Rapid diagnostic tests (RDTs) for malaria provide a practical alternative to light microscopy for malaria diagnosis in resource-limited settings. Three-band RDTs incorporating two parasite antigens may have enhanced diagnostic specificity, relative to two-band RDTs with a single parasite antigen .Phase 1: 2,000 children, two months to five years of age, admitted to a referral hospital in Jinja, Uganda, with acute febrile illness were enrolled. A WHO highly rated three-band RDT was compared to light microscopy of thick peripheral blood films read by local expert microscopists.Phase 2: the three-band RDT was used as a screening tool for inclusion of patients in a clinical trial, and subjects with three positive RDT bands were tested by microscopy using blood samples drawn in parallel. Discordant results were adjudicated by PCR.Phase 1: 1,648 children had both a RDT and peripheral blood smear performed. The specificity of a RDT with all three bands positive was 82% (95% CI: 79-85%) compared to 62% (95% CI: 59-66%) for HRP2 alone. The sensitivity was 88% (95% CI: 85-89%) and 94% (95% CI: 92-95%) for three-band positive RDT and HRP2 antigen, respectively. 119 patients (7.2%) had a positive HRP2 band, but negative parasite lactate dehydrogenase (pLHD) band and negative peripheral smear, and 72 (61%) of these had received pre-treatment with anti-malarials, suggesting a false positive HRP2 result (p = 0.002).P. falciparum by PCR in all cases, suggesting that the PPV was in fact higher.Phase 2: the positive predictive value (PPV) of the three-band RDT was 94% (95% CI 89%-97%) using microscopy as the reference standard. However, microscopy-discordant results were shown to be positive for The pLDH antigen on three-band RDTs, used in combination with HRP2, provides added diagnostic specificity for malaria parasitaemia and may be useful to distinguish acute infection from recently treated infection. In situations where diagnostic specificity is desirable , a three-band RDT should be considered in a sub-Saharan African setting. Immunochromatographic rapid diagnostic tests (RDTs) are practical and accurate tools to guide case management of clinical malaria, particularly in resource-constrained settings where routine microscopy is absent or of poor quality. In addition to their clinical utility, RDTs have been used for epidemiologic surveillance,2, and fPlasmodium falciparum, pLDH and parasite aldolase are common to all Plasmodium species. Test cartridges or strips may incorporate two bands , or three bands . The use of three-band RDTs allows the distinction of P. falciparum from Plasmodium vivax in settings, such as Asia and South America, where both species co-circulate[P. falciparum, and two-band RDTs incorporating HRP2 antigen as the sole antigen are sensitive tests for malaria diagnosis in clinical practice[The test principle underlying malaria RDTs is the detection of parasite antigens, most commonly histidine-rich protein 2 (HRP2), parasite lactate dehydrogenase (pLDH), and/or parasite aldolase through lateral flow immunochromatography. Whereas HRP2 is specific to irculate. On the practice. One welpractice,10. In epractice,12. For P. falciparum in an African context. Test performance characteristics of this three-band RDT were examined in a large cohort of children hospitalized with febrile illness in Africa. Furthermore, this approach was validated using the three-band RDT as a screening tool for patient inclusion in a randomized controlled trial of a novel adjunctive treatment for severe malaria[The objective of this study was to examine whether a three-band RDT, combining both HPR2 and pLDH antigens, would have increased diagnostic specificity for the diagnosis of  malaria.P. falciparum and the second was validation of the three-band RDT for recruitment of patients with falciparum malaria in the context of a randomized clinical trial[This study involved two phases: the first was a prospective cross-sectional comparison of a three band RDT with microscopy for the diagnosis of al trial. The comal trial. Previoual trial and Yemeal trial support For the first phase, febrile children presenting to Jinja Regional Referral Hospital, Uganda, were enrolled. Inclusion criteria were: age 2 months to 5 years; parental report of fever within the past 48 hours or axillary temperature higher than 37.5°C; and hospitalization indicated based on the admitting physician’s judgment. Patients were excluded if the accompanying parent or guardian did not provide written informed consent. Demographic and clinical information was collected, and a peripheral blood sample was drawn for testing by both RDT and microscopy. Study personnel consisting of experienced paediatric nurses and medical officers, were trained in the safe use and interpretation of the RDT from a small volume (15 μL) of capillary or venous blood. A simultaneously collected EDTA-anticoagulated sample was spread as a thick smear on a glass slide, dried, stained with Field’s stain, and examined by an experienced technician using a light microscope using a single reading at the Jinja Regional Referral Hospital (paediatric campus). The clinical personnel were not aware of the microscopy result at the time of RDT interpretation, and technician performing the microscopy was not aware of the RDT result. Test performance characteristics  of the RDT were computed, using microscopy as the reference standard. A sample size calculation using standard formulae for the confidence interval on a proportion  indicated that approximately 1,400 patients would be required to estimate both the sensitivity and specificity within a range of 5% at α = 0.05 level of significance.For the second phase, patients with suspected severe malaria were screened from the emergency department at Jinja Regional Referral Hospital, and 180 patients were finally recruited for a randomized controlled trial of a novel adjunctive treatment for severe malaria (nitric oxide). The hypothesis, rationale, and design of the trial, together with inclusion and exclusion criteria are described in detail elsewhere,16. The The accompanying parent or caregiver of all paediatric patients in this study provided full, written, informed consent for participation. Ethical oversight was provided by the Makerere University in Kampala, Uganda and the University Health Network, in Toronto, Canada. The study was conducted in accordance with the declaration of Helsinki and following Good Clinical Practice guidelines.For the first phase of the study, 2,000 febrile children were prospectively enrolled from the Emergency Department of the Jinja Regional Referral Hospital, from 15 February 2012 to 11 April 2013. The median (IQR) age was 1.3 (0.75-2.1) years and 886 (45%) were female. Common clinical diagnoses at presentation were malaria, pneumonia, sepsis, meningitis in 1518 (76%), 318 (16%), 220 (11%), and 33 (1.7%) patients, respectively. 65 (3.3%) patients in the cohort had a fatal outcome of their febrile illness.1,646 children had both a RDT and peripheral blood smear performed. The microscopy result was missing in 284 cases (14%) and the RDT result was missing in 83 (4.2%). Patients with missing data were similar in age and gender to those with available results for both tests: age (median (IQR) 1.4 (0.75-2.0) vs 1.3 (0.75-2.2), p = 0.77) and gender . HRP2 antigen was detected in 1199 (73%), pLDH in 1033 (63%), and light microscopy was positive in 1031 (63%). Test performance characteristics of the HRP2 line, the pLDH line and the combination of both antigens is given in Table 119 patients (7.2%) had a positive HRP2 line, but negative pLDH and negative peripheral smear. Of these, 72  had received pre-treatment with anti-malarials, suggesting that a significant proportion were false positive HRP2 results. Treatment with an anti-malarial agent prior to presentation was common in the cohort , and was associated with a negative microscopy result (p = 0.001), a negative pLDH result (p < 0.001), but was not statistically significantly associated with a negative HRP2 result (p = 0.09), consistent with persistence of HRP2 antigenaemia in recently treated patients.P. falciparum by this method, giving a positive predictive value of 94% (95% CI 89%-97%).For phase 2 of the study, we used the three-band RDT as a screening tool for enrolment of febrile children in a clinical trial conducted at the same centre. 727 patients were screened for eligibility and 180 were enrolled, all of whom had a RDT result with all three lines positive. A CAP quality-assured reference laboratory examined peripheral blood films by light microscopy, and 169 were found to be positive for Plasmodium ovale based on morphologic appearance of parasites under microscopic examination. These samples were tested by PCR for Plasmodium species: all were positive for P. falciparum, and none were positive for other Plasmodium species. Further clinical details for these 10 cases are provided in Table P. ovale read on blood smear who was positive for P. falciparum by PCR also likely had severe malaria from P. falciparum and not P. ovale.Ten patients had all three RDT lines positive , yet had no parasites visualized on microscopy. One further patient with three positive lines had a diagnosis of This study demonstrates that a three-band RDT improves specificity for the diagnosis of falciparum malaria among febrile children hospitalized at a referral hospital in sub-Saharan Africa. The additional specificity provided by the combination of HRP2 and pLDH antigen positivity over HRP2 positivity alone likely relates to the persistence of HRP2 antigen in children recently treated for malaria. This finding was validated in an independent cohort of hospitalized febrile children, using the three-band RDT as a rapid screening tool for a clinical trial, showing that this diagnostic strategy has a high positive predictive value for falciparum malaria.P. falciparum infection in this study were 94% and 62%, respectively. The high sensitivity is consistent with numerous previous studies in various populations, including a meta-analysis of patients screened for uncomplicated malaria (sensitivity = 95%)[The sensitivity and specificity of the HPR2 antigen for y = 95%). The pooy = 95%), but is y = 95%) and 85%y = 95%)) and neay = 95%),22.P. falciparum and P. vivax co-circulate, such as Asia and South America[Typically, three-band RDTs are used in areas where  America. Beyond  America. Other s America, and tha America. The comLimitations of this study include the use of an imperfect reference standard: light microscopy of stained peripheral smears. This approach may have led to misclassification of disease state, as illustrated by the second phase of this study, where discordant results adjudicated by PCR showed in all 11 cases that the RDT-based diagnosis  was likely correct, and the microscopy result likely represented false negative results or incorrect speciation. The first phase of the study employed Field’s stain, which is quicker and less labor intensive than the gold standard Giemsa stain, but has been associated with lower diagnostic sensitivity. TechnolIn summary, the findings of this study indicate that a three-band RDT incorporating both HPR2 and pLDH antigens can improve diagnostic specificity for falciparum malaria over a two-band RDT  in a sub-Saharan African context, by excluding false positive HRP2 results due to recently treated infection. Added specificity may be desirable when rapid identification of true cases is required, as for screening patients for inclusion in clinical trials. The three-band RDT has a positive predictive value of 94% in this context.RDT: Rapid diagnostic test; HRP2: Histidine-rich protein 2; CAP: College of American Pathologists; PCR: Polymerase chain reaction; PPV: Positive predictive value; NPV: Negative predictive value; pLDH: Parasite lactate dehydrogenase.The authors declare that they have no competing interests.MH conceived and designed the study, participated in patient care and data collection, analysed the results, and wrote the manuscript. ALC participated in the study design, supervision of study conduct, and helped to draft the manuscript. ROO participated in study design and logistical planning, and obtained ethical approval from the Ugandan bodies. SN participated in study design and logistical planning. WCL participated in the study design and helped to draft the manuscript. CCJ participated in the study design and helped to draft the manuscript. KCK conceived the study, participated in the study design and helped to draft the manuscript. All authors read and approved the final manuscript.


