Sir,As we all are aware, transfusion-transmitted malaria is emerging as a major problem especially in developing countries, which fall in the zone of endemicity. Transmission of malaria by blood transfusion was one of the fi rst recorded incidents of transfusion-transmitted infection. The freqMicroscopic detection of blood though considered the gold standard for malaria diagnosis for decades is quite labor-intensive and requires adequate technical skill and manpower. This has spurred the development of several nonmicroscopic malaria rapid detection tests (RDT) based on the detection of malaria parasite antigen in whole blood.P. falciparum), and p-lactate dehydrogenase (pLDH-P. vivax)  since March 2008. Thick and thin smears were made of all positive cases to corroborate the results of RDT. Total of 11,736 units of donated blood were screened between March 2008 and September, 2009. Three (0.03%) units were found to be positive, 2 for P. vivax, and 1 for P. falciparum. All the three cases were found positive microscopically. None of the donor had given a history of fever/malaria during predonation screening [We at our blood bank, Regional Blood Transfusion Centre, have been routinely screening all donated units of blood for malaria using RDT, based on immunochromatographic methods detecting antigens, histidine-rich protein 2 , and Clinotech Malaria cassette (detects surface protein of merozoites and sporozoites). Microscopy revealed parasitemia in 79 (20.0%) of potential donors, mean level of parasitemia being 445/μl. The corresponding prevalence of malarial parasitemia detected using RDT were 3.8% for OptiMAL and 57.8% for Clinotech, with the results for microscopy being the gold standard.[Falade et al. in 2008 evaluated the usefulness of a new RDT, pLDH/HRP2 malaria card test  for malaria diagnosis in the forested belt of central India. Analysis revealed that RDT was 93% sensitive, 85% specifi c with a positive predictive value of 79%, and a negative predictive value of 95% (microscopy being used as the gold standard).[et al. studied the operational accuracy and persistent antigenicity of HRP2 RDTs for Plasmodium falciparum malaria in a hyperendemic region of Uganda. Using a cross-sectional study design, a total of 357 febrile patients of all ages were tested using RDT out of which 40% (139) had positive blood smears for P. falciparum. RDT had overall sensitivity of 98%, specifi city of 72%, positive predictive value of 69%, and a negative predictive value of 98%. In the children followed-up after successful antimalarial treatment, the mean duration of persistent antigenicity was 32 days, this duration varied signifi cantly depending on pre-treatment parasitemia.[Bharti tandard). KyabayizImportance of recognizing transfusion-transmitted malaria lies in the fact that it can lead to febrile transfusion reaction which can falsely simulate a hemolytic transfusion reaction. It can lead to the widespread dissemination and spread of drug-resistant malarial parasite. The utmost importance is derived from the presumption that the screened blood units are transfused to patients ranging from newborns (requiring exchange transfusions) to the geriatric group. These along with the immune-suppressed (cancer and leukemia therapy) patients are at a higher risk of falling prey to units not effectively screened for malarial parasite. Thus clinicians will waste valuable time manage on lines of hemolytic transfusion reaction. Nonetheless, no matter what strategy is adopted, it is likely that cases of transfusion-transmitted malaria may still occur, so malaria must always be considered in any patient with a febrile illness post-transfusion.Thus we would like to recommend that use of rapid detection devices with peripheral smear screening of positive cases is reasonably reliable method to prevent transfusion-transmitted malaria in India.


