Plasmodium falciparum malaria. But recent evidence suggests that other risk factors are involved.Endemic Burkitt's lymphoma (eBL) has been associated with Epstein–Barr virus (EBV) and holoendemic n=258) from two locations in Nyanza Province, Kenya, with higher-than-expected and lower-than-expected incidence of eBL. The study participants were malaria asymptomatic children from the community.We hypothesised that selenoprotein glutathione peroxidase (GPx), a surrogate of nutritional status, is an important biomarker for eBL risk. We measured plasma GPx, anthropometric markers of malnutrition, EBV viral loads and malaria parasitaemia in children aged 1–9 years (P<0.001). Additionally, GPx levels were significantly lower in children with the highest EBV viral load and for those with P. falciparum infections .Children from eBL high-incidence areas had significantly lower GPx levels, high EBV viral load and more evidence of chronic malnutrition than children from eBL low-incidence areas (all These results suggest that selenium deficiency may be a risk factor for eBL. Endemic Burkitt's lymphoma (eBL) is the most prevalent paediatric cancer in sub-Saharan Africa , althougNyanza Province in western Kenya is an area with a high burden of both eBL and malaria . Howevern=258) who were permanent residents of a sub-location that experienced higher-than-expected cases of eBL  and sub-locations with lower-than-expected cases of eBL  . Study participants were enroled using written informed consent forms through home visits or at organised centres within the study sites. The approved ethical form detailing the purpose of the study and contact persons was availed and duly signed after conducting study participants or their guardians through the reading of the form using a language of choice. Inclusion criteria were residence in the study areas for the past 6 months, absence of severe illness and consenting to the study. The exclusion criteria were presence of severe chronic anaemia (haemoglobin <5 g per 100 ml), blood transfusions within the last 6 months, fever >37.5 °C and residence in an orphanage. The study was approved by the institutional review boards of the SUNY Upstate University and the ethical review committee for the KEMRI .Plasmodium parasites per 200 leukocytes was determined and parasite density was calculated by multiplying with the actual white blood cell count.Data collection included a questionnaire, completed using face-to-face interviews with guardians as well as a venipuncture blood sample, auxilliary temperature (°C) and height and weight measurements of study participants. Non-fasting blood was collected in the field between 2006 and 2008. Variations were minimised by using the same types of blood collection and processing tubes. Between 5 and 9 ml of blood was collected by venipuncture into heparinised tubes. Plasma was separated by centrifugation under subdued light in a Biosafety hood in the KEMRI laboratory. The samples were then frozen at −80 °C in freezers. Haemoglobin measurements (g per 100 ml) were conducted on site with a portable Hemocue (USA) before confirmation by Beckman Coulter AcT diff2  in the laboratory. Finger-prick blood samples were collected in EDTA tubes and stored at −80 °C for later DNA extraction. Thick and thin blood smears were prepared for malaria parasite screening and density calculation following the WHO criteria. The number of asexual μg per 100 ml) were determined in a subset of participants, n=65 and n=93, from the low and high eBL incidence regions, respectively.Levels of GPx were determined in plasma samples by enzyme-linked immunosorbent assays (ELISAs) using a commercially available kit  and following the manufacturer's protocol. The ELISA plate was read through a computer-aided ELISA reader. Results were interpolated from standard curves generated with recombinant human GPx-selenium protein supplied by the manufacturer. GPx levels  according to the manufacturer's protocol. DNA was eluted off the column in an equivalent volume of H2O and stored at −20 °C. DNA (5 μl) was used to determine EBV viral loads by real-time Q-PCR and the viral loads were calculated as viral copies per μg total DNA as previously described (n=104 from the low eBL IR area vs n=127 from the high eBL IR area.DNA was extracted from 200 Z-scores <−2. Height-for-age (stunting), weight-for-height (wasting) and weight-for-age (underweight) Z-scores were calculated using growth reference curves developed by the NCHS  using Epi Info 6.04 .Weight and height measurements were taken for use in the determination of whether the children were wasted, stunted or underweight, based on t-test and one-way analysis of variance (ANOVA), medians were compared with the Mann–Whitney U, whereas proportions were evaluated using Pearson's χ2 test. Statistical associations between variables were further examined using Pearson's correlation coefficient. Variables with significant skewness were transformed towards normality. The P-values of <0.05 were considered statistically significant for all analyses. Multivariable analyses were also conducted using logistic regression to evaluate associations between possible cofactors  and locale  with control for child age. Variables evaluated in this model included continuous GPx (μg per 100 ml), malaria parasite positivity , log-transformed EBV levels and child age in years. GPx was also categorised into quartiles to investigate a possible nonlinear association between GPx levels and locale. Logistic regression models included odds ratios (ORs) and 95% confidence intervals (CIs).The statistical analyses were performed using SPSS ; for windows (versions 17.0 and 18.0) (IBM CO.) and GraphPad Prism for windows software (version 5.01) . Means were compared using the n=258) are presented in P=0.68). Children residing in a high eBL incidence region were found to have lower haemoglobin concentration compared with those from the eBL low-risk area (P=0.016) and more cases of anaemia  (P<0.001) compared with children from a low eBL incidence area.We took advantage of disparate incidence rates of eBL in proximally located villages in Nyanza Province Kenya to ask whether there were other biological factors that would affect eBL incidence rates. We recruited children between the ages of 1 and 9 years with an equal distribution of children between children aged 1–4 and 5–9 years . We chosn=65) and high-incidence areas (n=93). The demographic and clinical characteristics of study participants analysed for GPx levels are shown in μg per 100 ml, respectively, were observed . When comparisons between sites were made analysing unique indicators of malnutrition, we observed a significantly higher proportion of children with stunting from the eBL high-risk area than the eBL low-risk area (P=0.003). The proportion of children who were wasted or underweight was comparable between eBL low- and high-risk regions . Further analyses showed that children aged 1–4 years had a greater proportion of individuals with wasting condition than those aged 5–9 years (P=0.006). The rest of the malnutrition conditions did not differ significantly between the two age groups. The proportion of children who were wasted, stunted, underweight or had any of the malnutrition conditions did not differ significantly based on gender.Because GPx is a selenium-based enzyme, we wanted to further examine these children for evidence of malnutrition, as decreased GPx could be an indicator of underlying malnutrition. We evaluated the n=104) and high-incidence regions (n=127) (P<0.001). Among those with detectable EBV, the log viral loads were significantly higher in children residing in the region with high eBL incidence (n=59) compared with the low eBL incidence area (n=74); the log viral load means were 3.33 (s.d. 1.56) and 2.57 (s.d. 0.81), respectively (P<0.001).In areas where malaria transmission is perennial and intense, EBV persistence is altered, resulting in elevated EBV viral loads in young children compared with children with minimal exposure to malaria ( (n=127) . DetectaP<0.001), with 62% of children from high eBL incidence areas positive for malaria parasites compared with 22% from the low eBL area. The mean temperature of children between both sites was within the normal range (36.4 vs 36.6°C), indicating that the children included in the study were experiencing asymptomatic parasitaemia. Of the children who were positive for parasites on the blood smear, the geometric mean parasite density was not statistically different between the sites . Similar, but nonsignificant, findings were observed in the low and high eBL region as well; mean log EBV viral loads for children with and without malaria parasites residing in the low eBL region were 2.86 (0.87) and 2.49 (0.78), P=0.11, whereas mean viral loads among children residing in the high eBL region, with and without malaria parasites, were 3.59 (1.63) and 2.92 (1.38), P=0.11.Because we observed significant differences in three parameters  between the children residing in the low and high eBL risk locations, we wanted to determine if there were correlations between these parameters. We found that plasma GPx levels were significantly lower among participants with the highest EBV viral load  and among those with malaria parasites in peripheral blood shown in . For chivs high eBL regions and GPx levels while controlling for child age, malaria positivity and log EBV load, higher GPx levels were associated with a decrease (69%) in the likelihood of residing in the high eBL region; the OR (95% CI) for a one-unit increase in GPx (in μg per 100 ml) was 0.31 . Malaria smear positivity was associated with a seven-fold increase in the likelihood of residing in the high eBL region . Log EBV load was not significantly associated with residence in the high eBL region (P=0.99). A similarly fitted model using quartiles of GPx rather than the continuous version indicated that the significant decrease in the likelihood of residing in the high eBL region was observed only for the highest GPx quartile relative to the lowest (data not shown).In a multivariable logistic regression model evaluating residence in the low The identification of microgeographic variation in eBL incidence in western Kenya  were previously combined as one district (Kisumu district), which had been a district classified as having Lake Endemic malaria transmission , but ouret al, unpublished results). Thus, elevated viral loads could result in increased ROS via EBNA-1. In a population with reduced GPx levels, this could be one potential mechanism for an increased risk for eBL. Repeated malaria infections also induce oxidative stress and could potentially enhance the effects of EBNA-1 in B cells.We found that the children with the lowest GPx levels had the highest EBV viral loads. In addition, there was a strong trend that found that children with stunting had high EBV viral loads. There is little information on potential links between EBV and micronutrient deficiency or malnutrition. Interestingly, however, is a recent report that shows that the EBV Epstein–Barr nuclear antigen (EBNA)-1 protein can induce ROS that promote genomic instability . We haveIn this study, we identified reduced plasma levels of the selenoprotein GPx, increased frequency of chronic malnutrition, elevated viral loads and a higher malaria burden in Kenyan children at high risk for eBL compared with those with a lower risk for eBL. Increased GPx levels were associated with a significantly decreased likelihood of residing in the high eBL region after controlling for child age, malaria positivity and log EBV load. A major limitation of our cross-sectional study is that it cannot indicate whether reduced GPx levels is a causal factor for eBL or a marker of oxidative stress resulting from increased infectious burden due to malaria and EBV. Nonetheless, our study highlights the complex relationships between malnutrition and infectious diseases, and is the first to explore whether micronutrients and malnutrition could have a role in eBL aetiology.