Plasmodium infection was conducted early in 2010.Increased control has produced remarkable reductions of malaria in some parts of sub-Saharan Africa, including Rwanda. In the southern highlands, near the district capital of Butare , a combined community-and facility-based survey on A total of 749 children below five years of age were examined including 545 randomly selected from 24 villages, 103 attending the health centre in charge, and 101 at the referral district hospital. Clinical, parasitological, haematological, and socio-economic data were collected.Plasmodium falciparum infection  was identified by microscopy and PCR in 11.7% and 16.7%, respectively; 5.5% of the children had malaria. PCR-based P. falciparum prevalence ranged between 0 and 38.5% in the villages, and was 21.4% in the health centre, and 14.9% in the hospital. Independent predictors of infection included increasing age, low mid-upper arm circumference, absence of several household assets, reported recent intake of artemether-lumefantrine, and chloroquine in plasma, measured by ELISA. Self-reported bed net use (58%) reduced infection only in univariate analysis. In the communities, most infections were seemingly asymptomatic but anaemia was observed in 82% and 28% of children with and without parasitaemia, respectively, the effect increasing with parasite density, and significant also for submicroscopic infections.Plasmodium falciparum infection in the highlands surrounding Butare, Rwanda, is seen in one out of six children under five years of age. The abundance of seemingly asymptomatic infections in the community forms a reservoir for transmission in this epidemic-prone area. Risk factors suggestive of low socio-economic status and insufficient effectiveness of self-reported bed net use refer to areas of improvable intervention. Recent years have seen a substantial increase in malaria control activities. Particularly in East Africa, growing evidence suggests a decline in malaria transmission, morbidity and mortality over the last decade -5. ContrRwanda is a prime example for the impact malaria control can have. Since 2000, several million insecticide treated nets (ITNs) have been distributed (mostly LLINs) increasing the percentage of the population (10 million) covered by nets to potentially ≥70%. In parallel, ACTs have been dispensed on a large scale. In 2007, 56% of households were considered to own a net and 56% of children to sleep under one . SurveilWhile this progress does not appear to be questionable, the extent of the declines as deduced from facility-based data might differ at community level. For instance, community-level case management programmes  have beePlasmodium infection and malaria in this population.One aim of the present study was therefore to provide up-to-date malariologic data at the levels of community, health centre, and district hospital for a highland area in southern Rwanda from where no published material exists so far. In addition, the study aimed at identifying (modifiable) factors associated with Butare  is the capital of Huye district, southern province of Rwanda. Located on the central plateau of Rwanda  is the most widespread. At an annual cost of 1,000 Rwandan Francs  per capita, treatment of common diseases is basically free of charge including utilization of district and provincial hospitals provided there is adherence to a strict referral system [Centre Hospitalier Universitaire de Butare ).Rwanda has a mandatory health insurance system in which the mutual health insurance scheme l system . Governmsector ; population approximately 20,000) was chosen  on Giemsa-stained thick blood films, while the patient was waiting. Children with malaria parasites were treated with artemether-lumefantrine. Other diseases were treated according to Rwanda health authority guidelines [per 200 WBCs at the CHUB central laboratory and the Institute of Tropical Medicine & International Health in Berlin, the definite parasite density was calculated on the basis of a putative mean WBC count of 8,000/μL. These data were used for analysis. Malaria was defined as any microscopically visible parasitaemia plus fever or a history of fever within the preceding 48 hours. DNA was extracted , and Plasmodium species and submicroscopic infections were identified by semi-nested multiplex polymerase chain reaction (PCR) assays [Plasmodium falciparum merozoite surface protein 1 (msp1 ) block 2  and of msp2 block 3  were amplified in five separate PCR assays [® -agarose gels  and analysed using GeneSnap software . In case of negative or inconclusive PCR results, assays were repeated maximally twice. Multiplicity of infection (MOI) was calculated as the highest number of fragments for either msp1 or msp2. Residual (pre-treatment) concentrations of chloroquine and pyrimethamine (indicative of sulphadoxine-pyrimethamine) in plasma were determined by ELISA with limits of detection of 5 ng/mL and 10 ng/mL, respectively [Brief questionnaires were filled in on socio-economic aspects of the children's families including household assets; specification of bed nets, e.g. impregnated or not, was omitted. All children were examined by a physician, a medical history obtained, and a venous blood sample collected. Age, sex, weight, height, mid-upper arm circumference (MUAC), and fever (axillary temperature ≥37.5°C) were documented. Haemoglobin (Hb) levels were measured by a HemoCue photometer . Anaemia was defined as an Hb level < 11 g/dL. Intestinal parasites were screened for by direct wet mount microscopical stool examination, and urinary tract infection by dipstick . Malaria parasites were counted idelines . Followi) assays . For allR assays . Size vaectively .2 test or Fisher's exact test. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were computed. Non-parametric ordinal regression analysis was performed to assess significantly differential effects of independent factors, e.g. infection, on a dependent variable, e.g. Hb levels, between groups. Despite non-parametric comparisons and for tangibility, parasite densities and Hb concentrations are displayed as geometric mean parasite densities (GMPDs) and means, respectively. Evaluation of determinants of P. falciparum infection and malaria was performed by logistic regression analysis. Stepwise backward selection was performed, and final models included those factors that retained statistical significance. A p-value < 0.05 was considered statistically significant.Data analysis was performed using Statview 5.0 (SAS Institute Inc.). Continuous variables were compared between groups by the non-parametric Mann-Whitney or Kruskal-Wallis tests, and proportions by χP < 0.0001). In community children, the leading one was gastro-intestinal affection including gastroenteritis, amoebiasis and helminthiasis. In health centre and hospital, the leading primary diagnosis was respiratory tract infection  and higher in those using bed nets as compared to non-users .The three groups showed large differences in the socio-economic characteristics of the children's families Table . In the P. falciparum , 11.7% had microscopically visible parasitaemia, and 5.5% malaria. All microscopically positive samples were also positive by PCR . The prevalences of P. falciparum infections detected by PCR  and of microscopically visible parasitaemia  did not differ between the groups (Table P = 0.02) or, non-significantly, to district hospital .Overall, 16.7% of all 749 children were found by PCR to harbour ps Table . HoweverP. falciparum infection (PCR) ranged from 0 to 38.5% , of microscopically visible parasitaemia (P = 0.03), and of malaria (P = 0.02) increased with age . GMPDs (95% CIs) did not show a clear trend: in the above age groups, they were 308 (143-663), 1,374 , 1,162 , 3,491 , and 2,061  parasites/μL, respectively (P = 0.29).In the villages, the prevalence of P. falciparum isolates. MOI ranged from one to five ; 65% (72/111) of infections were polyclonal (MOI >1). MOI did not differ between community children and those attending health facilities  but was increased in microscopically visible parasitaemia as compared to submicroscopic infections , correlated positively with parasite density , and tended to be reduced in former artemether-lumefantrine (AL) recipients as compared to children without such reported intake .Multiplicity of infection (MOI) was successfully typed for 88.8% (111/125) of all es Table , did notP. falciparum infection. The odds of P. falciparum was found to increase with increasing age, decreasing MUAC, low educational level, absent father, absence of several household assets, a low family income, lacking use of a bed net, intake of AL within the preceding two weeks , and the presence of chloroquine in plasma. For multivariate analysis, univariately associated factors, adjusted for study sub-groups, were entered into a logistic regression model, and subjected to stepwise backward removal , absent father (6.53 (2.15-19.83), P = 0.0009), presence of radio (0.30 (0.13-0.65), P = 0.003), recent intake of AL (4.40 (1.24-15.57), P = 0.02), and chloroquine in plasma (4.87 (1.43-16.57), P = 0.01), adjusted for attendance at health centre (15.93 (6.47-39.26), P < 0.0001) or district hospital (9.16 (3.16-26.52), P < 0.0001). The role of village among community children lost significance in multivariate analysis . In the above final models, self-reported bed net use showed no association with P. falciparum infection , P = 0.58) or malaria , P = 0.98).For malaria, the same analysis produced the following independently associated factors (aOR (95% CI)): age , P. falciparum infection. In multivariate analysis of current malaria in community children, chloroquine in plasma  lost significant association, and that of age (months) became borderline significant .Limiting multivariate analysis to community children produced basically the same results. However, household possessions of cupboard or bicycle lost significant association with P. falciparum infection (PCR) in the three groups had significantly differing effects on Hb  = 0.25; P = 0.02) and body temperature , the main analysis was performed for community children , 2.77 (0.90-8.55), P = 0.08) or microscopically visible parasitaemia (2.69 (0.80-9.0), P = 0.11). In contrast, at the district hospital, these figures yielded statistical significance , P = 0.02; microscopy, 10.3 (1.89-55.88), P = 0.007). For fever, the opposite was seen: Parasitaemia, e.g. detected by microscopy, increased the risk of fever nine times in the health centre (age-adjusted OR (95% CI), 8.88 (2.54-31.04), P = 0.0006) but showed no association in the hospital (1.27 (0.30-5.39), P = 0.74).In children attending the health centre, there were trends only of an association between anaemia and P. falciparum prevalence rates of 5% to 15% [Plasmodium lactate dehydrogenase tests. In the present study from the vicinity of Butare , 11% and 16% of community children were infected with P. falciparum based on microscopy and PCR, respectively. Data from the 2010 