Cerebral malaria, a disorder characterised by coma, parasitaemia, and no other evident cause of coma, is challenging to diagnose definitively in endemic regions that have high rates of asymptomatic parasitaemia and limited neurodiagnostic facilities. A recently described malaria retinopathy improves diagnostic specificity. We aimed to establish whether retinopathy-positive cerebral malaria is a risk factor for epilepsy or other neurodisabilities.Between 2005 and 2007, we did a prospective cohort study of survivors of cerebral malaria with malaria retinopathy in Blantyre, Malawi. Children with cerebral malaria were identified at the time of their index admission and age-matched to concurrently admitted children without coma or nervous system infection. Initially matching of cases to controls was 1:1 but, in 2006, enrolment criteria for cerebral malaria survivors were revised to limit inclusion to children with cerebral malaria and retinopathy on the basis of indirect ophthalmoscopic examination; matching was then changed to 1:2 and the revised inclusion criteria were applied retrospectively for children enrolled previously. Clinical assessments at discharge and standardised nurse-led follow-up every 3 months thereafter were done to identify children with new seizure disorders or other neurodisabilities. A Kaplan-Meier survival analysis was done for incident epilepsy.vs 38·5°C [1·1]; p=0·01) and acute seizures , and male sex was a risk factor for new neurodisabilities .132 children with retinopathy-positive cerebral malaria and 264 age-matched, non-comatose controls were followed up for a median of 495 days (IQR 195–819). 12 of 132 cerebral malaria survivors developed epilepsy versus none of 264 controls . 28 of 121 cerebral malaria survivors developed new neurodisabilities, characterised by gross motor, sensory, or language deficits, compared with two of 253 controls . The risk factors for epilepsy in children with cerebral malaria were a higher maximum temperature (39·4°C [SD 1·2] Almost a third of retinopathy-positive cerebral malaria survivors developed epilepsy or other neurobehavioural sequelae. Neuroprotective clinical trials aimed at managing hyperpyrexia and optimising seizure control are warranted.US National Institutes of Health and Wellcome Trust. Cerebral malaria, a disorder characterised by coma, parasitaemia, and no other evident cause of coma,9Most previous studies of neurological sequelae related to cerebral malaria have been limited by poor retention rates, short duration of follow-up, and assessments that were stopped when the results of neurological examinations were normal. Delayed cerebral malaria sequelae, such as epilepsy, have been reported in studies designed to identify them: a retrospective cohort study in KenyaMalaria retinopathy, recently described in children, enhances the potential for diagnosis with 95% sensitivity and 90% specificity as compared with autopsy findings as the gold standard.To establish whether retinopathy-positive cerebral malaria is a risk factor for epilepsy or other neurodisability and, if so, whether there are modifiable risk factors that offer potential opportunities for early intervention, we did a prospective cohort study of children with retinopathy-positive cerebral malaria. Follow-up assessments were designed to identify interim seizures and new neurodisabilities.Plasmodium falciparum. In Blantyre, about 83% of caregivers of children younger than 5 years cited fever and malaria as the impetus for seeking medical care in 2006–07.P falciparum parasitaemia, and no other evident cause of coma. We used the Epilepsy Screening Questionnaire to exclude children with a history of previous unprovoked seizures. This questionnaire has been previously used with 79·3% sensitivity and 92·9% specificity for detection of epilepsy.We did a prospective cohort study of cerebral malaria survivors who were admitted to the paediatric research ward of Queen Elizabeth Central Hospital (QECH), Blantyre, Malawi, during the malaria season (January–June) from 2005 to 2007 and who resided within the catchment area . Because QECH is in a region endemic for malaria, which is one of the commonest causes of illness in Malawian children, all comatose children admitted to QECH have a thick blood film reviewed to screen for Throughout recruitment, all patients with cerebral malaria had an ophthalmological assessment on admission. In 2006–07, enrolment criteria for cerebral malaria survivors were revised to limit case inclusion to children with retinopathy-positive cerebral malaria on the basis of indirect ophthalmoscopic examination through a dilated pupil by a qualified ophthalmologist. Patients without malaria retinopathy and a small number of children who did not have a retinal examination while comatose were excluded from this analysis. These patients have been described elsewhere.P falciparum parasitaemia were eligible as controls, although the presence of parasitaemia was not a requirement and not all control children were tested for malaria. The comparison group was also screened acutely with the Epilepsy Screening Questionnaire and the Ten Questions screen to identify children with a history of previous unprovoked seizures and those with pre-existing neurodisabilities. The findings