding lectin deficiency variant  increased the odds of placental malaria . In contrast, a common MASP2 mutation , which reduces the activity of MBL-MASP2 complexes occurred in 33% of non-affected women and in 22% primiparae with placental malaria .Placental malaria was significantly associated with SNPs in the lectin pathway genes Excessive complement activation is of importance in the pathogenesis of placental malaria by mediating inflammation, coagulation, and endothelial dysfunction. Mutated MBL and MASP2 proteins could have direct intrinsic effects on the susceptibility to placental malaria, in addition to their roles in regulation of downstream complement activation. Plasmodium falciparum infection during pregnancy is a major cause of maternal anaemia, preterm delivery (PD), low birth weight (LBW) and infant mortality. In areas endemic for P. falciparum, 85 million pregnancies occur annually, and malaria-associated LBW in Africa results in an estimated 100,000 indirect infant deaths each year [P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1) adhere to the placental endothelium lining the intervillous space which results in placental sequestration of infected red blood cells. The local accumulation of infected red blood cells and of malaria pigment (haemozoin), i.e. placental malaria, triggers the infiltration of inflammatory cells and a profound pro-inflammatory response [In sub-Saharan Africa, ach year . Pregnanresponse ,5. This response -8.P. falciparum [In conditions of lacking or weak acquired immunity, e.g. early childhood and pregnancy, innate immune responses may play a predominant role in host defense against malaria . Toll-lilciparum , and varlciparum ,12. Overlciparum . These mmbl2) is a serum protein involved in the initiation of innate immune responses by binding to microbial surface oligosaccharides, and activating the lectin pathway. Upon binding, MBL forms a complex with mannan-binding lectin serine peptidase 2 (MASP2) that cleaves C4 and C2 to form the C3 convertase (C4b2a). Subsequent complement activation leads to opsonization and phagocytosis of the target microbes, as well as formation of membrane attack complexes [MASP1) are still debated [Mannose-binding lectin  and other important complement system genes  were included in a novel "complement-chip". Because the coagulation system may also be involved in adverse pregnancy outcomes [thrombomodulin, thrombospondin, FLT1).The aim of the present study was to analyse the influence of lectin pathway polymorphisms on the susceptibility to placental malaria and its manifestation. Following a pathway-oriented approach and using Sequenom's MassaARRAYoutcomes , we alsoP. falciparum parasites and/or haemozoin in placental thick blood films. In addition, P. falciparum-specific PCR assays were performed [A total of 893 women attending for delivery were recruited between January 2000 and January 2001 at the Presbyterian Mission Hospital in Agogo, located in the hyper-to holoendemic Ashanti Region of Ghana. The study protocol was reviewed and approved by the Committee on Human Research Publication and Ethics, School of Medical Sciences, University for Science and Technology, Kumasi, Ghana, and informed consent was obtained from all participants. Diagnostic procedures, malariologic indices and clinical characteristics have been described earlier ,24. For erformed . Haemoglerformed , and lowMBL2 polymorphisms with severe malaria in children [thrombomodulin, thrombospondin) and pre-eclampsia (FLT1). In total, 17 genes were chosen for genotyping . Twenty-seven of the samples from 304 primiparous women were excluded from genotyping based on low DNA quality or quantity. Among the 277 samples genotyped, 8 were removed because of a genotyping call rate < 0.9 leaving 269 samples for the final analyses.SNP genotyping was performed using Sequenom's MassARRAY MALDI-TOF Mass Spectrometry Compact platform and iPLEX Gold chemistry  with standard protocols. A total of 98 SNPs were assayed in four multiplexes of 15 to 34 markers each. Eight SNPs that failed to meet a call rate of > 0.8 were removed. 