Rwanda Health Management Information System indicate that 13.4% of patients attending health facilities in the Huye district had microscopically confirmed malaria. In the present study, this figure was 12.3%.Malaria transmission in Rwanda varies widely. Traditionally, the central plateau  is considered as one of four distinct ecological zones with overall % to 15% . While t% to 15% , a linea% to 15% . Likely,% to 15% ,7,8. Dat% to 15%  indicateP. falciparum infection in southern highland communities and in health facilities serving this population. In contrast, routine health facility based surveillance has clear limitations in providing complete or representative data on e.g., malaria in the community, also because patients lacking access or choosing alternatives are not registered [Given the scarcity of published community-based data, the reasons for the discrepancy in the prevalence of infection reported in the DHS and observed in the present study are difficult to appraise. Higher sensitivity of PCR as compared to microscopy or rapid test devices ,21 may pgistered . A low hgistered , a figurgistered . Communigistered ,11.P. falciparum infection including submicroscopic ones had an impact on Hb levels, which exceeds the one commonly seen in children in high-endemicity areas [P. falciparum infections has important implications for malaria control in highland areas. In two sites in highland Kenya, both, high and low levels of asymptomatic P. falciparum infections have been observed among children and adults [In the communities, infection prevalence increased from 10% to almost 25% at four years of age, which was not accompanied by a decline in parasite density with age or increase in MOI. The additional age-dependent increase in malaria and trend for declining asymptomatic infections indicate that semi-immunity did not develop to the extent observed in highly endemic areas . In linety areas -29. On td adults ,31. Studd adults ,33; in hd adults ,34,35. Id adults -39. Targd adults ,31 illusP. falciparum infected. Irrespective of the better socio-economic status of the patients' families as compared to the communities . Latest cure rates of AL in Rwanda from 2006 have been reported as 97% [At the health facilities, roughly half of the children had respiratory tract infections or gastrointestinal problems. Every sixth child at the health centre had malaria and every fifth was es Table  this indd as 97% . NeverthIntake of chloroquine was stated by none of the respondents but the drug was present in plasma in 1.4% and 15.4% of non-infected and infected children, respectively. With the assay applied, chloroquine intake can be detected for several weeks, depending on the dose; cross-reactivity with amodiaquine is negligible . Likely,P. falciparum infection was a decreasing MUAC. This crude proxy parameter for malnutrition was, however, not associated with malaria itself. Chronic malnutrition affects every second child in Rwanda [Among the age-adjusted risk factors for n Rwanda  and compn Rwanda . Lackingn Rwanda ,44 but cn Rwanda ,45,46. PP. falciparum prevalence. This may reflect increased access to malaria-related information, improved awareness and increased usage of curative services. Given the latter is true, this points to accomplishable ways of reducing P. falciparum infection in the area, i.e. health communication and education.At variable statistical significance, several parameters reflecting low socio-economic status were associated with increased risks of infection. Remarkably, possession of a radio or a bicycle were independently associated predictors of reduced Lastly, although self-reported bed net usage was in the previously observed range ,19,22, tP. falciparum infection was observed in one out of six children under five years of age, without much variation between community and health facilities. While facility-based, most infections were symptomatic, the opposite was seen in the communities. These seemingly asymptomatic infections greatly contributed to anaemia and form an unrecognized source of transmission in the epidemic-prone highland area. Improved nutrition, identification and elimination of causes of low bed net effectiveness, and reinforced health education are promising and tangible measures to further reduce P. falciparum in this area of Rwanda. In parallel, community-based surveillance of malaria should be included to monitor the progress of malaria control.In this community and facility based survey on malariologic parameters in southern highland Rwanda, The authors declare that they have no competing interests.JBG, FPM, AM, and GH designed the study. JBG, CSt, CSh, NCA, CHL, and CK were responsible for patient recruitment, clinical and laboratory examinations, and logistics. IZ did the PCR analyses, TEA the ELISA assays, and ID, JBG, AU and FPM the statistical analyses. JBG and FPM wrote the paper with major contributions of the other authors. All authors read and approved the final manuscript.


It is unclear why some children with falciparum malaria develop acute seizures and what determines the phenotype of seizures. We sought to determine if polymorphisms of malaria candidate genes are associated with acute seizures.Logistic regression was used to investigate genetic associations with malaria-associated seizures (MAS) and complex MAS  in four MalariaGEN African sites, namely: Blantyre, Malawi; Kilifi, Kenya; Kumasi, Ghana; and Muheza, Tanzania. The analysis was repeated for five inheritance models  and adjusted for potential confounders and multiple testing.Complex phenotypes of seizures constituted 71% of all admissions with MAS across the sites. MAS were strongly associated with cluster of differentiation-ligand-rs3092945 in females in Kilifi (p = 0.00068) and interleukin (IL)-17 receptor E-rs708567 in the pooled analysis across the sites (p = 0.00709). Complex MAS were strongly associated with epidermal growth factor module-containing mucin-like hormone receptor (EMR)1-rs373533 in Kumasi (p = 0.00033), but none in the pooled analysis. Focal MAS were strongly associated with IL-20 receptor A-rs1555498 in Muheza (p = 0.00016), but none in the pooled analysis. Prolonged MAS were strongly associated with complement receptor 1-rs17047660 in Kilifi (p = 0.00121) and glucose-6-phosphate dehydrogenase-rs1050828 in females in the pooled analysis (p = 0.00155). Repetitive MAS were strongly associated with EMR1-rs373533 in Kumasi (p = 0.00003) and cystic fibrosis transmembrane conductance receptor-rs17140229 in the pooled analysis (p = 0.00543). MAS with coma/cerebral malaria were strongly associated with EMR1-rs373533 in Kumasi (p = 0.00019) and IL10-rs3024500 in the pooled analysis across the sites (p = 0.00064).We have identified a number of genetic associations that may explain the risk of seizures in >2,000 cases admitted to hospitals with MAS across four sites in Africa. These associations differed according to phenotype of seizures and site. Acute seizures are common in children admitted with falciparum malaria to hospitals in sub-Saharan Africa , and the phenotypes of MAS using 4,472 children admitted to hospitals with falciparum malaria in four African countries  as part of a case–control study for MalariaGEN  consortial project , with two seasonal peaks and the community prevalence of P. falciparum in children aged 2–5 years in the study area recorded as high as 88.2% in 2002. Additional detailed description of the sites is provided in The entomologic inoculation rate (EIR) for  be >100 . In Kilim), and/or severe normocytic anemia (<5 g/dl), with exclusion of other coincidental causes of severe illnesses such as pneumonia , hypoglycemia (whole blood glucose < 2.2 mChildren with MAS (whose DNA samples were available for genotyping) admitted to hospital between 1996 and 2001 in Blantyre, 2002 and 2008 in Kilifi, 2006 and 2009 in Kumasi, and 2006 and 2007 in Muheza were selected. Details about various seizure semiologies and or malaria were extracted from the clinical notes and databases at each site, and the notes were further examined in order to categorize cases and controls.We classified MAS into two major phenotypes namely: simple and complex, with complex further subdivided into focal MAS, repetitive MAS, and prolonged MAS , based uMAS in cerebral malaria  are in this study referred to as “MAS with coma” because the occurrence of prolonged seizures may have contributed to the depressed conscious level in some children.The genetic associations with MAS were investigated for six phenotypes, namely, all MAS, all focal MAS, all prolonged MAS, all repetitive MAS, all complex MAS, and all MAS with coma. In addition to being common in malaria, complex MAS are thought by some authors to form a more homogeneous group for genetic studies . Quality control procedures were applied to the genotype data across both individuals and polymorphisms. For example, polymorphisms with >10% genotypes missing duplicated records and individuals with excessive missing genotypes (>10%) were removed. We did not exclude polymorphisms on the basis of Hardy-Weinberg equilibrium deviation because both cases and controls were children admitted to hospital.Logistic regression models were used to measure polymorphism's association with MAS or complex MAS (odds ratios). The model could not run the associations for pure focal seizures in all sites and pure prolonged seizures in Muheza because of lack of power due to small numbers. Models included the polymorphism of interest assuming several related genotypic mechanisms  and general models (mutant homozygous and heterozygous gene against wild homozygous gene). Based on an a priori assumption of potentially confounding effects of country ethnic groups in each site (accounts for between site differences), age, sex, and adverse perinatal events  on the genetic susceptibility for seizures in malaria, we adjusted for these factors in the genotypic logistic regression models. We report the minimum p-value from the correlated genotypic tests or models. However, significant models with very large odds ratios (>100) were not reported as this could have introduced sparse-data bias due to infrequent genes in some categories.Pooled estimates for genotypic tests were computed by performing a logistic regression on combined raw data across the four sites. We chose the raw data method after establishing that its findings were similar to the individual site estimates combined using the meta-analysis method that utilizes both fixed effects (inverse-variance weighting) and random effects models  overall, and the proportion was highest in Blantyre  . The proThe proportion of cases who met the WHO definition of cerebral malaria . The proportion of cases with a previous history of seizures was 320/2,095 (15.3%) for all sites, 190/770 (24.7%) for Blantyre, 100/900 (11.1%) for Kilifi, 29/250 (11.6%) for Kumasi, and 1/175 (0.6%) for Moshi.The minor allele frequencies for the polymorphisms considered in the final analysis are summarized in 2 > 0.9, data not