from these questionnaires were used in the unexposed comparison group exactly as they were in the exposed group. Children with a history of unprovoked seizures were excluded from enrolment. Those with a history of neurodisability were eligible for inclusion but were excluded from analyses of the neurodisability outcome.The control group consisted of the first eligible child who was admitted to the general paediatric service after each case was enrolled and who was age-matched to within 6 months. Inclusion criteria for age-matched controls were residence within 1·5 h travel from QECH by four-wheeled drive vehicle, a normal level of consciousness, and no history of unprovoked seizures before the index admission. Children with a brief febrile seizure before admission who were conscious on admission were eligible. Children with Our study was approved by the University of Malawi College of Medicine Research Ethics Committee and Michigan State University Biomedical Institutional Review Board. Signed informed consent was provided by the parents or guardians of the study participants.We planned to recruit 225 cerebral malaria survivors matched 1:1 with unexposed controls. The addition of malaria retinopathy as an inclusion criterion in 2006 reduced the number of qualifying participants. Therefore, matching was increased to 1:2, to increase precision in estimates of differences between groups. Recruitment was continued through the 2007 malaria season with the goal of enrolling as many children as possible and assessments continued through to December, 2009, to allow at least 18 months of follow-up for the last children enrolled. Therefore, children enrolled earlier in the cohort were followed up for longer than children enrolled later.For both cases and controls, several clinical characteristics, including established risk factors for seizures and epilepsy, were documented at enrolment. A research nurse interviewed caregivers  with the Epilepsy Screening Questionnaire and the Ten Questions screen, and recorded demographic data including age and sex. Potential pre-existing risk factors for epilepsy and adverse neurological outcomes were assessed including family history of epilepsy,On admission to the paediatric research ward, we recorded details of the general and neurological examination. We did not commence enrolment in the long-term follow-up study until survival was probable, usually on day 2 or 3 of the admission after acute data had already been collected. Laboratory investigations included full blood count, packed cell volume, lactate and glucose concentration, and thick and thin blood smears (with Field's stains) to identify the species and quantify malaria parasitaemia. Temperature and coma score were recorded serially. Seizures related to cerebral malaria were recorded on the basis of clinical identification as well as electroencephalogram (EEG) findings.For patients with cerebral malaria, digital EEGs were obtained on the day of admission and daily thereafter until recovery from coma. EEGs were recorded with a BioLogic Ceegraph digital machine  with a modified 10–20 system and met the American Electroencephalography Society guidelines for EEG.At discharge, neurological assessment was completed by the attending physician (a paediatrician or internist), and for all study participants discharge diagnoses and body-mass index were recorded. Children were then reviewed by a study nurse at 1 month after discharge and then every 3 months until the study was completed. At follow-up assessments, which were done at the hospital or via home-based visits led by nurses, study nurses repeated the Epilepsy Screening Questionnaire and the Ten Questions screen to identify any interim seizures or newly evident neurodisabilities. All children who were discharged with neurological sequelae, had interim seizures, or screened positive on the Ten Questions screen during follow-up were assessed by a neurologist (GLB) to characterise their seizure disorder or the nature of the neurological disability. Any additional concerns or issues from either the research nurses or the parents also triggered a physician-level review of the child. The examining neurologist and study nurses were all involved in the acute care of the study participants and thus exposure status was not masked. Children who initially screened negative on the Ten Questions screen and who later exhibited motor, sensory, or language impairment were deemed to have a new neurodisability. Interim seizures that happened during follow-up were deemed provoked seizures if health care was sought and a fever was documented, the parent reported a tactile temperature or measured a fever around the time of the seizure, the parent reported an illness preceding the seizure, or parasitaemia was recorded on the day of the seizure even in the absence of acute fever. More than one unprovoked seizure during follow-up was needed for a child to meet the diagnostic criteria for epilepsy. If a child's family moved outside of our defined catchment area, the child was no longer followed up unless the parents brought them to the paediatric research ward for their quarterly assessments.Several children without seizures or neurodisability were referred for physician-level assessment because of parental