Control of malaria in pregnancy remains a public health challenge. Improvements in its correct diagnosis and the adequacy of protocols to evaluate anti-malarial drug efficacy in pregnancy, are essential to achieve this goal.Plasmodium falciparum was assessed by real-time (RT) PCR in 284 blood samples from pregnant women with clinical complaints suggestive of malaria, attending the maternity clinic of a Mozambican rural hospital. Parasite recrudescences in 33 consecutive paired episodes during the same pregnancy were identified by msp1 and msp2 genotyping.The presence of Prevalence of parasitaemia by microscopy was 5.3% (15/284) and 23.2% (66/284) by RT-PCR. Sensitivity of microscopy, compared to RT-PCR detection, was 22.7%. Risk of maternal anaemia was higher in PCR-positive women than in PCR-negative women . Genotyping confirmed that recrudescence after malaria treatment occurred in 7 (21%) out of 33 pregnant women with consecutive episodes during the same pregnancy (time range between recrudescent episodes: 14 to 187 days).More accurate and sensitive diagnostic indicators of malaria infection in pregnancy are needed to improve malaria control. Longer follow-up periods than the standard in vivo drug efficacy protocol should be used to assess anti-malarial drug efficacy in pregnancy. Plasmodium falciparum infection and disease [Pregnant women are at higher risk of  disease , frequen disease ,3. Infec disease .P. falciparum infections impose serious difficulties in the identification of malaria episodes. Until recently, malaria diagnosis relied on the microscopic detection of Plasmodium on Giemsa-stained blood smears. Lately, circulating antigen [P. falciparum parasitaemia levels below the threshold of microscopy [Control of malaria in pregnancy remains a challenge due to a number of factors. The low specificity of signs and symptoms ,6 and th antigen  and poly antigen -12 have croscopy -12. Realcroscopy ,14, has croscopy ,16.P. falciparum parasites [The declining efficacy of classically recommended anti-malarial drugs due to the increase of anti-malarial-resistant arasites , resultsarasites -21. CritIn this study, a PCR-based approach to assess the impact of sub-microscopic infections on disease manifestation of pregnant women attending the maternity clinic of a rural hospital in Mozambique was used. Genetic characterization of parasites isolated from consecutive episodes during the same pregnancy allowed to determine the extent of parasite recrudescences after anti-malarial treatment.P. falciparum. Anopheles funestus is the main vector, and the estimated entomological inoculation rate for 2002 was 38 infective bites per person per year [The study was carried out at the Centro de Investigação em Saúde da Manhiça (CISM) in Manhiça District, southern Mozambique. Adjacent to the CISM is the Manhiça District Hospital (MDH), a 110 bed health facility, which provides curative and preventive services. The characteristics of the area have been described in detail elsewhere . Perenniper year . The mosper year .P. falciparum parasitaemia and haematocrit, and blood on DNA filter paper  were collected if at least one of the pre-defined clinical criteria suggestive of malaria was met. A random selection of 10% of the filter papers collected, and all the available filter papers from women with two consecutive malaria episodes during the same pregnancy, were analysed by RT-PCR.This study was conducted in the context of a health facility-based descriptive study that aimed to characterize the clinical presentation of malaria in Mozambican pregnant women and to evaluate the adequacy of case management based on clinical complaints . A roundFollowing national guidelines at the time of the study, women were treated with SP plus CQ in case of non-complicated malaria, except for the first trimester when CQ alone was given. Oral quinine (Q) for seven days was given as rescue treatment. Women with complicated malaria were admitted at the maternity ward, and treated with parenteral quinine followed by SP. Anaemia was treated with oral ferrous sulphate and folic acid for one month. The study protocol was approved by the National Mozambican Ethics Committee and the Hospital Clinic of Barcelona Ethics Review Committee.P. falciparum asexual stages per 200 leukocytes, according to standard, quality-controlled procedures [Thick and thin blood films, in duplicate, were stained with Giemsa and read to determine parasite species and density of ocedures . The hae® Universal PCR Master Mix (Applied Biosystems), a 300 nM concentration of each primers specific for 18S ribosomal RNA gene of P. falciparum, and a 150 nM concentration of probe labelled with 6-carboxy-fluorescein (FAM) as a reporter and 6-carboxytetramethylrhodamine (TAMRA) as a quencher. Amplification and detection were performed under the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The results were automatically analysed by the ABI Prism SDS2.1 software. Each specimen was run in duplicate. A standard curve was prepared from titrated samples containing known numbers of ring-infected erythrocytes diluted in whole blood. The highest dilution of the curve that gave reproducible results in all the optimization assays was the equivalent to 50 parasites per ml of blood. The standard curve was run in triplicate for each test. RT-PCR efficiency was validated by comparing the slope of the standard curve to the theoretical optimum of -3.32 which reflects 100% efficient amplification, and was found to be -3.21 ± 0.13, R2 = 0.99 (n = 5).Parasite DNA was extracted from 50 μl blood on filter paper by using the ABIPrism 6700 Automated Nucleic Acid Work Station (Applied Biosystems), according to the manufacturer's instructions. Negative extraction controls consisting of water or non-infected erythrocytes were included in the process. Purified DNA templates were amplified in an ABI PRISM 7900 HT Real-Time System (Applied Biosystems) following a previously described method . BrieflyPlasmodium falciparum infections from consecutive malaria episodes during the same pregnancy were characterized on the basis of the fragment size of the amplified msp1  and msp2  loci using a previously described PCR approach [msp1 and mp2 alleles circulating in the study area was calculated by measuring the proportion of each detected genotype, as defined into 20-base pair ranges, in 60 isolates from pregnant women [approach . Each baP. falciparum parasitaemia of any density, and presence of any sign and/or symptom suggestive of malaria  [P. falciparum infections were grouped into the following categories: a) microscopically confirmed parasitaemia; b) RT-PCR confirmed parasitaemia; c) sub-microscopic infections (negative samples by microscopic examination which gave a RT-PCR positive result); d) no infection (negative by microscopy and RT-PCR). In this study, the duration of a single malaria episode was estimated as 28 days so as to differentiate between visits on the same episode and a new episode. Multiplicity of infection (MOI) was defined as the maximum number of msp1/msp2-amplicons detected in the sample. To eliminate the possibility of misclassifying patients with common msp1 and msp2 variants as treatment failures, recrudescence was only considered when all strains in the second episode were present in the previous episode, and no new genotypes were found . Infections in the second episode that consisted on different strains not present in the previous episode were designated as new infections. When the second malaria episode contained strains present in the previous episode but also new strains, infections were categorized as mixed [A malaria episode was defined as a  pallor) . Fever wmsp1 and msp2 genotypes detected in 60 pregnant women [p-value [p-value < 0.05 was considered statistically significant.Double data entry, validation, and cleaning were done using Microsoft Visual FoxPro 5.0, and statistical analysis was performed using STATA.9 . To evaluate the association between the presence of signs and symptoms suggestive of malaria, age, parity, gestational age, number of previous malaria episodes and season with parasitaemia, univariate logistic regression models were used. Sensitivity and specificity of microscopic determination was calculated considering RT-PCR as the gold standard. The cumulative probability of two parasites in two independent episodes being by chance the same for a particular 2-loci genotype was calculated from the frequencies of nt women . Days be[p-value . A permuDuring the study period (August 2003–November 2005), 3,129 filter papers corresponding to first visits with criteria for blood collection and complete laboratory results were collected from pregnant women attending the MDH. Three hundred out of these samples were randomly selected. Sixteen filter papers were not found. Among the women from whom the 284 filter papers were available, 70.9% (200/282) were 20–34 years of age, 59.4% (168/283) were in their second to fourth pregnancies, and 48.2% (137/284) had anaemia.P. falciparum malaria, both by microscopy and RT-PCR, was equal across age and parity groups  and 66 (23.2%) had malaria parasitaemia as detected by microscopy and RT-PCR, respectively. Fifty-one (18.9%) of the 269 women found to be aparasitaemic by microscopy were positive by RT-PCR (sub-microscopic infections). Compared with RT-PCR detection, microscopy showed a sensitivity of 22.7%. Prevalence of There was no evidence of an association between signs or symptoms suggestive of malaria and the presence of microscopically detected peripheral malaria Table . Risk ofmsp1 and 20 msp2 genotypes were found in 60 pregnant women, with allelic frequencies ranging from 0.008 to 0.18  occurred in 7 (21.2%) of the 33 women. In 22 (66.7%) of the women, the second episode consisted of P. falciparum isolates with msp1 and msp2 genotypes not present in the previous episodes (new infections). Mixed infections (both recrudescent and new strains present) were present in 4 (12.1%) of the women. Mean duration of the interval between consecutive episodes was similar in recrudescent  and new infections  (p = 0.84). No statistical difference was found in the mean multiplicity of infection (MOI) from first  and second episodes  (p = 0.33), and from recrudescent  and new infections  (p = 0.82).Sixty-six filter papers were analysed from 33 women with two consecutive malaria episodes during the same pregnancy  and the high prevalence of sub-microscopically infected women (18.9%). In other settings, sub-patent malaria infections have been found in up to 55% of the pregnant