shown). Others shown in All-MAS encompasses all MAS phenotypes and two genes were significantly associated with all-MAS in at least two sites, IL10-rs1800896 in Blantyre , 0.18–0.74], p = 0.00531, recessive model) and Kumasi  and epidermal growth factor module-containing mucin-like hormone receptor (EMR)1-rs373533 in Blantyre  and Kumasi . A second SNP (rs461645) in EMR1 was also significant in Blantyre and Kumasi due to high linkage disequilibrium with EMR1-rs373533 in all the ethnic groups  and IL-17 receptor E-rs708567 . We also looked for associations in the sub-phenotypes of MAS to reduce heterogeneity of the phenotype, reported below, although there would be some loss of power in the sample sizes. Meta-analysis of association estimates of IL10-rs1800896 in the all MAS phenotype showed that there was considerable heterogeneity across the sites (68.7% [95% CI 34.8–93.3%]).This phenotype comprises the prolonged, focal, and repetitive phenotypes that together define the majority of the malaria-associated seizures. We analyzed them together, since they have some degree of overlap . SeveralSeveral polymorphisms, none of which appeared in MAS and complex MAS, were significantly associated with focal MAS, particularly IL-20 receptor A-rs1555498  in Moshi and CD36-rs3211938  for Kumasi. However, there was no significant association with focal MAS in a pooled analysis across the sites.Three polymorphisms that appeared significant in MAS and/or complex MAS were associated with prolonged MAS  and 4, ETwo polymorphisms identified as significant in MAS, complex MAS, or prolonged MAS  and 4, wTwo polymorphisms associated with most of the above phenotypes –6 were aThis is the first study to investigate the effect of candidate genes for malaria infection on the risk of acute seizures across multiple sites in Africa. Using established definitions for seizure phenotypes , our datThe classification of seizures is largely based on phenotypes found in febrile seizures , possible differences in documenting the three phenotypes of complex MAS, haplotypic  differences between populations, and/or the effects of population structure. We tried to minimize the latter by adjusting for ethnicity within sites. Some of the heterogeneous associations in the sites may be due to linkage disequilibrium. Because we adjusted for multiple testing, such site-specific associations could be due to strong linkage disequilibrium with actual causal polymorphisms not investigated in this study. The finding that a gene was protective in one site and increased risk in another can be explained by the different inheritance models in the affected sites, or could suggest that the associations are related to a gene region rather than the effect of the particular polymorphism.SCN1A, SCN1B, or SCN2A), γ-amino-butyric acids genes (GABA(A) or GABRG2) (Nakayama & Arinami, In these associations, we studied polymorphisms that were genotyped as part of MalariaGEN consortium project (MalariaGEN, Some polymorphisms had stronger associations with acute seizures with complex phenotypes, suggesting that these three phenotypes may provide insights into the epileptogenesis of malaria. The finding that specific polymorphisms were replicated across the different complex phenotypes supports the role of these genes in the epileptogenesis of malaria. The complex phenotypes are particularly associated with malaria (Kariuki et al., The biologic functions of some polymorphisms have not been fully investigated, and can only be speculated. EMR1 is sometimes elevated in placental malaria (Muehlenbachs et al., P. falciparum infected erythrocytes to uninfected erythrocytes (rosetting; Nagayasu et al., P. falciparum–infected erythrocytes onto brain microvasculature (Berendt et al., Many of the polymorphisms genotyped in this study have previous published associations with severe malaria, and we have analyzed them in the context of association with seizures. CR1 is postulated to be involved in the adhesion of Our findings have demonstrated that children admitted with falciparum malaria may have a genetic predisposition to acute seizures, but the polymorphisms associated with seizures in malaria vary with the phenotype and sites across Africa. These polymorphisms were particularly associated with the three complex phenotypes of MAS, which could also be important in epileptogenesis of severe malaria. Studies from other malaria-endemic settings are required to confirm these findings, and genome-wide association studies are required to study if other seizures-specific genes determine the risk of acute seizures.


Objective: To study some hematological parameters in adult patients with complicated severe malaria and their relations to clinical outcome. Materials and Methods: This was a prospective study, including 77 patients from Aden Governorate with complicated severe malaria over the course of 2 years (2010-2011). Results: The common form of severe malaria in Aden was cerebral malaria (25.9%), followed by renal failure (18.2%), severe anemia (16.9%), and hepatitis (14.3%), with a case fatality rate of 7.8%. Hemoglobin concentration was significantly different among the various complications of severe malaria, while platelet and white blood cell counts did not show such differences. The mean age was older among patients that died. Hematological parameters did not significantly differ among dead or surviving patients. Thrombocytopenia was seen in 42.9% of patients and 18.2% of these had platelet counts of <50.0x109/L. However, none of them developed bleeding.Conclusion: This study concluded that hematological changes are common complications encountered in severe malaria, but they are not related to the clinical outcome. Conflict of interest:None declared. Malaria is an important cause of death and illness, especially in tropical countries . The mosThe World Health Organization (WHO) established criteria for severe malaria that assisted clinical and epidemiological studies. This project was begun in 1990  and was In Yemen, malaria remains a significant health problem, with Plasmodium falciparum as the predominant species that is responsible for 90% of malaria cases . DiffereThe previous studies on severe malaria in Yemen did not focus on the hematological parameters of complicated severe malaria in adults; therefore, this study was conducted to evaluate the main hematological parameters in adult patients with complicated severe malaria in Aden, where a large central referral hospital and a branch of the National Center of Public Health Laboratories (NCPHL) are present. This was a prospective study that included 77 patients from Aden Governorate with complicated severe malaria admitted to Al-Gamhouria Modern General Hospital  and treated at the NCPHL in Aden during 2 years (2010-2011). Patients were selected according to the following criteria: adult Yemeni patients with severe malaria (aged 18 years or more), confirmed to have Plasmodium falciparum malaria parasites in the peripheral blood or bone marrow, and presenting with severe malaria as according to the WHO definition . Severe Exclusion criteria included severe malaria patients of <18 years of age, severe malaria patients who were not residents of Aden , and severe malaria patients who absconded or were discharged against medical advice. The hematological data were collected from the NCPHL in Aden. The clinical data were obtained directly from patients or relatives as well as from physicians in the intensive care unit of the internal medical department of the hospital, where complicated severe malaria patients are admitted. In the NCPHL, malaria is diagnosed by thick and thin blood films. These films were stained with Giemsa for the detection and characterization of Plasmodium falciparum malaria and the parasite load was determined for each film. Diagnosis of severe malaria was established according to the WHO criteria published in 2000 : - Severe anemia was defined as normocytic normochromic anemia with hematocrit of <15% or hemoglobin of <5 g/dL in the presence of parasitemia at >1000/µL. In the case of microcytic/hypochromic anemia, iron deficiency, thalassemia, and hemoglobinopathy were ruled out. - Cerebral malaria was defined as unrousable coma not attributable to any other cause, with a Glasgow Coma Scale score of ≤9, or coma that persisted for at least 30 minutes after a generalized convulsion. - Renal failure was defined as urine output of <400 mL/24 h and serum creatinine of >3.0 mg/dL despite adequate volume repletion. - Circulatory collapse  was defined as systolic blood pressure of <70 mmHg with cold, clammy skin or a core-skin temperature difference of >10 °C. - Black-water fever was defined as the passage of dark red, brown, or black urine secondary to massive intravascular hemolysis and resulting hemoglobinuria. - Hepatitis was defined as the presence of clinically detected jaundice with serum bilirubin concentration of >3 mg/dL and ultrasonographic evidence of enlarged, inflamed liver with elevated liver enzymes. - Hypoglycemia was defined as plasma glucose level of <40 mg/dL. - Prostration and weakness was defined as a patient who could not sit or walk with no obvious neurological explanations. Statistical AnalysisThe collected data were analyzed with SPSS 18. Quantitative variables were presented as mean values, standard deviations, and ranges. Results were tested by the Mann–Whitney or Student t-test with a 95% level of significance and p≤0.05 was considered statistically significant. Ethical ConsiderationsThis study was approved by the ethics committee of the hospital as well as by the ethics committee of the NCPHL in Aden. During data collection from patients or relatives, verbal consent was obtained, and names and personal data were completely secured and transferred to codes to keep patients’ identities private. A total of 88 patients were diagnosed with complicated severe malaria, and 11 of these were excluded due to leaving the hospital early against medical advice. The remaining 77 patients were followed until discharge with medical advice or death.The most common form of complicated severe malaria among the studied 77 patients in Aden was cerebral malaria (25.9%), followed by renal failure, severe anemia, and hepatitis at 18.2%, 16.9%, and 14.3%, respectively . The mean hemoglobin concentration showed a highly significant statistical difference in relation to the type of complications in severe malaria (p=0.00001); it was lowest in those with severe anemia (3.5 g/dL), increasing to 8.2 g/dL in cases of renal failure, to 9 and 9.1 g/dL respectively in hepatitis cases and in prostrated patients, and up to 11.4 and 11.6 g/dL in cerebral malaria and algid malaria patients, respectively . Platelet and white blood cell (WBC) counts did not show significant statistical differences in relation to the type of complications in severe malaria  . The studied patients were followed until complete improvement and discharge, while 6 of them died, representing a case fatality rate of 7.8% . The