concerns about new behavioural problems. Assessment of these children by a neurologist (GLB) in two different settings  were done and all met Diagnostic and Statistical Manual of Mental Disorders (4th edn) criteria for hyperactive-impulsive attention-deficit hyperactive disorder (ADHD).At the time of enrolment, neither acute neuroimaging nor ventilatory support was available on the QECH paediatric research ward. All children with cerebral malaria received treatment for acute severe malaria according to national guidelines that included blood transfusions if indicated, intravenous quinine, and benzodiazepines or paraldehyde for acute symptomatic seizures. For refractory seizures, additional antiepileptic drugs, usually phenobarbital, were given.Recruitment of 225 cerebral malaria survivors matched 1:1 with unexposed controls would have provided 87% power to detect an 8% difference in epilepsy development. Data were originally recorded on paper forms with subsequent double data entry into Microsoft Office Excel 97–2003 software. This was then imported into EPI INFO (version 3.5) for analysis. Calculations of geometric means for parasitaemia were completed using Dimensions Research online software.t tests and odds ratios (ORs). Owing to the revised inclusion criteria in 2006–07 for cerebral malaria cases, and to use data obtained from all controls, we did an unmatched analysis comparing the group of cerebral malaria survivors versus controls. We assessed the ORs of incident epilepsy, new neurodisability, disruptive behavioural disorders, any adverse neurological outcome, and death. We did a Kaplan-Meier survival analysis for incident epilepsy, our primary outcome of interest. Any children lost to follow-up were censored from the data on the day after their last follow-up. To identify potential risk factors for adverse neurological outcomes in cerebral malaria survivors, we also assessed acute clinical characteristics associated with adverse neurological outcomes  with the t test or OR with 95% CIs. For any χ2 assessment with less than five items of data in a cell, Fisher's exact test was used. For t-test comparisons, Bartlett's test for population variance was assessed and where indicated  the Kruskal-Wallis (non-parametric) test was used. Because of the variance from our initial recruitment and analysis plan, we also did a sensitivity analysis of the risk of each adverse neurological outcome with matched pairs that included the first matched control selected for each case.The admission characteristics of retinopathy-positive cerebral malaria survivors and controls were compared with The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.vs 446 [374]; p=0·13). However, follow-up every 3 months was maintained through to December, 2009, in 101 (77%) of 132 cases and 172 (65%) of 264 controls (p=0·02). Attrition was due to death in six cases (5%) and three controls (1%), to withdrawal in seven cases (6%) and 12 controls (5%), and to family relocation outside of the tracking area in 18 cases (14%) and 77 controls (29%).The duration of follow-up from recruitment to an adverse outcome or attrition did not differ significantly between cases and controls .We provided ongoing medical care to the 42 children who had experienced adverse neurological outcomes. Among these children, 14 had families who relocated outside of the tracking area during this study, but parents for all 14 of these children continued to bring them in for assessments. Had these children been withdrawn from the study at the time of the relocation, the rate of study completion in the cerebral malaria survivors would have been 53% (70 of 132). To our knowledge, there were no children in the control group whose parents continued to bring them for quarterly assessments after family relocation outside the tracking area. When we compared the age, sex, enrolment date, and outcome of children who completed follow-up through to December, 2009, with those of children who did not complete follow-up, more recent enrolment and having an adverse neurological outcome were both associated with higher completion rates. There was no difference in the proportion of cases versus controls who continued follow-up for the full duration of the study once the children with adverse neurological outcomes were excluded from the comparison  provides prospective longitudinal outcome data on a population of patients with retinopathy-positive paediatric cerebral malaria. Findings show that cerebral malaria is a risk factor for subsequent epilepsy, disruptive behavioural disorders, and other neurodisabilities. The epilepsy syndrome that comes after cerebral malaria seems to be associated with localisation-related, often refractory, epilepsy. The risk factor analysis provides information on potential neuroprotective interventions, which include improved acute seizure control during cerebral malaria and more aggressive fever management.Our prospective study describes neurological outcomes for a population of well characterised paediatric cerebral malaria survivors. Our acute clinical data provide information on the clinical characteristics predictive of brain injury in survivors . Acute aSeveral limitations of our study deserve a mention. Overall, cases were followed up longer than