14 SNPs (for which no previous frequency data were available) were excluded due to a minor allele frequency < 0.05, leaving 76 SNPs for the final analyses  between markers. Haplotypes were constructed and tested with PLINK using 2-to 6-marker sliding windows over all areas where adjacent SNPs were located less than 10 Mb from each other.Analyses were performed with PLINK v1.06 , SPSS (SOdds ratios (OR) along with their confidence intervals (CIs) were estimated with PLINK and R scripts . Joint Oepistasis, which tests all pairs of SNPs separated by at least 1 Mb and in different chromosomes. A p-value threshold of 0.001 was used to avoid most false positives. We acknowledge that our data is small for making robust inference on potential interactions, therefore these results should be considered preliminary.Finally, interactions were screened with PLINK procedure P. falciparum-PCR was positive in 66% and maternal peripheral blood P. falciparum-PCR positive in 59% of cases. Maternal hemoglobin and child birth weights were lower in cases with placental malaria than in uninfected mothers (p < 0.001 and p = 0.023). Gestational age did not differ between the groups.The characteristics of the 269 primiparous Ghanaian women are shown in Table MBL2, MASP2, Ficolin 2 (FCN2) and Complement factor properdin (CFP) genes , the main African MBL variant, which is associated with malaria in children [p = 0.01). The results suggested a dominant effect for an increased susceptibility to placental malaria  than in non-affected women  resulting in an OR of 1.8  for the susceptibility allele in our data. MBL*C is significantly associated to microscopy positivity of placental blood samples, whereas other diagnostic tests show non-significant trends in the same direction  and rs1033638 , remained significantly associated with placental malaria after permutation tests. These exon 11 variants are located only 930 bp apart ; and most of the risk increase could be attributed to R439H. This amino acid residue is located in the activation peptide of MASP2 , but was not associated to microscopy or PCR positivity of the placenta .With regard to t Figure . In this2 Figure , betweenMASP2 gene, the region rs3765900-rs56392418-R99Q as a whole was associated with placental malaria at p = 0.05. Specifically, haplotype TGG was at present in 53% and 21% of women with and without placental malaria, respectively  is a positive regulator of the alternative complement pathway. It had one SNP associated with placental malaria .Complement factor properdin  or MASP2 (R439 wildtype), compared to 65% of women with risk alleles at both loci . These results suggest an additive effect of both risk alleles, but an excess risk due to interaction could not be shown.As MBL forms an active complex with MASP2, the potential interaction between the MBL2, MASP2, FCN2 and CFP genes were shown to be potential risk factors for placental malaria in Ghana. These results thus suggest a relevant role for the lectin pathway of the complement system in the human defense against placental malaria.Recent genome-wide association studies on malaria have had difficulties in identifying causal gene variants in African populations due to low LD ,32. In tMBL2 mutations interfere with the formation of oligomers and result in low serum levels of high molecular weight MBL and impaired MBL function [MBL2*C mutation [MBL2 SNPs examined were found to be associated with placental malaria [MBL2*C was not included. The absence of association of the other SNPs can probably be explained by the low LD in Africans. These results suggest that the MBL2*C variant is a risk factor for placental malaria, in addition to infant malaria. It remains unclear, however, why such a disadvantageous disposition is maintained at a high frequency in malaria-endemic Africa; respective issues have been discussed elsewhere [Functional function . Their rfunction -22,35, a malaria , but MBLlsewhere .MASP2 mutation R439H (rs12085877) is absent in European and Asian populations but according to a previous study [MASP2*R439H occurred in 27% of the women. In contrast to MBL2*C, it was protective against placental malaria, especially chronic infection, and a trend towards protection from low birth weight was discernible. MASP2 with the R439H mutation is able to bind to MBL, but the enzymatic activity of the complex is considerably reduced [Protection from malaria provides an evolutionary benefit in Africa and respective SNPs can be expected to reach polymorphic frequencies. The us study  and HapMus study . In the  reduced . At pres reduced . MASP2 o reduced . MASP2 cMASP2*R439H protecting against placental malaria could involve a curbed production of C5a. Recent work has demonstrated that placental parasitaemia induces increased levels of this potent pro-inflammatory peptide in primiparae [Another explanation for imiparae . Elevateimiparae . Curbed Alternatively, these findings on a common MASP2 variant show some resemblance with deficiency in the closely related complement receptor 1 (CR1). CR1 is also involved in the activation and regulation of C4, and patients with CR1 deficiency show greatly reduced rosetting, i.e. binding and aggregation of infected and non-infected erythrocytes . CytoadhFCN2 gene showed a suggestive association with placental malaria. These FCN2 variants are located in introns and are thus not likely to be functional themselves. On the contrary, they might be involved in gene expression regulation or splicing, or in LD with the actual predisposing mutations.As for the ficolins, two SNPs on the Downstream complement amplification of the lectin pathway is normally activated when MASP2 cleaves C4 and C2 to form the C3 convertase (C4b2a). C2 bypass of the lectin pathway is also possible, and involves alternative pathway amplification where complement factor properdin (CFP) and complement factor D (CFD) are involved . This coMASP2*R439H mutation makes MASP2 an interesting target for drug and vaccine development. MASP2 inhibitors need to be studied as potential adjunctive therapy for the various manifestations of malaria [Excessive complement activation has been suggested to play a role in the pathogenesis of severe malaria, including cerebral malaria as well as severe malarial anaemia, and placental malaria . Through malaria .The authors declare that they have no competing interests.VH designed the study, participated in the statistical analyses and drafted the manuscript. PO carried out the statistical analyses. EL participated in the genotyping. GBA participated in the recruitment of study participants and sample collection in Ghana. FPM coordinated the sample collection and participated in drafting the manuscript. SM participated in the study design and drafting the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria is likely to be mediated by T-cell recognition of malaria epitopes presented on infected host cells via class I and II major histocompatibility complex (MHC) antigens. To test for associations of human leukocyte antigen (HLA) alleles with disease severity, we performed high-resolution typing of HLA class I and II loci and compared the distributions of alleles of HLA-A, -B, -C and -DRB1 loci in 359 Malian children of Dogon ethnicity with uncomplicated or severe malaria. We observed that alleles A*30:01 and A*33:01 had higher frequency in the group of patients with cerebral disease compared to patients with uncomplicated disease  and , respectively. The A*30:01 and A*33:01 alleles share some sequence motifs and A*30:01 appears to have a unique peptide binding repertoire compared to other A*30 group alleles. Computer algorithms predicted malaria peptides with strong binding affinity for HLA-A*30:01 and HLA-A*33:01 but not to closely related alleles. In conclusion, we identified A*30:01 and A*33:01 as potential susceptibility factors for cerebral malaria, providing further evidence that polymorphism of MHC genes results in altered malaria susceptibility.Pre-erythrocytic immunity to  The importance of HLA haplotypes in protection from natural infection was first indirectly shown by the association of HLA-B53 and the HLA class II block DRB1*13:02-DQB1*05:01 with protection against severe malaria in West Africa Plasmodium falciparum, we performed pair-wise group comparisons in a population of ethnically homogenous, Malian Dogon children with severe malaria who were age- and residence-matched to children with uncomplicated malaria. Differences in the distributions of alleles at the loci, A, B, C and DRB1 of the HLA system were assessed and the relationship between HLA polymorphisms and cytokine production was examined. Our results help define the association of defined HLA alleles with clinical outcomes as a result of the selection force of malaria on the human genome, and illustrate further the complexity of African genetic diversity as it relates to the development of malaria T-cell vaccines. This study also establishes an association between HLA polymorphisms and IL-10 production in severe malaria.To examine previously described associations and to investigate if other HLA factors play a differential predispositional role in the development of clinically distinct disease courses after infection with Malian children aged 3 months to 14 years presenting with clinical symptoms consistent with malaria were enrolled into a matched