mean age of deceased patients with severe malaria was statistically significantly older than the mean age of those who survived after severe malaria: 45.2 years versus 29.3 years, respectively (p=0.015). However, the mean values of the studied hematological parameters  were not significantly different among those who died or survived after severe complicated malaria . Thirty-three patients from the studied 77 with severe complicated malaria had thrombocytopenia (platelet count of <150x109/L) at variable degrees; this rate represents 42.9% of all severe complicated malaria cases in this study. However, none of them developed bleeding. In these patients, the count was repeated by manual method. They were classified into 3 grades: grade 1 thrombocytopenia with counts of <150 to 75x109/L, grade 2 thrombocytopenia with counts of <75 to 50x109/L, and grade 3 thrombocytopenia with counts of <50x109/L. The studied cases of thrombocytopenia were classified as 57.6% in grade 1, 24.2% in grade 2, and 18.2% in grade 3 . Three of the 6 deceased patients had grade 1 thrombocytopenia, 2 had grade 2, and 1 had grade 3 . Five ouThis study demonstrated some hematological parameters in complicated severe malaria due to Plasmodium falciparum, which is the most common type of malaria infection in Yemen, accounting for 90% of malaria cases . Cerebral malaria was the most common complication in severe malaria in adults, representing a quarter of the studied cases (25.9%). Several hypotheses have been proposed to explain the pathophysiology of cerebral malaria, but none have been completely satisfactory. Moreover, there is no association of cerebral malaria with altered hematological parameters . This isHemoglobin concentration showed lowest mean values reaching 3.5 g/dL in the studied patients with severe anemia. Plasmodium falciparum malaria is one of the most common causes of anemia. The other Plasmodium species rarely cause anemia because only select red cell populations  are invaded. Multiple mechanisms cause anemia in severe malaria, the most important being hemolysis of infected and uninfected red blood cells (RBCs), splenic sequestration of RBCs, dyserythropoiesis, and bone marrow suppression; such factors can culminate in the chronically low hemoglobin values observed in patients residing in holoendemic regions ,9. Studies found that anemia is also correlated with the severity of malaria infection . In thisThrombocytopenia was detected in 42.9% of the studied patients with complicated severe malaria. This is a common hematological alteration in malaria, which may be a result of peripheral platelet destruction and consumption. Studies showed that immune complexes generated by malarial antigens lead to sequestration of the injured platelets by macrophages in the spleen . The preIn this study, thrombocytopenia was graded to look for the percentage of lower platelet counts; 18.2% of the studied patients showed a count of <50.0x109/L. In a study by Khan et al. , a quartIn regard to mortality associated with complicated severe malaria, neurological manifestations were the major determinant of morbidity and mortality in severe malaria cases in adults. All 6 of the deceased patients in this study suffered cardiopulmonary arrest and died. Postmortem autopsy is not a routine in Yemen; it is only done in legal cases. Five out of the 6 deceased patients in this study (83.3%) had cerebral malaria and the sixth 16.7%) had renal failure. However, hematological findings were not seriously deteriorated in the studied patients with cerebral malaria. This finding is similar to that reported by Mengistu and Diro in Ethiopia , as well.7% had rThis study concluded that hematological changes are common complications encountered in any patient with severe malaria. Hemoglobin concentration is associated with significant changes in relation to the type of complications of severe malaria; however, it is not associated with the clinical outcome after severe malaria. The total WBC count cannot be used as a predictor for severity. Thrombocytopenia can implicate complications, but it is usually asymptomatic and platelet transfusions are generally not required because patients recover quickly. It is recommended that physicians should rely on the clinical presentation and complaints of patients with severe malaria and not hurry to conduct transfusion of blood or blood components based on the findings of hematological parameters alone .The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


Plasmodium vivax infection with a time interval suggesting relapse. It is uncertain whether the two infections are acquired simultaneously or separately. To determine whether mixed species infections in humans are derived from mainly from simultaneous or separate mosquito inoculations the literature on malaria species infection in wild captured anopheline mosquitoes was reviewed.In patients with malaria mixed species infections are common and under reported. In PCR studies conducted in Asia mixed infection rates often exceed 20%. In South-East Asia, approximately one third of patients treated for falciparum malaria experience a subsequent The biomedical literature was searched for studies of malaria infection and species identification in trapped wild mosquitoes and artificially infected mosquitoes. The study location and year, collection methods, mosquito species, number of specimens, parasite stage examined (oocysts or sporozoites), and the methods of parasite detection and speciation were tabulated. The entomological results in South East Asia were compared with mixed infection rates documented in patients in clinical studies.Plasmodium falciparum parasites only was 0.51% (95% CI: 0.44 to 0.57%), for P. vivax only was 0.26% (95% CI: 0.21 to 0.30%), and for mixed P. falciparum and P. vivax infections was 0.036% (95% CI: 0.016 to 0.056%). The proportion of mixed infections in mosquitoes was significantly higher than expected by chance (P < 0.001), but was one fifth of that sufficient to explain the high rates of clinical mixed infections by simultaneous inoculation.In total 63 studies were identified. Individual anopheline mosquitoes were examined for different malaria species in 28 of these. There were 14 studies from Africa; four with species evaluations in individual captured mosquitoes (SEICM). One study, from Ghana, identified a single mixed infection. No mixed infections were identified in Central and South America . 42 studies were conducted in Asia and Oceania . The proportion of anophelines infected with P. vivax infection follows P. falciparum malaria in one third of cases, the available entomological information suggests that the majority of these mixed species malaria infections are acquired from separate inoculations.There are relatively few data on mixed infection rates in mosquitoes from Africa. Mixed species malaria infections may be acquired by simultaneous inoculation of sporozoites from multiply infected anopheline mosquitoes but this is relatively unusual. In South East Asia, where  Plasmodium falciparum malaria infections are followed soon afterwards by an infection with Plasmodium vivax. Indeed in endemic areas in this region, the main clinical complication of falciparum malaria is subsequent vivax malaria, far outweighing the incidence of recrudescent falciparum malaria [Where transmission of both vivax and falciparum malaria is high in parts of Oceania, mixed species infections in humans are common. With frequent infection from repeated inoculation it is not surprising that infections accumulate and so comprise a multiplicity of genotypes and species. In South-East Asia where transmission of malaria is low, seasonal, and unstable people typically receive one infected mosquito bite each year or less, yet a remarkably high proportion 30 to 50%) of acute  malaria -5. The i0% of acu malaria ,5. This P. falciparum. In endemic areas, where the prevalence of malaria revealed by sensitive PCR methods is much higher than evident from microscopy, high rates of mixed blood stage infection (~20%) have been reported, presumably because both infections are carried chronically [P. falciparum malaria in these same patients. Simultaneous infection studies in the malaria therapy of neurosyphilis and in volunteers clearly showed that mixed infection could occur from simultaneous inoculation, that pre-erythrocytic development of P. falciparum was more rapid, and that in the blood stage infection P. falciparum tended to suppress P. vivax, so low level P. vivax parasitaemia might still be missed by current diagnostic methods [Plasmodium vivax may also suppress P. falciparum; up to 10% of acute vivax malaria episodes in Thailand are followed shortly after by P. falciparum infections without reinfection [Mixed species infections in patients are often under reported, with a tendency to over report the more dangerous onically ,7. Lower methods -10. Plasnfection ,11. Bloonfection . To resoThe NLM PubMed was searched since 1987 for publications in English describing studies on anopheline vectors in malaria endemic areas in which malaria parasite identification had been performed using immunological or molecular methods. The search terms were; anopheles AND sporozoite OR oocyst; Subheadings: Asia OR Africa OR America. Studies of both trapped wild mosquitoes and artificially infected mosquitoes were included. After compilation the lists were checked manually. The location of study and year, the collection method, mosquito species, number of specimens, parasite stage examined (oocysts or sporozoites), parasite detection method, and the results of the species identification were tabulated. Investigators in the South East Asian region were contacted for further information. ELISA and more recently PCR methods were used generally for species identification. The sensitivity of these different methods were also reviewed.In total, 42 studies were identified which were conducted in Asia and Oceania (11 were from Thailand) and 21 studies were conducted in other areas  between 1987 and 2011. Three of the American and three of the Asian studies involved blood feeding of anophelines and later dissection. In the remainder of the studies trapped wild anophelines were examined.Table P. falciparum and 184,744 were examined for P. vivax. Only one study reported mixed infections but, because of pooling, it cannot be ascertained whether this was in single mosquitoes. In 27 studies of trapped wild mosquitoes 167,465 individual anophelines were examined for P. falciparum sporozoites and in a further 2,049 the whole body was assessed. P. falciparum parasites only were identified in 1087 . For P. vivax 334,698 anophelines were assessed and 544 were positive  for P. vivax alone. Mixed P. falciparum and P. vivax infections were assessed in 41,325 mosquitoes. If there was no association between the parasites then none of these would have been expected to have mixed species infection, whereas 17 (0.036%: 95% CI: 0.016 to 0.056%) had evidence of infection with both