controls. A longer follow-up and higher retention rate among cases might have caused us to overestimate the effects of cerebral malaria. However, the duration of follow-up from recruitment to initial adverse outcome or attrition did not differ significantly between cases and unexposed controls, and when children with adverse neurological outcomes were excluded from the comparison, the proportion of cases versus controls who continued follow-up for the full duration of the study did not differ. These findings suggest that the additional medical services provided to children with adverse neurological outcomes represented an incentive for ongoing follow-up visits, and confounding is probably limited.We used the Epilepsy Screening Questionnaire to exclude children with a history of unprovoked seizures; however, it has not been validated for use in Malawian children. We used the Ten Questions screen to assess neurodisabilities. This screen lacks sensitivity in children younger than 2 years; 79 children (20%) in our study were younger than 2 years at the time of their first follow-up. We are not aware of any screening methods for identifying neurodisabilities in children younger than 2 years that have been validated for use by non-physician health-care workers in an African setting. Owing to age-based matching, extent of use of the Ten Questions screen in children younger than 2 years did not differ for cases versus controls and 382 (97%) of 396 children were screened at least once after reaching the age of 2 years.Before we started our study in 2005, behavioural disorders had not been reported as sequelae of cerebral malaria and behavioural disorders were not a pre-specified outcome. Recent retrospective clinic-based studies from MaliOur findings show that retinopathy-positive cerebral malaria is a risk factor for several adverse neurological outcomes including epilepsy, disruptive behavioural disorders, and neurodisabilities characterised by motor, sensory, or language deficits. Most sequelae were delayed in presentation and hence were not evident at the time of discharge from the hospital after the initial illness. Cerebral sequestration of parasites is the hallmark of cerebral malaria and the only methods available for confirming CNS sequestration require analysis of brain tissue. As such, it is difficult to know how representative this population of children with retinopathy-positive cerebral malaria is of all children with cerebral malaria. If the findings of our study are generalisable, then about 135 000 African children younger than 5 years have neurological sequelae due to cerebral malaria-induced brain injury each year and cerebral malaria might be one of the more common causes of epilepsy in malaria-endemic regions.The syndrome of epilepsy after cerebral malaria described in our cohort of cerebral malaria survivors is primarily localisation-related epilepsy that is often associated with other neurodisabilities and refractory to treatment with a single drug. Early mortality in two of 12 children who developed epilepsy suggests that retrospective studies might not identify epilepsy that results in early deaths.The finding that boys were at greater risk for new neurodisability was unexpected and might suggest that the Ten Questions screen has some sex-based bias in the Malawi population. However, recent epidemiological studies have established that boys experience higher stroke mortality rates across all paediatric age rangesMost of the risk factors for adverse neurological outcomes after cerebral malaria identified in this study are consistent with data from other causes of CNS injury. Hyperthermia is a risk factor for adverse outcome in stroke and traumatic brain injury,


Intermittent preventive treatment (IPTi) with sulphadoxine-pyrimethamine (SP) in infants resulted in different estimates of clinical malaria protection in two trials that used the same protocol in Ifakara, Tanzania, and Manhiça, Mozambique. Understanding the reasons for the discrepant results will help to elucidate the action mechanism of this intervention, which is essential for rational policy formulation.A comparative analysis of two IPTi trials that used the same study design, follow-up, intervention, procedures and assessment of outcomes, in Tanzania and Mozambique was undertaken. Children were randomised to receive either SP or placebo administered 3 times alongside routine vaccinations delivered through the Expanded Program on Immunisation (EPI). Characteristics of the two areas and efficacy on clinical malaria after each dose were compared.The most relevant difference was in ITN's use ; 68% in Ifakara and zero in Manhiça. In Ifakara, IPTi was associated with a 53%  reduction in the risk of clinical malaria between the second and the third dose; during the same period there was no significant effect in Manhiça. Similarly, protection against malaria episodes was maintained in Ifakara during 6 months after dose 3, but no effect of IPTi was observed in Manhiça.The high ITN coverage in Ifakara is the most likely explanation for the difference in IPTi efficacy on clinical malaria. Combination of IPTi and ITNs may be the most cost-effective tool for malaria control currently available, and needs to be explored in current and future studies.Manhiça