women [This study described the prevalence of d women 1.9%. In ont women . This sunt women ,31. Howent women ,32,33.P. falciparum, as detected by microscopy (94.7%) and even by RT-PCR (76.8%). Thus, in this context, presumptive malaria treatment would lead to unnecessary exposure to anti-malarial drugs in the majority of pregnant women presenting with "malaria-like" clinical complaints [P. falciparum infection both in pregnant women [In this study, the majority of women presenting clinical complaints suggestive of malaria were not infected with  RT-PCR 7.8%. Thus RT-PCR 7.8%. Thusnt women .In this study, anaemia was the only symptom associated with parasitaemia as detected by RT-PCR. This association was not found for microscopical parasitaemia in this subset of samples, maybe due to limitations of the sample size. Women with sub-microscopic infections were also at higher risk of anaemia compared with women with negative microscopy and RT-PCR test, suggesting a role of low-density infections in the aetiology of maternal anaemia. A similar association has been found in some studies ,34, but P. falciparum parasites during pregnancy can occur as late as 187 days. In other studies, reappearance of parasites has been shown to occur 35, 49, 85 and 119 days after treatment [PCR-confirmed recrudescences were found in 21% of the women experiencing two successive episodes during the same pregnancy. The rate of recrudescences found in this study raises important questions for drug efficacy in pregnancy. First, it suggests that the efficacy of anti-malarial drugs during pregnancy may be reduced. This can be due to inadequate drug levels, limited clearing capacity of placental parasites, or to immunosuppresion and physiological changes during pregnancy which are likely to alter the pharmacokinetics of most anti-malarials -21,36. Sreatment ,38. In Treatment . Thus, treatment  may not reatment -42 to 63reatment ,38 afterThese results point out two important issues for malaria control during pregnancy; on the one hand, the need to apply more accurate and sensitive measures to detect malaria infections in pregnancy. On the other hand, it might be necessary to extend the follow-up period for in vivo tests to correctly identify anti-malarial drug failures during pregnancy. Appropriate, standardized genotyping methods should be applied to unequivocally identify recrudescent parasites. This is essential to optimize the drug regimens needed to eliminate the plasmodial biomass in pregnant women, and to correctly measure the efficacy of the existing and new anti-malarials to be used in pregnancy.The authors declare that they have no competing interests.AM and ES carried out the molecular genotyping study, the analysis and interpretation of data and prepared the manuscript. LP and PC contributed to the molecular genotyping study and the interpretation of data. SS and JA carried the statistical analysis and helped to draft the manuscript. AB and IM carried out the sample collection and contributed to the analysis and interpretation of data. BS contributed to the interpretation of the data and drafting of the paper. CM and PA conceived and coordinated the study, participated in the analysis and interpretation of the data, and contributed to the preparation of the manuscript. All authors read and approved the final manuscript.


Severe anaemia is a common cause for hospitalization in children in sub-Saharan Africa. Malaria plays an important aetiological role, resulting in a substantial burden of paediatric transfusion in hospitals. A decline in malaria and paediatric admissions to the Kilifi District Hospital has been reported recently. This study aimed to investigate whether this trend affected clinical burden, clinical severity of anaemia and requirements for paediatric transfusion.Eight-year retrospective review of paediatric admissions to Kilifi District Hospital, Kenya describing the frequency of moderate and severe anaemia, blood transfusion and case fatality over time. Definitions for severe anaemia were Hb <8 g/dl for newborns and <5 g/dl for other age groups and for moderate anaemia was Hb 8 to <11 g/dl for newborns and 5 to <9.3 g/dl for other age groups. Life threatening anaemia was defined as severe anaemia (Hb <5 g/dl) complicated by either deep breathing or prostration or profound anaemia (Hb <4 g/dl) alone.2 for trend = 134, P < 0.0001). The number and proportion of admissions transfused also declined significantly over this time . Of the 2,265 children with severe anaemia 191 (8%) died. Case fatality remained unchanged during this period (P < 0.26) and was largely explained by the unchanged proportion with life-threatening anaemia, present in 58-65% of cases throughout the study period.Of the 35,139 admissions 13,037 (37%) had moderate anaemia and 2,265 (6%) had severe anaemia; respiratory distress complicated 35% of cases with Hb <5 g/dl. Concurrent with the decline in malaria there was a marked decline in the prevalence of severe anaemia between 2002 (8%) and 2009 (< 4%) (chiThe impact of reduced malaria transmission on child morbidity has positive public benefits on the demand and use of blood for paediatric transfusion. Despite an overall reduction in paediatric transfusion requirement, case fatality of severe anaemia remained unchanged over this decade. Further research is required to improve outcome from severe anaemia, particularly in the high-risk group with life threatening features. In sub-Saharan Africa severe anaemia is a leading cause of paediatric admission to hospital. The community prevalence of anaemia in African children, defined as a haemoglobin (Hb) <11 g/dl, ranges from 49% to 89% and severe anaemia  affects between 1%- 6% of children, with infants being the most vulnerable age group,3, resulPlasmodium falciparum malaria infection[The aetiology of anaemia is frequently multifactorial, commonlinfection and nutrinfection,7. The rinfection. Despiteinfection. Blood tinfection,9. Howevinfection-12. WHO infection. Post-diinfection.et al evaluated the practice between 1998 and 2000 and found that 13% of all paediatric admissions (excluding newborns) were transfused[Despite these guidelines being in place for many years in Africa, up to 50% of hospitalized children in malaria endemic receive a blood transfusion. Malariaransfused. This re® database, which is password-protected and has limited access. Data on the following were retrieved; age, sex, diagnosis, falciparum malaria parasitaemia, haemoglobin (Hb) level, respiratory distress, level of consciousness, nutritional status, capillary refill > 2 seconds (as a sign of hypovolvaemic shock) together with data on blood transfusion, length of stay and mortality as outcome variables.A retrospective study of hospital admissions to address the following specific objectives. First, to describe the proportions of anaemia and blood transfusions secondary to malaria and describe the changes over time. Secondly, to examine compliance with the threshold for blood transfusion, established by the World Health Organization (WHO) current transfusion guidelines, and finally, to determine in-hospital survival. Retrospective data review of the clinical surveillance database covering all child and neonatal admissions to Kilifi District Hospital (KDH), Kenya, over an 8-year period, from January 2002 to December 2009. Clinical data are routinely collected prospectively at admission and directly entered into a Filemaker ProKilifi District is one of the poorest area in Kenya, where malaria is endemic, with 40% of children under five years presenting anthropometric measures of malnutrition, half with biochemical markers of iron deficiency, 14% of children have a sickle cell trait and nearly 60% have α+ thalassaemia deletion genotypes .Proportions of transfusions due to malaria and other diagnoses were calculated for each year and the proportions compared over time. Since transfusion thresholds are different for neonates, we analysed the trends in neonates separately from those of older children. Children outside the neonatal period (≥ 1 month) were further sub-grouped into infants (1-12 months), 1-4 years, and older then 5 years since the prevalence of severe malaria anaemia varies considerably over these age groups. The children were further categorised by their clinical severity features.Severe anaemia was defined as Hb <8 g/dl for neonates and <5 g/dl for other age groups; profound anaemia in non-neonatal groups was defined as Hb <4 g/dl. As defined by WHO criteria for anaemia: moderate anaemia was a Hb 8 - <11 g/dl for newborns and 5 - <9.3 g/dl for other age groups; .mild or no anaemia as Hb >11 g/dl for newborns and > 9.3 g/dl for other age groups. Respiratory distress was defined as either the presence of deep breathing  or indrawing. Nutritional status was measured by the parameter birth weight or gestational age for neonates . For the other age groups severe malnutrition was defined as either mid upper arm circumference (MUAC) <11 cm and/or presence of oedema (kwashiorkor). Impaired consciousness indicated the presence of prostration (inability to sit unsupported if > 9 months or inability to breast feed if ≤ 9 months). Life-threatening anaemia was defined as either profound anaemia (Hb <4 g/dl) irrespective of clinical severity or severe anaemia (Hb <5 g/dl) complicated by deep breathing or prostration. Stata version 11  was used for data analysis for descriptive statistics.2 test for trend = 2440, P < 0.0001), which is covered in more detail in a previous report[P. falciparum malaria (46%) and all types of malnutrition with 13%  was available for 35,139 and missing in 1482 (3.9%). Moderate anaemia occured in 13,037 (37%) and 2,265 (6%) had severe anaemia . Overall, 8% of admissions in 2002 had severe anaemia which decreased to < 4% by 2009. The effect was the most dramatic in infants and young children where proportions with severe anaemia declined from 10% of admissions to 2%. A steep decline was present in most age groups except children 5-12 years who showed an initial steep decline in prevalence in the years 2002-2005, which plateaued then rose again in 2008.Concurrent with the decline in malaria there was a marked decline in the prevalence of severe anaemia Figure  from 2002 for trend = 152, P < 0.0001) mirroring the reduction in severe anaemia . The majority of transfusions, 55% , were received by children with severe anaemia, 29% (789) were received by children admitted with moderate anaemia Hb 5.0-9.3 g/dl) and 14% (375) received by children who had mild or no anaemia (Hb >9.3 g/dl) at admission. Two