case–control study evaluating the risk and protective factors for severe malaria. The study was conducted at the Bandiagara Malaria Project research clinic in Bandiagara, a rural town of 13,634 inhabitants in the Dogon country in northeast Mali. Malaria transmission is seasonal and heavy with children aged less than 10 years having an average of 2 (range 0–4) clinical malaria episodes per transmission season The trial was conducted in compliance with the International Conference on Harmonization Good Clinical Practices, the Declaration of Helsinki and regulatory requirements of Mali. Study protocols were reviewed and approved by institutional review boards of the University of Bamako Faculty of Medicine, the University of Maryland School of Medicine and the National Institute of Allergy and Infectious Diseases. Village ‘permission to enter’ was obtained from village chiefs, government officials and traditional healers prior to study initiation as described Blood was collected into sterile ethylenediaminetetraacetic acid (EDTA) tubes and Eppendorf tubes on admission and before antimalarial therapy, refrigerated and processed within 2 h of acquisition. Peripheral blood mononuclear cells (PBMC) were processed by density centrifugation using lymphocyte separation medium  following standard techniques. PBMC were linear-rate frozen using isopropyl alcohol containers to −70°C at the field site before transferring to liquid nitrogen storage containers for transportation to the University of Maryland. HLA typing was performed sequentially at the University of Maryland and at Georgetown University. Similarly, sera were processed, frozen and transported to the University of Maryland for cytokine analysis as previously described dna Sequencing Analysis software, AutoAssemblerdna Sequence Assembly software, mt Navigator software and Matchtools software (PE Applied Biosystems). In the present study, we did not test for polymorphisms outside exons 2 and 3 for class I and exon 2 for class II; therefore, some groups of alleles with sequence differences only in the other exons were not distinguished. Indistinguishable alleles were assigned as the allele with the lowest number followed by a letter ‘G’ indicating that this is a group of alleles  as detailed in our previous study Genomic DNA was obtained using the QIAamp 96 DNA blood kit  for each individual. HLA-A, -B, -C and -DRB1 alleles were initially typed at intermediate or low-resolution level using the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe (SSOP) hybridization as described previously for class I P. falciparum proteins of interest. HLA class I peptides derived from the P. falciparum 3D7 CSP, LSA-1, LSA-3, merozoite surface protein-1 (MSP-1), thrombospondin-related anonymous protein (TRAP) and P. falciparum variant erythrocyte Membrane Protein 1 (PfEMP1) with a predicted high to medium binding affinity [500 IC50 (nM) or less] for HLA-A*30:01, HLA-A*30:02 and HLA-A*33:01 were identified with the Average Neural Networks (ANN), Stabilized Matrix Method (SMM) and Average Relative Binding (ARB) algorithms http://tools.immuneepitope.org/main/). The amino acid sequences of the P. falciparum 3D7 proteins were obtained from the National Center for Biotechnology Information (NCBI): CSP (XP_001351122.1), LSA-1 (XP_001347640.1), LSA-3 (XP_001349701), MSP-1 (XP_00 1352170.1) and TRAP (XP_001350088.1). Var gene sequences were obtained from collaborations with the University of Edinburgh To determine whether malaria peptides could bind preferentially to closely aligned HLA subtypes that differed by minor amino acid sequence differences, a computer algorithm was used to assess HLA class I binding affinity of peptides from known Serum levels of IL-6 and IL-10 were determined using cytometric bead array technology  and fluorescence detection by flow cytometry. Briefly, bead populations with discrete fluorescent intensities of peridinin chlorophyll protein complex (PerCP)-Cy5.5 and coated with cytokine-specific capture antibodies were added to individual patient sera and phycoerythrin (PE)-conjugated antihuman inflammatory cytokine antibodies. Simultaneously, standards for each cytokine (0–5000 pg/ml) were likewise mixed with cytokine capture beads and PE-conjugated reagent. The vortexed mixtures were allowed to incubate for 3 days enhancing the lower limit of detection. Flow cytometric analysis was performed and analyzed by a single operator and standard curves were derived from the cytokine standards. The