parasites (p < 0.001)  and 24 P. vivax (0.21%) infections, but no mixed P. falciparum and P. vivax infections were found , one of which was also infected with P. falciparum. Only one of the P. malariae was from mainland SE Asia. One mixed P. falciparum, P. vivax and Plasmodium knowlesi infection was identified in Vietnam. No Plasmodium ovale was identified. The VK210 and 247 P. vivax genotypes were distinguished in most studies; there were 207 VK210 and 279 VK247, and six were mixed genotype infections. None of the four artificial feeding studies in which different vectors were fed on parasitaemic blood yielded any mixed infections despite high rates of positivity for the individual parasite species.In four studies, sporozoites were examined but not speciated; 525 of 85,359 mosquitoes were positive. In ten studies some or all of the anophelines caught were pooled in groups before analysis. In these ten studies 155,497 anophelines were examined for et al [Studies conducted on the treatment of falciparum malaria during and immediately after the first world war indicated that vivax malaria commonly followed falciparum malaria acquired in Europe or Asia. In India rates ranged from seven to 40% . Betweenet al  reportedet al .et al [P. falciparum monoinfection and 1,164 (11.0%) had mixed P. falciparum/P. vivax infections according to microscopy at presentation. The cumulative proportion with P. vivax recurrence by day 63 was very similar to that documented in Bangkok; 31.5% . However the overall rate of P. vivax infection following P. falciparum monoinfection may have been underestimated as the cumulative risk of vivax malaria was 51.1%  after treatment with rapidly eliminated drugs , 35.3%  after treatment with intermediate half-life drugs (t1/2 1 to 7 days), and 19.6%  after treatment with slowly eliminated drugs (t1/2 > 7 days). Some late relapses suppressed by the slowly eliminated drugs may therefore have emerged after day 63. The cumulative 63 day relapse/recurrence rate for vivax malaria at the same location was 63% (143/227)[Anopheles dirus, Anopheles minimus, Anopheles maculatus) examined individually five carried P. vivax and two carried P. falciparum. There were no mixed infections .Douglas et al  recently(143/227). Two entet al [P. vivax infection during follow-up. Of the 679 patients with P. falciparum infections only, 235 (35%) had subsequent P. vivax malaria. Children were nearly three times more likely to develop intercurrent vivax malaria than adults.Smithuis et al  recentlyP. falciparum infected mosquitoesTaking the more conservative data from Bangkok in Thailand where reinfection could be excluded, then if all vivax infections are acquired from simultaneous inoculation it would be expected that the mixed falciparum and vivax infection proportion in mosquitoes would be 32% of all the P. falciparum positive mosquitoes  7,125 individual anophelines were examined. P. falciparum sporozoites only were identified in 3 (0.042%), P. malariae in 1 (0.014%), and 50 were positive (0.70%) for P. vivax alone (47 Pv VK 210 and 3 Pv VK 247). No mixed P. falciparum and P. vivax infections were identified.In five of the seven studies identified some or all the mosquitoes were pooled before analysis and in three studies anopheline mosquitoes were infected artificially. One of these studies examined only for P. falciparum. In the artificial feeding study conducted in Guinea-Bissau where all species were investigated 510 anophelines were examined and infection rates with single species ranged from 5 to 15% for P. falciparum, 1 to 2% for P. malariae and 0.54 to 5% for P. ovale. Mixed infection rates ranged from 0.4 to 15%. In the 11 studies in which individual anophelines were examined 1654 of 27738 (6%) had P. falciparum sporozoites although rates for individual vectors in different studies varied between 0.7% and 78%. In the four studies where mixed infections were studied specifically, two studies examined only for P. vivax. In the remaining two studies one from Cameroon did not identify mixed infections. In the other study from Ghana overall infectivity rate was 7.7% for Anopheles gambiae (s.s) and 4.9% for Anopheles funestus. Of the sporozoite positive mosquitoes 5.7% contained P. malariae only and 5.7% contained P. ovale only and a single A. gambiae (s.s) was identified carrying mixed P. falciparum and P. malariae. The remainder were positive for P. falciparum only. No mixed P. falciparum and P. vivax infections were identified. P. vivax was reported only from Ethiopia where 14 (1.8%) of 796 mosquitoes were positive for P. vivax and 4 (0.5%) were positive for P. falciparum. No mixed infections were identified in this study.In one of the 14 studies identified mosquitoes were artificially infected and in two studies mosquitoes were pooled before analysis. Eight studies examined the mosquitoes only for P. falciparum infections are common, but there are relatively few studies which have examined the prevalence of other infections. In Asia and Oceania, these areas of intense transmission tend to be very small focal forest or forest fringe areas, and much of the coastal part of the Island of New Guinea. In those areas where both P. falciparum and P. vivax are prevalent mixed species infections with both species are common but maybe also be below the level of microscopy detection [In areas where malaria transmission is stable and intense the majority of the human population is constantly infected, although older children and adults often have parasite densities below the level of microscopy detection. Many parts of Africa have this pattern of malaria transmission. Mixed genotype etection . Both inetection . Sensitietection  althoughP. vivax infections may still be asymptomatic, P. falciparum malaria is usually accompanied by illness and treatment seeking. Symptomatic infections are seen at all ages, although the burden of symptomatic vivax malaria is in young children. In this epidemiological context mixed species infections are found in 10% or less of acute infections, yet approximately one third of patients still experience vivax malaria following falciparum malaria. The high efficacy of initial falciparum malaria treatment against P. vivax and the time interval between initial presentation and the subsequent vivax malaria episode suggests that it is a relapse. This presumed relapse was thought to result from simultaneously acquired mixed species infection, but the evidence reviewed here does not support this hypothesis. The proportion of P. falciparum infected mosquitoes overall which also carried P. vivax in Asia and Oceania was only 6.6% overall. As expected most mixed infections in mosquitoes were found in studies on the island of New Guinea where transmission of both species is intense. None were found in Thailand where one third of P. falciparum malaria is followed by P. vivax. Thus, the proportion of mixed infections in anopheline mosquito vectors in Asia and Oceania is approximately one fifth of that expected. These findings suggest that simultaneous inoculation of the two malaria species does occur, and indeed is more frequent than would be expected by chance alone, but that the majority of mixed species infections do not derive from a single mosquito bite.In contrast in much of the remainder of Asia transmission is generally low and seasonal and, although Several explanations are possible. The entomology sampling frameworks in these studies may not have been representative. The vectors might not have been adequately representative of those transmitting malaria. The laboratory methods may have been insensitive Table  or not dP. falciparum and P. vivax gametocyte carriage at infectious densities in the blood of the parasitaemic population. This may well occur in high transmission settings but is rare in low unstable transmission settings and is not supported by the mosquito feeding studies reported here. As P. vivax develops more rapidly than P. falciparum in the anopheline vector, it also demands mosquito longevity. If the two infections were acquired separately then the close and reproducible temporal association between the infections needs explanation. Although the distribution of infected mosquito biting undoubtedly clusters it would require a remarkable degree of association for one third of all P. falciparum inoculations to be accompanied within a day or two by a P. vivax inoculation -when average inoculation rates are less than one infected bite per person per year! Nevertheless mixed species infections in anopheline mosquitoes do occur in Asia significantly more frequently than expected by chance so it is clear that simultaneous transmission does occur and does contribute to mixed infections. It is likely to account for the significant proportion of acute symptomatic malaria in low transmission settings where both species are identified in the blood at presentation.If the mixed species infections are all acquired from simultaneous inoculation it would also requires very high rates of concomitant P. falciparum and P. malariae infection rates were also very low this negative finding has limited significance. There are surprisingly few data from Africa on species other than P. falciparum. Plasmodium vivax was identified in Ethiopia as expected. In Ghana where transmission is intense mixed infection was identified. Artificial infection studies in Guinea-Bissau suggested that mixed infection of mosquitoes would be frequent, but these data are too few to draw firm conclusions on the entomology of mixed malaria infection in Africa.The data from Central and South America where transmission is generally low and unstable also showed low rates of mosquito infection. No mixed infections were found but as P. vivax infections follows P. falciparum malaria in one third of cases, the published data suggests that the majority of these mixed infections are not acquired by simultaneous inoculation. A more plausible explanation is that P. vivax relapse is stimulated by symptomatic falciparum malaria infection.This review is almost certainly an underestimate of the available information. For example, much of the Central and South American literature is in Spanish or Portuguese and some of the African literature is in French. The search strategy employed may well have missed studies in English, which lacked the key words. Nevertheless because of the large apparent discrepancies between mixed species infection rates in the anopheline mosquito vectors and humans this comprehensive sample of the literature provides a convincing message. Mixed infections of anopheline vectors do occur and account for some of the mixed infections in humans. However in South East Asia where The authors declare that they have no competing interests.NJW, MI and NJPD conceived of this review. MI and SN carried out the review. NJW and NJPD conducted the analysis. MI, SN, NJPD and NJW participated in the interpretation and presentation. MI and NJW drafted the manuscript. All authors read and approved the final manuscript.