percent of transfusions were given to children whose Hb values were not recorded on admission. These relative proportions were constant throughout the period of the study. The number and proportion of admissions transfused declined significantly over time (chi g/dl andOf the 2265 children with severe anaemia 191 (8%) died. The highest case fatality was in the neonatal group 31%)  and demand on transfusion services we found that this had little effect on the overall case fatality rate of children admitted with severe anaemia, with mortality remaining unchanged at 8% throughout the study period. This was largely explained by the proportion of children with severe anaemia with life threatening complications, which remained constant over the period of study, affecting over 60% requiring transfusion.These data suggest that the impact of reduced malaria transmission on child morbidity are greater than those measured by changes in malaria admissions alone. This is reinforced by Gerritsen and colleagues study in South Africa (Limpopo) which found a significant reduction of malaria from 1998 to 2007 . The draThere are a number of potential explanations why reducing the burden of malaria disease and transfusion requirements did not result in a reduction in case fatality. First, that the proportion with life threatening anaemia did not decline and thus the proportion requiring emergency transfusion remained unchanged. Previous work in Kilifi in 2000 by English and colleagues demonstrated that respiratory distress, prostration and profound anaemia, were the clinical features most associated with death, odds ratios of 4.1 (95% CI 2.2 - 7.4), 7.4 (95% CI 4.2-13.1) and 2.5 (95% CI 1-4 - 4.5) respectively. This daWhilst most groups showed a decline in anaemia and transfusion overtime, there was an increase in children aged 5-12 years in the years 2008-2009. One possible suggestion is that other causes of anaemia were becoming more prominent as a cause of admission in this age group. Unfortunately, in this retrospective study the degree of fine resolution offered by the recorded final diagnosis of severe anaemia does not permit a greater insight into causality.The effect of introduction of guidelines or protocols that include rationale transfusion practice, restricting blood transfusion use to only those at greatest risk has been examined in several randomized studies in adults, children and newborns. A review of these meta-analyses concludes that restrictive strategies reduce the need for transfusion, in the UK, without increasing morbidity and mortality. CompliaThe consequence of a reduction in the number and proportion of cases admitted with falciparum malaria was a decrease in the burden of severe malarial anaemia and a reduction in the demand for paediatric transfusion. Although the reduced malaria burden leads to a welcome reduction in the transfusion requirement the case fatality for those admitted with severe anaemia remains unacceptably high. The most recent systematic reviews,21 indicThe authors declare that they have no competing interests.RP did the primary data analysis and prepare the initial and subsequent versions of the manuscript. SA supervised the data analysis and prepartion of the results table and commented on versions of the manuscripts. KM conceived the study, reviewed the data and draft versions of the manuscript. All authors read and approved the final manuscript


D vaccine has been shown to have a promising safety profile, to be immunogenic and to confer protection against malaria in children and infants.The RTS,S/AS02D given at 10, 14 and 18 weeks of age staggered with routine immunization vaccines in 214 Mozambican infants. The study was double-blind until the young child completed 6 months of follow-up over which period vaccine efficacy against new Plasmodium falciparum infections was estimated at 65.9% . We now report safety, immunogenicity and estimated efficacy against clinical malaria up to 14 months after study start. Vaccine efficacy was assessed using Cox regression models. The frequency of serious adverse events was 32.7% in the RTS,S/AS02D and 31.8% in the control group. The geometric mean titers of anti-circumsporozoite antibodies declined from 199.9 to 7.3 EU/mL from one to 12 months post dose three of RTS,S/AS02D, remaining 15-fold higher than in the control group. Vaccine efficacy against clinical malaria was 33%  over 14 months of follow-up. The hazard rate of disease per 2-fold increase in anti-CS titters was reduced by 84% .We did a randomized, controlled, phase I/IIb trial of RTS,S/AS02D malaria vaccine administered to young infants has a good safety profile and remains efficacious over 14 months. A strong association between anti-CS antibodies and risk of clinical malaria has been described for the first time. The results also suggest a decrease of both anti-CS antibodies and vaccine efficacy over time.The RTS,S/AS02NCT00197028ClinicalTrials.gov  Plasmodium falciparum malaria is one of the most serious public health problems worldwideP. falciparum infection, uncomplicated malaria and severe disease, and that protection lasted for at least 45 months The last decade has witnessed important progresses in the development of a first generation malaria vaccine. GlaxoSmithKline's (GSK) RTS,S, formulated with the Adjuvant System AS02 or AS01, is currently the world's most clinically-advanced malaria vaccine candidate. This vaccine has been shown to be safe and efficacious against malaria infection and disease in adult naïve and semi-immune volunteers D in children less than 12 months of age. Vaccine efficacy (VE) against malaria infection was 65.9%  at the end of 6 months of follow-up Recognizing that malaria control strategies must prioritize protection in infants D in Tanzanian infants has recently shown very similar results [vaccine efficacy of 65.2% ] E in Tanzania and Kenya yielded a 53%  reduction of clinical malaria episodes over an 8 month follow up period A subsequent trial of the RTS,S/AS02This paper reports the safety, reactogenicity, immunogenicity and efficacy of the complete 14 months follow-up period of the Mozambican phase I/IIb proof-of-concept trial in infants, with particular emphasis on safety and reactogenicity, given that it was the first time that RTS,S formulated with AS02 was administered to infants.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study was carried out by the Centro de Investigação em Saúde de Manhiça (CISM) in the rural areas of Taninga and Ilha Josina Machel, 50 Km north of Manhiça village, Mozambique, from June 2005 to December 2007. Detailed description of the area can be found elsewhere D vaccine administered to infants at 10, 14 and 18 weeks of age, staggered with EPI vaccines (DTPw/Hib [TETRActHib™ Aventis Pasteur]) at 8, 12 and 16 weeks of age. The study was double-blind until the youngest child completed 6 months of follow-up. After the unblinding, the study was considered single blinded although both participants and field investigators remained blinded. Only a senior statistician had access to the treatment codes allocated to the subjects, and he was not involved in the children follow-up. Data provided to the field investigators did not include information of the allocated treatment per subject during the entire duration of the trial.This study was a phase I/IIb, randomized controlled trial to assess the safety, immunogenicity and efficacy of the RTS,S/AS02D or the control hepatitis B vaccine, Engerix-B™. Details of the malaria and control vaccines as well as the trial profile for the double-blind phase have been presented elsewhere A total of 214 children were enrolled and randomized to receive either RTS,S/AS02th, 2007, when the last recruited child completed 14 months of follow-up.Infants were screened between 6 and 12 weeks of age and a second written informed consent was obtained from parents/guardians of all participants. Infants were enrolled if they were born after a normal gestational period and in the absence of obvious medical abnormalities. Children born to Hepatitis B and HIV positive mothers were not included in the trial. Children were excluded as well from participation if BCG vaccine had not been given at least one week before the first study vaccination or if any other vaccinations, other than the first dose of oral polio vaccine (OPV) given at birth with BCG, had been given prior to enrolment. Identification cards were provided soon after recruitment. Study activities were completed on December 27The protocol (NCT00197028) was approved by the Mozambican National Bioethics Committee, the Hospital Clínic of Barcelona Ethics Review Committee and the PATH Human Subjects Protection Committee and implemented according to the International Conference of Harmonization and Good Clinical Practices guidelines. GSK monitored the study. A Local Safety Monitor and a Data and Safety Monitoring Board oversaw the design, conduct and results of the trial.The sample size for the original study was based on an evaluation of vaccine safety Safety endpoints included the occurrence of solicited and unsolicited symptoms within 7 and 30 days after each vaccination respectively and the occurrence of serious adverse events (SAEs) during the entire 14 month follow-up period. All SAEs were reported within 24 hours after detection.Vaccine safety was evaluated using active and passive follow-up. All study participants were observed for at least one hour after each vaccine dose by a physician equipped with an emergency kit. Children were visited daily in their homes for 6 days after vaccination where any adverse events (AEs), local or general, were registered on diary cards. Study physicians evaluated all suspected grade 3 AEs and guided clinical management.Passive follow-up was done through a health facility based morbidity surveillance system Participants with SAEs were followed-up until events resolved. Deaths occurring at home were investigated by a review of available medical records and by verbal autopsy, as described elsewhere 9/L, platelets ≥100×109/L, ALT ≤60 µmol/L, creatinine ≤45 µmol/L and bilirubin ≤34 µmol/L.Safety monitoring of hematological parameters  and biochemical parameters  were measured one week after dose 1 and 1, 3½, and 12 months after dose 3. Normality values considered were: hemoglobin ≥80 g/L, hematocrit ≥25%, WBC 5-17×10Biochemical, hematological and packed cell volume (PCV) tests were determined as described elsewhere P. falciparum circumsporozoite protein (anti-CS) at screening and 1, 3½ and 12 months post dose 3.Antibody titres were