lower limit of detection for the various cytokines evaluated ranged from 2.5 to 10 pg/ml.pypop (http://www.pypop.org), which is able to handle the high levels of polymorphism characteristic of the HLA loci Population analyses including calculations of gene frequencies, tests for fit to expectations under Hardy–Weinberg equilibrium (HWE) and estimations of haplotype frequencies by the expectation-maximization (EM) algorithm Tests for heterogeneity between specific groups and association analyses were performed using contingency table testing and a standard chi-squared measure. Alleles that were observed four or fewer times were combined for analysis. Degrees of freedom (dof) for the chi-squared analysis were calculated from the number of alleles observed five or more times, plus the combined category, minus 1. The relative predispositional effect (RPE) method was used to identify all heterogeneity in disease risk at the primary disease gene; alleles, haplotypes or genotypes with the strongest predisposing or protective effects were sequentially removed, based on their contribution to the overall chi-squared value, from the analysis until no further heterogeneity in risk effects was seen not been identified.To account for linkage disequilibrium within the HLA region, stratification by the disease-associated alleles using the conditional haplotype method (CHM) was used raphPad Prism 5, LaJolla, CA; 2007).Analyses of differences in individual cytokine levels between clinical groups with requisite HLA alleles were performed using Mann–Whitney Rank Sum analysis for populations not normally distributed (Gn = 182) without malaria infection. No allelic differences were noted between healthy volunteers and those with malarial disease, but full details of these results are to be reported elsewhere . HLA determinations were also examined between 237 Malian children with severe malaria and 239 with uncomplicated malaria. The median age of children with malaria was 32 months (range 3–135). Only those children who were self-reported as Dogon were included in the analysis [192 (81%) cases of severe malaria and 201 (84%) cases of uncomplicated malaria]. Of these, 171 children with severe malaria and 188 children with uncomplicated malaria were fully typed at loci, A, B, C and DRB1 of the HLA system .We examined the HLA results between all children with malaria infection and healthy controls (n = 71), (2) cerebral with malaria-associated anemia (n = 25), (3) severe noncerebral (n = 10) and (4) hyperparasitemia (n = 65). Severe malaria subgroups were compared against children of the Dogon ethnicity with uncomplicated malaria (n = 188). Owing to clinical and HLA similarities, groups 1 and 2  were combined (n = 96). Children with severe malaria but not categorized as either cerebral (groups 1 and 2) or hyperparasitemic (group 4) had a small sample size (n = 10) that was analyzed as a single, separate group (group 3). It was observed that the allele frequency distribution of HLA-A differed significantly (P = 0.016) between patients with cerebral and uncomplicated malaria, whereas there was no significant difference found for HLA-B, -C nor -DRB1 .The distribution of HLA alleles of pediatric patients of the Dogon ethnicity with severe malarial infection was compared to the allelic distribution in children with uncomplicated malaria. Severe malaria was subdivided into the following clinical groups: (1) cerebral malaria  = 3.17, 95% confidence interval (CI) (1.94–5.19), P = 0.004 and gf = 0.0781 vs gf = 0.0266, OR = 4.31, 95% CI (1.89–9.84), P = 0.005, respectively]  and uncomplicated malaria (gf = 0.0372) . The allA similar observation was made for the alleles A*33:01 and A*33:03. The allele A*33:03 had similar frequencies in patients with cerebral (gf = 0.0365) and uncomplicated malaria (gf = 0.0585). The alleles A*33:01 and A*33:03 belong to the same serotype (HLA-A33). A row by column comparison indicates that the increase in frequency of the allele A*33:01 in patients with cerebral malaria was not paralleled by an increase in frequency in patients with uncomplicated malaria. The alleles A*33:01 and A*33:03 differ by only one nonconservative amino acid replacement at residue 171; this residue points toward pocket A of the antigen recognition site .An analysis of the distribution of the phenotypes and genotypes of patients bearing the allele A*30:01 showed that this allele was present in 37 of 96 (38.5%) patients with cerebral malaria and in 40 of 188 (21.2%) patients with uncomplicated malaria. Only two patients with cerebral malaria and none of the patients with uncomplicated malaria were homozygous of HLA-A*30:01; this difference was not statistically significant and genotypic distributions conformed to expectations under HWE.n = 48) of the patients with cerebral malaria carried the A*30:01 or A*33:01 alleles, whereas they were detected in only 26.1% of the patients with uncomplicated malaria The allele A*33:01 was present in 15 of 96 (15.6%) patients with cerebral malaria and in 10 of 188 patients (5.3%) with uncomplicated malaria. There were no patients homozygous of HLA-A*33:01 in either of these groups. In the cerebral malaria group, there were four patients (4.2%) carrying the genotype A*30:01, A*33:01, while there was only one patient (0.5%) with this genotype in the uncomplicated malaria group; the difference of the distribution of this genotype in the cerebral and uncomplicated malaria groups was not statistically significant. In all, 50% ; this haplotype had frequencies of 0.0493 and 0.1042, respectively, in these groups of patients. In contrast, the allele A*33:01 was found in multiple haplotypes, all of them present at low frequencies (data not shown). P. falciparum 3D7-predicted peptides with a high to medium binding affinity for HLA-A*30:01 and/or HLA-A*33:01, but not HLA-A*30:02 . Sera were available from 46 of 48 children with HLA alleles A*30:01 and/or A*33:01 and 48 children with alternate alleles and cerebral disease . No significant differences were noted in median IL-6 ; however, a significant elevation in median IL-10 was noted between cerebral malaria patients bearing the A*30:01 and A*33:01 alleles (n = 46) as compared to cerebral malaria patients with other alleles (n = 48) .Serum cytokines (IL-6 and IL-10), which have previously been shown to be elevated in children with cerebral disease compared to children with alternate severe malaria diagnoses We performed high-resolution typing of HLA class I and II loci and compared the distributions of alleles of HLA-A, -B, -C and -DRB1 loci to test for associations of HLA alleles with malaria disease severity in Malian children of Dogon ethnicity. The strong selection force that malaria exerts on the human genome coupled with the genetic diversity noted in Africa suggests that regional differences may develop that contribute to variable immune response to disease. We did not find specific alleles to be broadly associated with malaria disease but we did find allelic disparities within subcategories of malaria disease. In a large study cohort controlled for residence and ethnicity, we have found an association with the alleles A*30:01 and A*33:01 and the development of cerebral disease as compared to a group of age-matched children with uncomplicated malaria, while we did not detect associations with either the well-known allele HLA-B53 (B*53:01) or with the HLA class II block DRB1*13:02-DQB1*05:01 with severe malaria or malaria subtypes. In all, 50% of patients with cerebral malaria carried the A*30:01 or A*33:01 alleles, whereas they were detected in only 26.1% of children with uncomplicated malaria.Supporting Information).A*30:01 and A*33:01 are common HLA alleles in populations of African descent. The concept of HLA class I superfamilies has been defined based on similar peptide-binding motifs, structural similarities in antigen-binding grooves and cross-reactive peptide binding. HLA A*33:01 along with the alleles A3, A11, A31 and A*68:01 have all been assigned to the A3 supertype vs Asp)  .P. falciparum variant erythrocyte surface antigens known as PfEMP1. None of the algorithms allowed for predictions using the comparator allele HLA-A*33:03. In addition, it is unclear whether peptide polymorphisms might lead to cross-strain variability between 3D7 strain high-affinity binding motifs for A*30:01 and A*33:01 and those of other malaria strains. To our knowledge, none of the proteins are known to have sequences correlating with susceptibility to cerebral malaria except for PfEMP1, which is known to precipitate adhesion and immunomodulation var gene transcription in Malian Dogon children with cerebral malaria as compared to alternate types of malaria. However, no allele binding motifs could be established, by computer modeling, within group A var genes in this study. In summary, the data suggest that very few amino acid changes can, in a computer-prediction model, profoundly influence the binding capacity within