P.falciparum infection impairs EBV-specific T-cell surveillance, allowing expansion of EBV infected B-cells from which BL derives. We re-examined the situation in the same area, The Gambia, after a reduction in malaria endemicity. Cellular immune responses to EBV were measured in children with uncomplicated malaria before (day 0) and after treatment (day 28), comparing EBV genome loads in blood and EBV-specific CD8+ T-cell numbers (assayed by MHC Class I tetramers and IFNγ ELISPOTS) with those seen in age- and sex-matched healthy controls. No significant changes were seen in EBV genome loads, percentage of EBV-specific CD8+ T-cells and IFNγ producing T-cells in acute versus convalescent samples, nor any difference versus controls. Regression assays performed also no longer detected any impairment of EBV-specific T-cell surveillance. Acute uncomplicated malaria infection no longer alters EBV-specific immune responses in children in The Gambia. Given the recent decline in malaria incidence in that country, we hypothesise that gross disturbance of the EBV-host balance may be a specific effect of acute malaria only in children with a history of chronic/recurrent malaria challenge.Burkitt's lymphoma (BL), an EBV-associated tumour, occurs at high incidence in populations where malaria is holoendemic. Previous studies in one such population suggested that acute  EBV is a gamma herpesvirus, ubiquitous in human populations, that is usually acquired early in life by the oral route, replicates locally through a virus productive (lytic) infection at oropharyngeal sites and is carried thereafter for life as a latent, largely asymptomatic, infection of the B-cell pool Indeed EBV first colonises the B-cell system through a growth-transforming infection, where coordinate expression of specific latent cycle genes drives the expansion of infected B-cell clones. These expansions are controlled by the EBV-specific T-cell response but it appears that some infected cells survive by extinguishing latent gene expression, thereby generating a reservoir of latently infected, resting B-cells through which the virus persists + cytotoxic T-cells + T-cell responses restricted through two HLA alleles, B*3501 and B*5301, were measured by IFN-γ ELISPOT in Gambian children with acute malaria and compared to results obtained up to 6 weeks post-treatment. With the caveat of a small sample size in mind, this comparison suggested that responses were impaired during acute disease, but it was not clear whether this reflected a reduction in the numbers or in the functional competence of EBV specific T-cells in the circulation Several studies have indeed implied that malaria exposure can cause a reduction in EBV-specific T-cell responses A further heterogeneous group of studies have looked at EBV viral load dynamics during malaria exposure + T-cell immunity in these new circumstances. To do so, we assayed EBV-specific CD8+ T-cell responses using the two functional assays (regression and IFN-γ ELISPOT) that had been used separately in earlier work P. falciparum infection in children aged 1–15 yrs and in healthy age- and sex- matched control children without malaria.Interestingly, the intensity of malarial infection in the Gambian population has reduced substantially P. falciparum parasites. All of the malaria cases and all except one of the controls who was excluded from the analyses were EBV positive by IgG VCA ELISA. Consistent with previous findings All malaria cases were both slide and PCR positive with a geometric mean slide parasite density of 135,878/ul . All controls were slide and PCR negative for + and CD8+ T-cell counts were significantly lower during acute (Day 0) P. falciparum infection compared to convalescence (Day 28) consistent with previous findings Absolute CD4P. falciparum malaria and age- and sex- matched healthy controls (p = 0.749). Likewise, the EBV genome loads did not change significantly between D0 and D28 (p = 0.2173) (In contrast to previous findings  0.2173) . Conside 0.2173)  it is un6 PBMCs in Day 0, 28 and control groups. Consistent with the observations made for viral loads EBV-specific IFN-γ ELISPOT counts didn't differ between children with acute (Day 0) P. falciparum malaria and age- and sex- matched healthy controls to both lytic (p = 0.096) and latent (p = 0.3738) peptide pools. Likewise, there was no significant difference between Day 0 and Day 28 to both lytic (p = 0.9374) and latent (p = 0.0998) peptide pools. (Of the 84% of donors with appropriate HLA types for the lytic peptide pool there were 7 (29%), 7 (30%) and 8 (31%) non-responders in the acute (Day 0), convalescent (Day 28) and control groups, respectively. Numbers of non-responders were of a similar magnitude for the latent peptide pool, being 6 (25%), 9 (39%) and 8 (31%) for acute (Day 0), convalescent (Day 28) and control groups, respectively. Background values were of a similar magnitude in all groups, with medians of 15 SFU/10e pools. . The resP. falciparum infected children (n = 9) during the following malaria season. Consistent with our current EBV genome, IFN-γ ELISPOT and MHC Class I Tetramer data, but in contrast to the original findings by Whittle et al. P. falciparum malaria. These assays were conducted on cryopreserved PBMCs but the magnitude of regressive capacity in acute (D0) P. falciparum infected children did not significantly differ from those detected using ex vivo assays on healthy control children  and CD4+ T-cells (p = 0.0085) was significantly higher than in convalescent samples  and CD4+ T-cells (p<0.001) were significantly higher than in healthy controls and during convalescence this number remained significantly higher than seen in the controls.During acute  samples . This tr B-cells . During + T-cells , supporting the hypothesis that PD-1 expression is associated with cellular activation and is not purely a marker of functional exhaustion in these cells. However, this was not found to be the case for CD19+ cells , data not shown.Consistent with recent data P. falciparum infection on the number and function of EBV-specific CD8+ T-cells ex vivo; in parallel with viral genome loads and traditional cell culture-based techniques measuring T-cell mediated regression of outgrowth of autologous B-cells. We show that the situation currently in The Gambia is different to that reported earlier P.falciparum malaria experience no elevation in EBV genome load in the blood compared to age- and sex-matched healthy controls, nor do they have any evidence of reduction in numbers or impairment of function of their EBV-specific CD8+ T-cells. We argue that these different findings could be due to a recent decline in chronic exposure to malaria in infancy and early childhood in the Gambia This work directly assesses the effect of acute Primary EBV infection, like Cytomegalovirus infection occurs in early childhood in the Gambia et al.) from malaria cases, both in acute and convalescent samples, are lower and not significantly different from those of age and sex-matched controls, although they remain significantly higher than the range typically seen for healthy virus carriers in the UK population et al. and Njie et al. and the current study is highlighted by the dramatic change in entomological inoculation rates from Brefet, a rural community where part of the current study was based: 3.21 in 1991 to 0.62 in 2006 P. falciparum infection in childhood was still sufficiently chronic/recurrent as to render the EBV-host virus balance susceptible to modulation during a renewed bout of acute malaria. By contrast, given the recent decline in malaria incidence and increasing peak age of P. falciparum related hospital admissions in the Gambia P. falciparum infection. We would argue that a single, primary P. falciparum infection is not sufficient to impair immunological control of EBV infection.Loss of regression of EBV-infected B-cell outgrowth during acute malaria was first demonstrated in Gambian children over 25 years ago The work by Moormann et al., who studied two cohorts with differential malaria exposure in the Kisumu and Nandi areas of Western Kenya, is relevant here. They noted higher EBV genome loads in children, one to four years of age, living in areas holoendemic for malaria compared to those only sporadically exposed + T-cells as measured by IFN-γ ELISPOT did not differ significantly between malaria cases, in acute stage or convalescence, and age- and sex- matched healthy controls. In a different study comparing children living under differing malaria exposure in Kenya, Moormann et al found that fewer children between five and nine years of age responded to pools of EBV peptides when coming from an area of high rather than sporadic malaria transmission + T-cell responses, we find that PD-1, a marker reported to signify cellular exhaustion, is significantly up regulated in acute malaria and does not decline to control levels by day 28. However, PD-1 expression significantly correlates with CD38 expression on CD4+ and CD8+ T-cells which supports recent findings in HIV that PD-1 is not purely a marker of cell exhaustion Furthermore, we found that the function of EBV-specific CD8Our study has limitations that are worth discussion. Although previously reported data suggest a dramatic decline in malaria exposure in the country as a whole, our study design precludes exact knowledge of the exposure history of our study participants. However, we do know that 95% of three year old children followed up longitudinally in a birth cohort adjacent to our study's catchment area had no serological evidence of malaria exposure P. falciparum infection and therefore suggest that alteration in EBV host balance is likely to be dependent on cumulative prior infections. A further longitudinal study of primary and cumulative acute P. falciparum infections and their impact on EBV host balance would be required to test this hypothesis.Previous case-control studies have demonstrated that acute malaria infection can alter EBV host balance leading to an increase in EBV genome loads Our findings, however, have more general implications adding to the mounting evidence that the decline in malaria seen in some parts of sub-Saharan Africa have wider implications than a reduction in morbidity and mortality from malaria alone. Additional changes have been seen in the rates of invasive bacterial diseases such as non-typhoidal salmonella in the Gambia The Gambian Government/ MRC Laboratories Joint Ethics Committee approved this study. Participants were enrolled after individual written informed consent was obtained from the participant's parent/guardian.P. falciparum malaria, defined as a temperature >37.5°C within the last 48 hours, and ≥5000 parasites/µl detected by slide microscopy. Thirty-two age- and sex- matched healthy aparasitaemic controls were recruited from Brefet, a rural community where malaria transmission has dropped considerably Thirty-two Gambian children (aged 1–15 years) enrolled into studies of severe and uncomplicated malaria were tested P. falciparum infected children were bled on admission (D0) and after 28 days (±3 days); controls were bled at one time point. 500 µl of blood was collected into an EDTA microcontainer for a full blood count (FBC) and DNA extraction. A further 4 mls was collected into heparinised vacutainers® (BD). PBMCs were separated on lymphoprep for use in ex vivo flow cytometric surface staining and IFN-γ ELISPOT assays.6 PBMCs correcting for the PBMC percentage using the haematology count.EBV genome loads were assayed by quantitative real-time PCR, as described elsewhere IgG reactivity to EBV Viral Capsid Antigen (VCA) was measured by a commercial ELISA kit .+ epitopes, two types of EBV can be distinguished. Epitopes used in this study have been defined based on work with type 1 EBV strains in Caucasians. While we did not sequence the resident strains of EBV in our donors, Njie et al. described previously that 90% of Gambian donors they studied carried type 1 EBV Low resolution class I HLA typing was performed using sequence-specific primers (SSP) for the HLA-alleles of interest, A2, B35, B8 and B53, as previously described 5 PBMCs per well in duplicate. Plates were incubated overnight at 37°C at 5% CO2. The cells were discarded the following day, and a biotinylated anti-IFN-γ mAb, 7-B6-1 biotin (MABTECH), was added at 1 µg/ml for 3 hours at room temperature, followed by streptavidin-conjugated alkaline phosphatase (MABTECH) for 2 hours. Cytokine-producing cells were detected after a 30-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazoliym using an alkaline phosphatase-conjugate substrate kit . Spots were counted using an automated AID ELISPOT plate reader©. PBMCs were screened for reactivity to EBV using pools of commonly recognised CD8+ lytic and latent peptides as shown in IFN-γ producing EBV specific lymphocytes were enumerated as previously described using the ELISPOT technique 6 PBMCs per MHC Class I tetramer  were stained for 1 hour at 4°C in the dark. MHC class I Tetramers containing GLCTLVAML (A2_GLC), CLGGLLTMV (A2_CLG), EPLPQGQLTAY (B35_ EPL) and RAKFKQLL (B8_RAK) peptides were used, conjugated to Phycoerythrin, PE, . Tetramer staining was followed by surface marker staining  according to the manufacturer's protocol (BD Biosystems), using the following antibodies: Programmed Death-1 (PD-1) Fluorescein Isothiocyanate (FITC), CD4 PerCP, CD19 PE-Cy7, CD38 Allophycocyanin (APC), (BD Biosystems) CD8 Pacific Blue, CD27 APC-Alexa750, (Ebioscience) CD28 ECD (Beckman Coulter) and CD3 cascade yellow (Dako). Appropriate isotype control antibodies, IgG1 FITC, IgG1 PerCP, IgG1 PE-Cy7, IgG1 APC, (BD Biosystems) IgG2a Pacific Blue, IgG1 APC-Alexa750, (Ebioscience) and IgG1 Cascade Yellow (Dako), were used. Samples were acquired on a Cyan™ ADP flow cytometer using Summit software (Beckman Coulter). Analysis was performed using FlowJo (Tree Star Inc.) and isotype controls were used to set the gating.Ex vivo, 1×10P. falciparum malaria cases recruited from the same study platform 4 to 1.2×103 cells/well. The strength of regression was expressed as the initial cell seeding necessary to give a 50% incidence of regression among replicate wells as calculated using the Reed-Muench formula.These were carried out on cryopreserved PBMCs in the following malaria season. PBMCs from uncomplicated and complicated Viral genome load, serology, ELISPOT and flow cytometry data were plotted and statistically analysed using GraphPad Prism software . Wilcoxon rank and Mann-Whitney U non-parametric tests were used for comparisons of matched and unmatched data respectively. STATA (version 11) was used to calculate Spearman's rank correlation coefficients.