measured against hepatitis B surface antigen (anti-HBs) and Anti-CS antibodies were measured by a standardized ELISA, using plates absorbed with recombinant R32LR with an assay cut-off of 0.5 EU/mL. Anti-HBs antibodies were quantified using the EIA kit from Abbott Laboratories and a GSK validated sandwich ELISA described elsewhere P. falciparum were ascertained by a combination of passive case detection (PCD) and active detection of infection (ADI) as described elsewhere Cases of clinical malaria and malaria infection by P. falciparum asexual parasitemia >500/µL. This definition has a sensitivity and specificity >90% in this age group P. falciparum parasitemia.The primary case definition of clinical malaria was the presence of fever (axillary temperature ≥37.5°C) with a P. falciparum malaria as well as multiple episodes of clinical malaria detected by PCD during 14 months after dose 1. An additional endpoint was first or only clinical episode of P. falciparum malaria detected by a combination of ADI and PCD.Exploratory efficacy endpoints were first or only clinical episode of 3–9) and study months 3–14 (ATP3–14). VE explored both first or only episode and multiple episodes of clinical malaria detected during the two study periods.Analyses were done for intention to treat (ITT) and according to protocol (ATP) cohorts, following a predefined analytical plan. The ITT cohort included all children who received at least one dose of the study vaccine. All safety analyses were based on ITT. The ATP cohort included participants that met all eligibility criteria, completed the vaccination course and contributed to follow-up time during the evaluation period. For exploratory analyses, the ATP cohort was split into two follow-up periods: follow-up over study months 3–9  with 95% confidence intervals (95% CI). Titres below the cut-off were assigned an arbitrary value of half the cut-off of the assay for the purpose of GMT calculation.Person years at risk (PYAR) accounted for absences from the study area and use of antimalarial drugs as previously described VE was defined as 1 minus the hazard ratio multiplied by 100 [(1 – HR)*100] and was adjusted for distance to health facility A test based on the Schoenfeld residuals was performed to assess whether the hazard was constant over the surveillance period, and alternative approaches were applied if the assumption of proportional hazards was not supported.D recipients who either did or did not experience at least one episode of clinical malaria meeting the primary case definition over ATP3–14 follow-up, using the Wilcoxon Rank Sum test. The hazard rate per 2-fold increase in post-vaccination anti-CS response was calculated for both ATP3–9 and ATP3–14 follow-up, along with their 95% confidence intervals.The risk of clinical malaria as a function of immune response was evaluated by comparing post-vaccination anti-CS titters for RTS,S/AS02Analyses were performed using SAS version 9.1  and STATA version 10 .D group (107) or the control group (107). A total of 177 children completed the 14 months follow-up period: 91 in the RTS,S/AS02D group and 86 in the Engerix-B™ group  experienced grade 3 solicited general symptoms following either the first or the second TETRActHib™ dose  (data not shown).Unsolicited AEs occurring within 30 days following vaccination were reported by 86.9% of participants in both vaccine groups. In both groups, the most frequently reported diagnosis was upper respiratory tract infection , bronchopneumonia (1), P. falciparum infection (4), pneumonia (1)] and seven subjects (6.5%) reported twelve grade 3 unsolicited AEs in the Engerix-B™ group  (data not shown).Grade 3 unsolicited events were rare, occurring with similar frequency in both comparison groups. Five subjects (4.7%) reported ten grade 3 unsolicited AEs in the RTS,S/AS02D and 34 in the Engerix-B™ group) as shown in D  and the control group . None of them were considered to be related to vaccination. The total number of SAEs classified according to the Medical Dictionary for Regulatory Activities (MedDRA)D and 82 in the control group).There were 69 children with at least one SAE . In the control group, there were 13 participants hospitalized with malaria, corresponding to 12.1% (95% CI 6.6–19.9). All cases fully recovered. The other main diagnoses of SAEs requiring admission were anaemia (15.9% vs 12.1%) gastroenteritis (12.1% vs 16.1%) and pneumonia (8.4% vs 7.5%) in the RTS,S/AS02D and Engerix-B™ groups, respectively (data not shown).During the entire follow-up period, 15 participants in the RTS,S/AS02D group, an eight month old girl died at home four months after having received a study vaccine. The presumptive diagnosis based on the verbal autopsy obtained from the mother was staphylococcal septic shock .Four deaths occurred during follow-up (two in each group). None of the deaths was judged to be related to vaccination. In the RTS,S/AS02Streptococcus pneumoniae pneumonia (confirmed by a positive blood culture) and anaemia. According to the verbal autopsy, the death occurred after about 3 weeks of fever, vomiting, diarrhoea, abdominal pain and pallor. The parents did not seek treatment at any health facility. The final diagnosis following verbal autopsy review was chronic gastroenteritis with severe dehydration.The second death in this group also occurred at home nine months after the child had received the last study vaccination. The 15 month HIV negative old boy had previously been admitted for a Engerix-B™ group, a 10 month old boy died at home six months after receiving the third dose of the vaccine. The child had been seen by a field worker 3 days before he died and he appeared to be in good health. The verbal autopsy performed to the mother revealed that 24 hours prior to the fatal event the child abruptly started with intense vomiting and diarrhoea. The mother took him to a traditional healer who administered him “traditional medication”. The child died shortly after. The probable cause of death was severe dehydration from gastroenteritis. The possibility of an adverse effect secondary to traditional medicine ingestion could not be excluded.In the Engerix-B™. According to the child's father, the child had diarrhoea and fever for 4 days, stopped eating, and progressively developed sunken eyes and pallor. The child was not brought to the health centre. Clinician's review of the verbal autopsy report concluded that the most probable cause of death was severe dehydration secondary to gastroenteritis.The second death was of an 11 month old girl, who died at home 7 months after the last vaccination with D group had a low platelet count (44×109/L) of grade 2 intensity one month after the last vaccination. This value was within the normal range (167×109/L) at month 6.Hematological values outside the normal range were infrequent. The majority of abnormal values of hemoglobin, white blood cells and platelets were of grade 1 intensity and occurred with similar incidence in the two groups. One child with concomitant malaria in the RTS,S/AS02D group had a grade 2 ALT value one week after the first dose (162 µmol/L) which dropped to 38 µmol/L one month after the third dose and to 39 µmol/L by study month 6. No creatinine values were outside the normal ranges.Biochemistry values outside the normal range were also infrequent. The majority of out of range ALT and bilirubin values were of grade 1 intensity, occurring with a similar incidence in the two groups. One participant in the RTS,S/AS02D and 78 in the control group). The anti-CS antibody GMTs declined from 199.9 EU/mL one month post dose 3 to 58.8 EU/mL and 7.3 EU/mL by 3.5 and 12 months post dose 3 respectively in the RTS,S/AS02D group. In the control group, anti-CS antibody GMTs were below the assay cut off (0.5 EU/mL) at all post vaccination time points.ATP analysis of vaccine immunogenicity at month 14 included 151 children  was 48.8%  against first or only clinical episodes and 53.7%  against multiple episodes.VE analysis between months 3 to 9 of follow-up (ATP3–14) was 33.0%  and VE against multiple malaria episodes was 25.9% . VE against first or only episodes of clinical malaria over the entire follow-up period up to month 14 (ATP3–9 and ATP3–14), respectively.Analysis of the relationship between anti-CS antibody levels and VE against clinical malaria suggested that within RTS,S recipients, the hazard rates of disease per 2 fold increase anti-CS titres at one month post dose 3 were significantly reduced by 84.1%  and 72.4%  for the two follow-up periods  also We observed no strong evidence of significant differences in the immunological responses to vaccination with RTS,S/AS02D than of the licensed Hepatitis B control vaccine probably reflecting the use of a different adjuvant system. HBs antibody titters also decayed over time. However, all children vaccinated with both RTS,S/AS02D and Engerix-B™ vaccines reached seroprotection levels for anti-HBs 12 months post Dose 3.Anti-HBs responses were higher throughout the follow-up in recipients of RTS,S/AS02Vaccine efficacy against clinical malaria over the 12 months follow-up period after dose three was 33% , whereas during the initial 3.5 months of double-blind follow-up the efficacy was 65.8% . This difference could be due to chance as the confidence intervals of the two estimates overlap, and the study is underpowered for such analyses. Nevertheless, together with the data showing that the hazard was not proportional over the follow-up periods, the results suggest that VE against clinical malaria may have waned over the 14 months follow-up period.Caution is needed when attempting to compare the results of this study with data reported from a previous phase IIb trial conducted in this same area among children aged 1 to 4 years While several previous trials have shown a relationship between anti-CS antibody responses and risk of malaria infection, this study provides the first evidence of a similar relationship between anti-CS antibodies and protection against clinical malaria. It is probable that in trials in older populations similar analyses have been confounded by the superimposed naturally acquired immunity.D malaria vaccine in African infants, as well as confirm protection against clinical malaria for at least one year. Together they support the rationale for the ongoing Phase III trial.In summary, these results confirm the good safety and immunogenicity profile of RTS,S/AS02Protocol S1Trial Protocol(0.92 MB PDF)Click here for additional data file.Checklist S1CONSORT Checklist(7.60 MB RTF)Click here for additional data file.