closely related HLA alleles. We realize that this computer modeling may not necessarily reflect in vivo HLA binding but it does provide proof of principle that differential binding is possible and should be pursued in future analyses. We speculate that this may result in altered immune responses that could influence the manifestations of malaria after infection. We hypothesize that HLA does not necessarily play a role in preventing infection, but rather appears to play a significant role in determining the disease course.To establish a proof of principle for this concept, it would be necessary to establish that malaria peptides restricted by particular alleles of interest would have binding patterns that differed from those of closely related alleles. As a practical measure, we could not perform this analysis using PBMC and synthesized peptides. With advanced computer algorithms, however, we could screen HLA class I-restricted nonamers and decamers within malaria antigens of interest such as LSA-1, LSA-3, MSP-1 and TRAP. The ANN, SMM and ARB algorithms have been shown to be accurate computer models enabling prediction of peptides that bind to common MHC molecules to identify T-cell epitopes + T cells may play a role in the development of cerebral malaria. Rodent models of infection with Plasmodium berghei show that brain-sequestered CD8+αβ T cells do contribute to experimental cerebral malaria + T cells contribute to human cerebral malaria has been noted. The finding of an association of defined class I alleles with cerebral malaria raises the possibility that CD8+ T cells may be involved in the pathogenesis of this manifestation of severe malaria. The logical next step would be to examine cellular immune responses to synthesized peptides corresponding to the computer-generated peptides of interest and to determine if altered immune responses occurred in individuals bearing the HLA alleles associated to susceptibility to cerebral malaria in comparison to individuals with other alleles. Prospective studies might be prohibitively complex and involve large sample sizes. However, we were able to examine data on cytokine production in these same individuals to establish whether alterations in cytokine secretion occurred. We previously examined a large panel of inflammatory cytokines and determined that IL-6 and IL-10 levels corresponded to both severity of malaria and cerebral malaria There is some evidence to suggest that activated CD8P. falciparum, to other host genetic differences that influence the manifestations of malaria or to distinct patterns of malaria epidemiology and transmission intensity, which in turn affect acquired immunity and disease progression. In addition, analysis may be subject to random fluctuations in allelic frequencies in a sample size of this nature; therefore, sampling error could skew results. Nevertheless, this data adds evidence that points to MHC-restricted CD8+ T-cell-mediated responses as being crucial in mediating immunity to malaria Our study did not identify protective effects of certain HLA alleles against severe malaria that were described previously HLA class II antigens may play a direct role in parasite clearance by mediating antibody formation. Malaria-associated severe anemia, which has been associated with ongoing, recurrent infection and failure to eradicate parasitemia, is relatively uncommon in Mali, possibly related to the sharply seasonal pattern of transmission. The HLA class II haplotype, DRB1*13:02-DQB1*05:01, was shown to correlate with protection from severe disease in the Gambia In this study, we found evidence of a possible correlation between two class I HLA alleles, A*30:01 and A*33:01, and the development of cerebral disease in a large cohort of Dogon children from Mali. We have established that a few amino acid changes within defined alleles can result in altered peptide binding in computer-prediction models. Moreover, we have shown a tangible immunologic effect (i.e. elevated IL-10 levels) that is seen among children with the alleles of interest as compared to other children with cerebral malaria. The regional differences that have been noted with respect to haplotypic variance and susceptibility or protection against manifestations of malaria could pose obstacles to the development of malaria vaccines aimed at eliciting CMI but speak to the remarkable plasticity of the human immunologic response and the effect that selective pressures play upon the diversity of the human genome.