Plasmodium vivax and Plasmodium falciparum) was analyzed in 1,468 women from three longitudinal cohort studies in which weekly malaria screening was done systematically during pregnancy. One-third of women  had at least one malaria infection in pregnancy. The ABO blood group phenotype was not associated with the species of infection, frequency of malaria attacks, symptoms of malaria, hematocrit, or parasitemia during pregnancy.In a few small studies an association between blood group O and placental malaria has been described. The relationship between blood group and malaria in pregnancy ( Plasmodium vivax and Plasmodium falciparum) in pregnancy and pregnancy outcome analyzing longitudinal data from three cohort studies in which regular malaria screening was done systematically during pregnancy.Only a few studies have addressed the association between the ABO blood group system and malaria during pregnancy. Four studies from Africa Gabon, were recOn the western border of Thailand, the Shoklo Malaria Research Unit has been conducting weekly antenatal clinics (ANC) since 1986. It has six established clinics that provide antenatal care and three clinics with delivery services for migrant workers and refugee populations from Myanmar. All pregnant women are encouraged to attend ANC on a weekly basis for malaria screening and early diagnosis and treatment, because there is no drug available for intermittent preventive treatment in this area of multi-drug resistant malaria.6Since the ANC was established three pregnant women cohort studies have been conducted, all with frequent and longitudinal follow-up for malaria screening and routine blood group testing. In all studies exclusion criteria were limited to women who refused to participate in the trial. Two studies commenced enrollment from the first trimester and one study before 24 weeks in the second trimester. The blood group was obtained routinely in the time frame between enrollment and completion of the study. Attached to the ANC clinics were labor and delivery rooms, where women could deliver with trained midwifes. Birth weight was measured on electronic Seca scales (accuracy 10 grams) for unit deliveries and with Salter hanging scales (accuracy 50 g) for home deliveries.P. vivax (only) group” or “P. falciparum (only) group” or both P. falciparum and P. vivax detected during pregnancy. The analysis of malaria in primipara and multipara was separated for the different species as differences with gravida groups and P. vivax have previously been reported.2 test and the Bonferroni correction was applied for multiple comparisons. Birth weight was only used for analysis if the infant was weighed within 1 day after birth.Data were analyzed using SPSS for Windows version 18.0 . In the analysis, women were categorized as “free of malaria” if all the malaria smears done at antenatal visits were negative, including malaria smears of the mother peripheral and placental blood at delivery. Women with a positive malaria smear were categorized into three groups: “Blood smears (thin and thick films) were prepared using the Giemsa coloration and were read for 200 fields before declared negative. Parasite count was given by 500 white blood cells or by 1,000 red blood cells. All stages of the parasites were recorded . The blood group was tested with the agglutination test using serum anti-A and anti-B test .All studies were approved by the Ethical Committee of the Faculty of Tropical Medicine, Mahidol University Bangkok, Thailand and Oxford Tropical Medicine Ethical Committee, Oxford University, England.P < 0.001), and were less likely to have been followed up until delivery  compared with women who were tested. Out of 1,468 women 447 (30.4%) had at least one malaria episode during pregnancy, and there was no significant difference in the proportion of women with malaria in the different blood groups , or severe P. falciparum malaria (one case), were so low that statistical comparisons were not performed. Of the 54%  women who delivered in the Shoklo Malaria Research Unit (SMRU) clinic, 487 (61.4%) placenta samples were available. Only 5 placentas (1.0%) were malaria positive: for blood group O 0.5% (1 of 205), A 0.9% (1 of 110), B 2.2% (3 of 137), and AB none of 35. Of the women who had malaria during pregnancy the proportion infected with different species was not significantly different between blood groups and this non-significant association was maintained when comparing symptomatic malaria infection, the median number of infections of P. falciparum or P. vivax, the geometric mean maximum parasitemia of P. falciparum or P. vivax, or the mean hematocrit at the time of P. falciparum or P. vivax  had a blood group phenotype available: group O was the most prevalent and group AB the least prevalent . The 6.5d groups . The numP. vivax .P. falciparum or P. vivax was not significantly different between blood groups  during pregnancy. There was no indication from this large cohort where nearly one-third of women were diagnosed with malaria during pregnancy that ABO blood group phenotype relates to 1) the species of infection, 2) frequency of malaria attacks, 3) symptoms of malaria, 4) hematocrit, and 5) parasitemia. This absence of association remained true within each gravida group.This is the first study to examine the relationship between ABO blood group phenotype in women prospectively and actively followed for malaria (P. falciparum randomized controlled treatment trial in this population, placental infection only occurred in women with peripherally detected malaria infection that occurred at the time of delivery.The findings presented are in contrast to those from The Gambia and Malawi, where a relationship between blood group O and placental malaria was observed.In addition, in this setting where immunity is low, symptoms and parasitemia are correlated with disease severity and these factors were also not significantly associated with ABO blood group phenotype. There was no relationship between birth weight and ABO blood group. This is in contrast to studies from The Gambia and Malawi, where besides a relationship with placental malaria an increased birth weight and a higher ponderal index were found in neonates from women with blood group O.3This cohort is likely to be less subject to bias than previous publications as pregnant women were followed longitudinally and actively; therefore, the diagnosis of malaria did not depend upon whether a placenta was available or not. Despite the large number of women some of the sub-group analysis, especially for blood group AB, had only a small number of women. A much larger cohort from this population would be required to definitively rule out differences with blood group AB. This study does not support an association between blood group and malaria during pregnancy.


Plasmodium falciparum is associated with the capacity of the infected red blood cell (iRBC) to adhere to uninfected RBCs, a process known as rosetting, which has been linked to the occurrence of severe malaria. The present study was carried out in three Ethiopian malaria endemic localities to investigate the relationship between blood group type and severe disease in falciparum malaria.The virulence of A total of 210 cases of malaria (70 severe and 140 uncomplicated) and 190 healthy controls participated in the study. Patients with at least one of the severe malaria syndromes  were considered as severe malaria cases.In the severe malaria category, there were 25 (35.7%), 15 (21.4%), 14 (20%) and 16 (22.9%) blood group A, B, AB and O patients, respectively. Blood group O was the dominant blood type in both uncomplicated malaria (45.7%) and healthy controls (41.6%). A case of severe malaria was almost twice as likely to be of type A as to be of type O (odds ratio (OR) 0.42, 95% confidence interval (CI) 0.20-0.88, P = 0.019), and more than twice as likely to be of type B as to be of type O . Furthermore, individuals with severe malaria were about six fold less likely to be of O as to be of type AB .P. falciparum malaria, patients with blood group O, which is less prone to rosetting have a reduced chance of developing severe falciparum malaria as compared to patients with other blood groups.The study revealed that on the basis of the three criteria  used to determine severity in  Plasmodium. Of the four species that infect humans , P. falciparum is responsible for virtually all deaths . Carlsol [et al  and Roweez et al  also sugThe present study does not adjusted for the effect of other determinants such as genetic polymorphisms that might play key roles in the severity of falciparum infection, so that the effect of these factors could not be discussed in this paper.P. falciparum malaria by mechanisms other than rosetting, further studies of rosette formation with Ethiopian strains of P. falciparum could be useful to test the role of rosette formation in disease severity.The study revealed that on the basis of the three criteria used to determine severity of falciparum malaria, patients with blood group O were less prone to severe malaria as compared to patients with other blood groups. However, since ABO blood group can also affect the severity of The authors declare that they have no competing interests.ZT was involved in all aspects of the project, data collection, analysis, interpretation and determined haemoglobin concentrations and in writing of the manuscript. BP has made a contribution in the design, data interpretation, work supervision and in critically revising the manuscript. All authors read and approved the manuscript.