In endemic areas, falciparum malaria remains the leading cause of seizures in children presenting to emergency departments. In addition, seizures in malaria have been shown to increase morbidity and mortality in these patients. The management of seizures in malaria is sometimes complicated by the refractory nature of these seizures to readily available anti-convulsants. The objective of this study was to determine predictors of anti-convulsant treatment failure and seizure recurrence after initial control among children with malaria.In a previous study, the efficacy and safety of buccal midazolam was compared to that of rectal diazepam in the treatment of prolonged seizures in children aged three months to 12 years in Kampala, Uganda. For this study, predictive models were used to determine risk factors for anti-convulsant treatment failure and seizure recurrence among the 221 of these children with malaria.Using predictive models, focal seizures , cerebral malaria  and a blood sugar ≥200 mg/dl at presentation  were independent predictors of treatment failure (seizure persistence beyond 10 minutes or recurrence within one hour of treatment). Predictors of seizure recurrence included: 1) cerebral malaria , 2) presenting with multiple seizures , 3) focal seizures , 4) recent use of diazepam  and 5) initial control of the seizure with diazepam .Specific predictors, including cerebral malaria, can identify patients with malaria at risk of anti-convulsant treatment failure and seizure recurrence. In endemic areas, falciparum malaria remains the leading cause of seizures in children presenting to emergency departments -4. In adUncontrolled seizures in malaria can damage the brain by aggravating hypoxia, hypoglycaemia and intracranial hypertension, and in children with cerebral malaria they have been shown to increase the risks of neurological complications, cognitive impairment and death ,7-10. InDiazepam administered intravenously or rectally is the recommended first-line treatment for seizures in children with malaria ,12. HoweIdentification of patients at risk of failing anti-convulsant treatment or experiencing recurrences of seizures after initial control is a practical step toward improving the care of children with malaria complicated by seizures. If identified early these children could be provided with optimal anti-convulsants to control seizures and minimize the risk of respiratory depression. In this study, risk factors for anti-convulsant treatment failure and seizure recurrence among Ugandan children with malaria and prolonged seizures were identified.Results of a randomized trial to compare the efficacy and safety of buccal midazolam versus rectal diazepam in children with persistent seizures were reported previously . The stuPlasmodium falciparum detected in the blood, and without another illness explaining the clinical state was considered to have malaria. Cerebral malaria was defined as a child unable to localize painful stimuli with persistence of coma (Blantyre coma score ≤ 2) for more than an hour after control of the seizure, with peripheral parasitaemia and after the exclusion of other causes of encephalopathy, in particular central nervous system infections and hypoglycaemia [Patients were randomly assigned to one of two treatment arms: rectal diazepam and buccal placebo or rectal placebo and buccal midazolam. Parenteral preparations of both diazepam  and midazolam  were prepackaged by a pharmacist in two boxes that corresponded to the two treatment arms. Boxes were stored at 5–10°C and emptied and refilled on a weekly basis. Diazepam and placebo were packaged in 2 ml glass syringes and buccal midazolam and placebo were packaged in 2 ml plastic syringes. Both re-packaged midazolam and diazepam are stable under these conditions for up to one month -26. Bothlycaemia . Seizurelycaemia .The Uganda National Council for Science and Technology and the institutional review boards of Makerere University, Kampala and the University of California, San Francisco approved the study.Statistical analysis was done using Stata 10.0 . Multivariate analysis using logistic regression was used to identify predictors of treatment failure. Associations between potential predictors for treatment failure were assessed using cross-tabulations with chi-squared tests. The final model was selected using the Stata 10.0 backward selection procedure set at p < 0.20 and it included: 1) duration of seizures prior to receiving treatment, 2) form of disease , 3) seizure type, 4) age and 5) blood sugar level. For survival analysis, differences in survival functions between potential explanatory predictors of seizure recurrence were initially compared using Kaplan Meier curves and the log rank test. The Cox proportional hazard model was applied to assess the independent roles of potential predictors of seizure recurrence. Covariates with a significant log rank test (p ≤ 0.004) were included in the final model. Anti-convulsant treatment received to terminate the seizure was included in both the logistic regression model and Cox model to determine any associations between treatment received and treatment failure or seizure recurrence, respectively. In the Cox model, time analysis was restricted to the first 12 hours after initial control, a period when most (92%) of the seizures had recurred, and the proportional hazard assumption was not violated.Of the 330 patients enrolled in the clinical trial, 221 (67%) had malaria and were analysed for outcomes of interest  (Table Predictors of seizure recurrence were also assessed using survival analysis Figure . In addiIn the final Cox proportional hazard model Table , the folPredictors of anti-convulsant treatment failure and seizure recurrence were evaluated in Ugandan children presenting to an emergency unit with malaria complicated by prolonged seizures. Presenting with cerebral malaria, focal seizures, or hyperglycaemia were found to be independent predictors of anti-convulsant treatment failure. Predictors of seizure recurrence after initial control with an anti-convulsant included presenting with cerebral malaria, focal seizures, or multiple seizures. In addition, compared to midazolam, prior use of diazepam or initial control of a seizure with diazepam were associated with an increased hazard of experiencing a recurrence. Of note, in children with malaria, focal seizures and cerebral malaria were found to be important markers of refractory seizures, considering that they were associated with both anti-convulsant treatment failure and seizure recurrence.In this study, children with malaria who presented with focal seizures were more likely to fail treatment than those with other seizure types. This finding is consistent with prior reports that focal seizures are a risk factor for refractory status epilepticus in adults . One posRisk factors for seizure recurrence in children with malaria have not been well characterized. Predictors of seizure recurrence in patients without malaria include presentation with multiple or focal seizures . In thisThese predictors can be used to identify children with malaria who may benefit from alternative therapy, in the particular the use of longer-acting and safer acting anti-convulsants, such as midazolam or lorazepam. More studies are urgently needed to evaluate the effectiveness and prophylactic benefit of longer acting agents in the treatment of seizures in children with malaria.The authors declare that they have no competing interests. The sponsors of this study and the donors of study drugs had no role in study design, the collection, analysis, or interpretation of data or in the writing of this report.AM contributed to study design and coordination, collected data, supervised patient enrollment and follow-up, and analyzed and interpreted data. JB contributed to study design, supervised coordination of the study, enrollment, and clinical care of the patients and contributed to interpretation of data. GN contributed to study design and coordination, supervised enrollment and follow-up of the patients and contributed to data interpretation. SS contributed to study design, analysis and data interpretation. PJR contributed to study design and coordination, analysis and data interpretation. All authors read and approved the final manuscript.PJR is a Doris Duke Charitable Foundation Distinguished Clinical Scientist.


Geohelminth infections are common in rural western Kenya, but risk factors and effects among pregnant women are not clear.During a community-based cross-sectional survey, pregnant women were interviewed and asked to provide a blood sample and a single fecal sample. Hemoglobin was measured and a blood slide examined for malaria. Geohelminth infections were identified using the concentration and Kato-Katz method.Ascaris lumbricoides, 39.5% with hookworm, and 29.0% with Trichuris trichiura. Infection with at least one geohelminth species was associated with the use of an unprotected water source  and the lack of treatment of drinking water . Geohelminth infections were not associated with clinical symptoms, or low body mass index. A hookworm infection was associated with a lower mid upper arm circumference . Hookworm infections with an egg count ≥1000/gram feces (11 women) were associated with lower hemoglobin . Among gravidae 2 and 3, women with A. lumbricoides were less likely to have malaria parasitemia  compared to women without A. lumbricoides, unlike other gravidity groups.Among 390 participants who provided a stool sample, 76.2% were infected with at least one geohelminth: 52.3% with Geohelminth infections are common in this pregnant population; however, there were few observed detrimental effects. Routine provision of antihelminth treatment during an antenatal clinic visit is recommended, but in this area an evaluation of the impact on pregnancy, malaria, and birth outcome is useful. Ascaris lumbricoides) were less likely to have malaria than other women in their second or third pregnancies who did not have this type of worm infection. Although our results suggest that it would be good advice to treat women with drugs for intestinal worm infections during their pregnancy in this area, the effect on maternal and infant health and malaria infection needs further study.In rural western Kenya, both malaria and intestinal infections with worms are common. Pregnant women are particularly vulnerable to infection with malaria, but the effect on pregnancy of intestinal infections with worms is not clear and may depend both on how heavy the worm infection is and on the type of worm. Additionally, it is not clear whether infections with worms may affect malaria infections. In this article, we begin to disentangle some of these issues. Intestinal infections with worms were diagnosed in three-quarters of 390 pregnant women in western Kenya who provided a stool sample. In these women, intestinal worm infections caused a modest decrease both in haemoglobin levels and indicators of nutritional status. Women in their second and third pregnancies who were diagnosed with one particular type of worm infection ( Geohelminth infections are a major global health burden, with an estimated 3.8 billion persons infected, 720 million clinical cases, and an estimated 135,000 deaths attributed to clinical complications annually Ancylostoma duodenale and Necator americanus. Mature hookworms can cause intestinal bleeding and protein loss proportional to worm burden; however, the severity of the effect is dependent on the host's underlying nutritional status Trichuris trichiura infection, but it is less prominent than in hookworm infection; however, it often occurs along with hookworm infections and so may accelerate the onset of iron-deficiency anemia. Ascaris lumbricoides infections are commonly asymptomatic, although clinical complications of extra-intestinal or high numbers of ascarids have been well described A. lumbricoides infection has been associated with impaired fat digestion, reduced vitamin absorption, and temporary lactose intolerance, and treatment has shown to improve nutritional status Hookworm disease is caused by A. lumbricoides and Plasmodium falciparum; studies have shown either an increase in clinical malaria in the presence of A. lumbricoidesA. lumbricoides and T. trichiura infections compared to non-pregnant women A. lumbricoides, and (asymptomatic) malaria in pregnancy The overlapping geographic distributions of geohelminth infections and malaria result in a high rate of co-infection A community-based cross-sectional survey of health and nutritional indicators among pregnant women in rural western Kenya allowed us to measure the prevalence of and to identify risk factors for geohelminth infections among pregnant women. We also examined effects of geohelminths on symptoms, anemia, and nutritional status, and explored the association of geohelminth infections with malaria.The study was conducted in Gem , Nyanza Province, western Kenya. Gem has a population of approximately 75,000 people residing in 142 villages. The vast majority of people are of Luo ethnicity (97.7%). This area is part of a health and demographic surveillance system. All births, deaths, pregnancies, in and out migrations, socio-economic information and educational level are regularly recorded and updated every four months. Based on previous estimates, we anticipated that at least 600 women in Gem would be pregnant at the time of the survey. All households received free insecticide-treated nets as part of a community-based trial (1998–2002) and continue to receive free retreatment As part of the health and demographic surveillance system, one or two health workers in each village assist in the collection of information on births and deaths. For this survey, village health workers invited all pregnant women to a central location in a village on a specified day. Women were asked to bring their antenatal card if they had one, and a fresh (<24 hours old) stool sample in a container provided by the study. Only pregnant women who were living in the demographic surveillance area and were willing to give informed consent could participate.A 5-day training workshop for all team members was held prior to the survey, which took place in July 2003, to standardize assessment methodology. Study staff administered a standardized questionnaire on medical and obstetric history, and blood was obtained for a malaria smear and a hemoglobin measurement. Weight was measured without shoes to the nearest 0.1 kg on a weighing scale and height was measured to the nearest centimeter on a locally made height instrument. The mid upper arm circumference (MUAC) was measured to the nearest 0.1 cm at the midpoint of the upper arm with an insertion tape, and axillary temperature was measured using a digital thermometer. Two independent observers performed all body measurements, and the average was used for data analysis. Socio-economic information at the household level was obtained from the health and demographic surveillance system. We used principal components analysis to generate weights for the following broad household characteristics: occupation of participant and spouse, source and quality of water, source of fuel for cooking, livestock and asset ownership, and dwelling/housing structure. From this we derived a wealth index, and scores were used to rank the study participants into socio-economic status quintiles All pregnant women received hematinic supplementation (200 mg ferrous sulfate 3 times per day and 5 mg folic acid once per day) for 30 days and, if appropriate, a dose of sulfadoxine-pyrimethamine as intermittent preventive treatment for malaria as per national guidelines. A clinical officer and a nurse treated additional complaints. All participants in their second or third trimester with a geohelminth infection received a single treatment dose of 400 mg albendazole.Thick and thin smears were stained with Giemsa and examined under oil immersion; parasite counts were determined against 500 white blood cells, assuming a white blood cell count of 8000/µl, and species were identified. A 10% sample of smears was reread for quality control. Hemoglobin was measured using a Hemocue hemoglobin detection system  on site. Stool samples were collected and microscopically examined for geohelminth infection using a modification of the formol-ether and ethyl acetate by concentration technique and by Kato-Katz method; hookworm egg counts were performed The study protocol was reviewed and approved by the Kenya Medical Research Institute  and the Centers for Disease Control and Prevention .All participants in this study gave written informed consent or indicated their approval with a thumb print in the presence of a witness if the participant was illiterate.th percentile of the body mass index by trimester of a Swiss reference population  Malaria was defined as the presence of any asexual blood stage parasite of any species in a thick smear independent of signs or symptoms. The body mass index was calculated as weight in kilograms/height (in meters) squared; a low body mass or underweight was defined as a body mass index less than the average 5t-test or a non-parametric test if not normally distributed. The following variables were examined for their association with geohelminth infection : socio-economic status, level of education, type of water source, household water treatment, marital status, young age, gravidity, trimester of pregnancy, malaria, a report of soil eating, and presence of animals in the house. In addition, in the models for separate geohelminth species, the association with the presence of other geohelminth species was examined as well. Odds ratios and their 95% confidence intervals (CI) were calculated. Adjusted odds ratios were obtained using a logistic regression whereby all factors significant in the univariate analyses were introduced (as independent variables), and factors with a P-value>0.05 were removed. However, we kept a malaria infection in the model for each geohelminth species, even if not significant, because this was our variable of interest. Linear regression was used to assess the relationship between geohelminths and both hemoglobin and MUAC. We examined effects and prevalence of geohelminths by gravidity group as well, whereby we divided gravidity group into primigravidae, gravidae 2 and 3, and gravidae ≥4. We decided for this division after inspection of the graphs of prevalence of geohelminths by gravidity, and because of our interest in the interaction of geohelminth infections with malaria. Malaria is known to be more common among primigravidae, and to decrease with increasing gravidity number in this area Differences in proportions were analyzed using the chi-square test or Fisher's exact test when appropriate. Differences in means were compared by Student's P = 0.009), and were more likely to be of higher gravidity number, and of lower socio-economic status and education level . Participants who brought a stool sample were older  than women who did not bring a stool sample  was most prevalent, followed by hookworm (39.5%) and T. trichiura (29.0%). Schistosoma mansoni was detected in the stool of 2 women; S. strongyloides was not detected. Overall, hookworm infections were low density, with egg counts of ≥1000 eggs/gram feces in only 7.1% of infections. Patterns of the prevalence of geohelminth infections seemed more distinct by gravidity compared to age ; there was no clear pattern for T. trichiura , or by combination of species. Geohelminth infections overall, or by species or combination of species, were not associated with a low body mass index or a hemoglobin <8 g/dl.  or MUAC . In lineA. lumbricoides was associated with anemia and lower hemoglobin level among primigravidae (independent of hookworm), but not overall or in any other gravidity group  and the lack of household treatment of drinking water . However, water source or treatment was not statistically significantly associated with any geohelminth species. A report of eating soil was not associated with geohelminth infection overall or by species.T. trichiura (increased risk) and being married (decreased risk) .Factors associated with a hookworm infection included ed risk) . T. tricA. lumbricoides included being in the first or second trimester of pregnancy and a stool infection with T. trichiura; conversely, age <30 years (or in a separate model including gravidity instead of age being gravidae 2 or 3) were associated with a decreased risk  was not associated with a positive blood smear. Prevalence of malaria by gravidity or age  than were those without A. lumbricoides; conversely, among gravidae 2 and 3, those with T. trichiura were more likely to have malaria  than those without T. trichiura. These relationships were not observed in other gravidity groups.Malaria was detected in the blood smear of 37.8% of the women: 95.9% of infections were y or age  showed tT. trichiura infection; for A. lumbricoides, there was a trend towards decreased malaria risk . When combining gravidity and A. lumbricoides infection in one variable and introducing this in the model for factors associated with malaria infection, compared to gravidae ≥4 without A. lumbricoides infection, gravidae 2 and 3 without A. lumbricoides infection had an AOR of 2.5 (95% CI 1.3–4.7) for malaria, whereas the same group with A. lumbricoides had an AOR of 0.9 (95% CI 0.4–1.9) for malaria.In multivariate analysis, malaria was not associated with either hookworm or No interaction was noted between malaria and any geohelminth on hemoglobin.A. lumbricoides, T. trichiura or hookworm were very common among pregnant women in rural Kenya; 3 out of 4 women had one or more parasites, and 1 out of 10 were infected with all 3. Of note, even these high prevalence figures may underestimate the true burden, because participants who did not bring a stool sample were more likely to use an unprotected water source, which was associated with a geohelminth infection in this survey. High geohelminth infection rates among pregnant women have been reported elsewhere; in Nepal  Intestinal infections with A. lumbricoides and T. trichiura have been reported to peak in childhood and decline thereafter A. lumbricoides infections after around 30 years. It is possible that pregnancy status may have affected this outcome. Pregnancy has been associated with an increase in A. lumbricoides and T. trichiura infections compared to non-pregnant women in a small study in Gabon A. lumbricoides and T. trichiura ; however, the prevalence of these two geohelminths was higher in the geographic location bordering our study area The prevalence of hookworm infection has been described to increase throughout childhood followed by a stable plateau in adulthood Geohelminth infections are transmitted through soil; however, in this study we did not find an association between soil eating and geohelminth infection, similar to findings from a study among pregnant women in Zanzibar A. lumbricoides.Although the prevalence of geohelminth infection was high, its effect of on maternal health was not straightforward. Compared to other studies, effects of hookworm infection on anemia and hemoglobin were limited, possibly because of the low density of infections P. falciparum malaria with successive pregnancies as they acquire antibodies to these special placental forms of the parasite T. trichiura in univariate analysis, but not in multivariate analysis or among the other geohelminths. Of note, the effect of A. lumbricoides on asymptomatic malaria parasitemia among gravidae 2 and 3 was the opposite, and remained significant in multivariate analysis. This would suggest that an A. lumbricoides infection may either prevent or control the development or improve the clearance of a P. falciparum infection. An increase in P. falciparum densities was reported among children aged 5–14 years treated with levimasole when participating in a randomized controlled trial to evaluate antihelminth treatment A. lumbricoides may affect the development of concomitant P. falciparum infections. Maeno et al. (1993) reported decreased sequestration of parasite infected red blood cells in the placentas of women with IgE deposits in the capillaries, which may be another indication of the negative interaction between a geohelminth and a malaria infection A. lumbricoides and a malaria infection, the population attributable fraction of malaria among gravidae 2 and 3 in this area due to not having an A. lumbricoides infection could be approximately 30% and among the total pregnant population about 10%. Again, the sample size in our study is small, and our findings would need to be confirmed in larger studies. Given the differing effects, it may not be advisable to categorize geohelminths as a group when analyzing their interaction with malaria; among pregnant women it may be advisable to explore the interaction by gravidity group. Similar to an observation from coastal Kenya, we did not note an interaction between malaria and any geohelminth on hemoglobin among pregnant women The interactions between malaria and geohelminth infections have been a subject of recent interest with regards to assessment of vaccine efficacy A. lumbricoidesLimitations of our study include the small sample size and use of a single stool specimen to assess infection status, which may have underestimated geohelminth burden. Also, we did not assess the HIV-status of the participants, and HIV-infected women are more likely to carry malaria parasites and to be anemic than HIV-uninfected women. In an adjacent rural area, an HIV prevalence of 21% has been described among women in the age group 13–34 years Mebendazole and albendazole are considered safe in pregnancy after the first trimester, and in areas with a high prevalence of hookworm infection (20–30% or more), anthelminth treatment once in the second trimester of pregnancy is recommended


Detection of malaria pigment -containing leukocytes may have prognostic relevance in malaria; however, studies reported conflicting results, with microscopic counts suggestive of being inaccurate and imprecise.Numbers of Hz-containing leukocytes from a malaria patient obtained with a flow cytometer counting 50.000 gated events were compared with thin film microscopy as applied under field conditions.Flow cytometry identified 5.8% Hz-containing monocytes and 1.8% Hz-containing neutrophils. The microscopic examination yielded 10% and 13% of Hz-containing monocytes, as well as 0% and 0.5% of Hz-containing neutrophils for observers one and two, respectively.Novel, robust and affordable cytometric methods should be evaluated in the field as they may assist in utilizing Hz-containing cells as clinically useful parameter. Plasmodium spp. However, pigmented monocytes remain in the circulation for a prolonged period of time and may also indicate a recent, successfully treated malaria episode [Malaria continues to be a major health threat to people living in endemic areas, and reliable early identification of patients at particular risk for progressing towards severe disease remains a challenge. During haemoglobin digestion, malaria parasites detoxify the remaining haem into the crystalline malaria pigment, haemozoin (Hz). When the parasites are liberated into the blood stream, the Hz crystal(s) are also released and removed from the blood by either resident macrophages; or monocytes and granulocytes in the blood. Thus, the presence of Hz-containing leukocytes in the circulation is an indicator of the presence of  episode ,2. Moreo episode ,4.®, Abbott, Santa Clara, California) also detects Hz-containing leukocytes during routine full-blood counts has revived this interest and led to a series of studies in endemic and non-endemic areas, as reviewed elsewhere [Hz has interesting physical properties; it is bi-refringent (depolarizing) and paramagnetic. The fact that Hz can be easily detected using dark field or polarizing microscopy led to some interest in the diagnostic value of pigmented leukocytes. The chance finding that one type of automated haematology analyzers  differential count . Basophi® analysers showed a sensitivity for the diagnosis of malaria of 90-95% in malaria-endemic regions, including several African countries [did have pigmented monocytes in the peripheral blood that were detectable - a result much higher than in reported studies using microscopy. Moreover, a large number, if not the majority of the patients in the Cell-Dyn® studies had only non-severe malaria; and thus were likely to have considerably lower numbers of pigmented leukocytes. These results are further corroborated by a study that employed image analysis of the Cell-Dyn® on-screen results [® instrument and by manual microscopy count of thick films [® instrument's on-screen results constitute a rather approximate representation of cells analysed. Factors like low screen resolution and representation algorithms may cause a data reduction by a factor of around 5 [Thus, it is not too surprising that, contrary to the mentioned reports, studies that used flow cytometric methods reported the detection of pigmented leukocytes in most, if not all malaria patients. The various Cell-Dynountries ,13,14. T results . FurtherP. falciparum malaria (0.2% parasitaemia), were labelled with CD14 (monocytes) and with CD16 (granulocytes)  and erythrocytes were lysed using a Lysis kit . 50,000 gated events were acquired with a flow cytometer  and Hz containing leukocytes determined by measurement of Side-Scatter-Depolarization, as described for the Cell-Dyn® instruments [Further support for this notion was lent by a simple comparison of a flow cytometric count of Hz-containing leukocytes with thin film microscopy. Leukocytes from a patient with truments . MicroscFigure Mycobacterium spp. and Plasmodium spp.; and it seems likely that it may also facilitate determination of Hz-containing leukocytes. Whereas rapid diagnostic tests (RDTs) for malaria are of utmost value to assist with establishing a diagnosis of malaria, simple, easily maintainable and affordable flow cytometers adapted to the needs and conditions of rural/semi-rural settings in the South could play an important role in swiftly assisting to predict outcome in malaria, in combination with other much-needed applications in full blood counting, CD4+ counting in HIV/AIDS patients and also TB diagnosis [These results indicate that microscopic counting of Hz-containing cells is prone to large errors. So, if there is an association of Hz-containing leukocytes and severity of malaria, is there any  point in counting them? It seems that counting them by microscopy may not yield sufficiently accurate and precise results to turn the number of pigmented leukocytes into a clinically useful marker. However, as flow cytometric methods show, it appears too early to dismiss Hz-containing leukocytes as potentially useful clinical markers of malaria severity. The question is: are there any other, less complicated and expensive methods than flow cytometry to determine these cells with higher accuracy than light-microscopy? Recently a prototype of a low-cost image cytometer has been described that may offer one possible answer to this question . It is ciagnosis ,17.In conclusion, robust and affordable, purpose-tailored 'no frills' flow cytometric field applications should be tested in the field for diagnostic accuracy and cost effectiveness in comparison with light microscopy as they may assist in overcoming practical limitations of microscopy otherwise so far indispensable for malaria diagnosis.The authors declare that they have no competing interests.TH conceived and designed the study, contributed to the experiments and to data analysis and drafted the paper. RF contributed to the experiments, data analysis and the writing of the paper. ML contributed to the underlying experiments, data interpretation and the writing of the paper. PGK contributed to the design of the study, the data analysis and the writing of the paper. MPG contributed to the design of the study, the data analysis and the writing of the paper. All authors read and approved the final manuscript.