study registration number: NCT00209795Ifakara study registration number: NCT88523834 Despite increased attention to malaria control by donors, researchers, clinicians, and communities, malaria continues to exact an intolerable toll, particularly in sub-Saharan Africa. The development of new tools such as combination of drug therapies and insecticide treated nets (ITNs) have offered hope, but their impact has been limited by low implementation and logistical and financial constraints. No single tool currently exists that can drastically reduce the malaria burden. Given this reality, and while awaiting new technologies, the malaria community must reexamine available data and interventions to look for creative and synergistic control strategies.It is well documented and accepted that the burden of malaria falls greatly on young children and infants . Even inIPTi with sulphadoxine-pyrimethamine (SP) has been shown to significantly reduce malaria episodes in randomized trials carried out in Tanzania, and more recently in Ghana and Mozambique -6. The eDespite the positive results for efficacy and safety, the determinants and underlying protection mechanisms of IPTi are not yet clear. The analysis of differences between clinical trials that use similar designs could provide insight into the potential determinants and possible underlying protection mechanisms, as well as facilitate planning for future policy recommendations. We had the unique opportunity to compare two very similar IPTi trials in two malaria endemic countries in Eastern and Southern Africa. We present results from a comparative analysis of the protective efficacy of IPTi, and examine the factors that may explain the different protection levels achieved ,6. The gPlasmodium falciparum malaria transmission has been intense in the area. The estimated mean annual entomological inoculation rate (EIR) was 300 bites/person/year in the late 1980's and early 1990's in villages surrounding Ifakara town [Anopheles gambiae, and to a lesser extent Anopheles funestus, are the main vectors. The Ifakara population was estimated to be 55.000 people.The Tanzanian study was based in Ifakara town, Kilombero District, in rural Tanzania and is described in detail elsewhere . Malariaara town . Howeverara town ,9 to an ara town . AnophelIn Ifakara, SP was not associated with any late treatment failures, had a in vivo parasitological sensitivity of 69% at day 14 , and wasP. falciparum. Anopheles funestus is the main vector and the EIR for 2002 was 38. The population under demographic surveillance was about 70.000 people.In Mozambique the trial was conducted in Manhiça town, Manhiça District, in southern Mozambique. The characteristics of the area have been described in detail elsewhere . The cliin vivo parasitological sensitivity of 78.6% at day 14 [During the Mozambique study, first line treatment of uncomplicated malaria changed from chloroquine to amodiaquine plus SP Most recent data from 2001 on the efficacy of SP in this area showed a combined therapeutic efficacy rate of 83% of children treated, with an t day 14 . CompliaDetailed descriptions of the Ifakara  and ManhAssessment of outcomes was similar in both studies. Comparable round-the-clock hospital-based clinical surveillance systems were operating in both sites; at each consultation a detailed standardized questionnaire was completed to document signs and symptoms ,17. BlooThick and thin blood films were stained and read according to standard, quality-controlled procedures . The pacThe impact of IPTi with SP on malaria morbidity was assessed through passive clinical surveillance and cross-sectional surveys carried out at ages12 and 18 months in Ifakara, and at ages 12 and 24 months in Manhiça.In the Ifakara study, first or only episodes of clinical malaria were reduced by 59% 95% CI, 41; 72%), and the incidence of severe anemia dropped by 50%  1; 72%, a. In ManhThe original data was re-analyzed at individual level using more stringent criteria than originally to include children in both cohorts that: i) received three doses of IPTi; ii) received the last IPTi dose between 9 and 10 months of age; and iii) had at least 1 month of follow-up after the last IPTi dose. Five periods were defined: i) 1 month after IPTi dose 1; ii) 1 month after IPTi dose 2; iii) 1 month after dose 2 until dose 3; iv) 1 month after dose 3; and v) 6 months, starting 1 month after dose 3.Plasmodium falciparum asexual parasitaemia. Cox regression models were used to compare the hazards for the time to first or only episode of clinical malaria in each period. The interaction between study and treatment effect was evaluated using the likelihood ratio test.Clinical malaria was defined as fever (axillary temperature ≥ 37.5°C) plus a Main characteristics of both trials are summarized in Table The analysis estimated the protective efficacy of SP for 30 days after each IPTi dose and found protection above 50% in both sites Table . The anaIntermittent preventive treatment with SP had different protective efficacies in reducing malaria and anemia incidence in these two malaria endemic settings in sub-Saharan Africa. The two trials were very similar in most aspects. Randomization, intervention, follow-up and assessment of outcome procedures were close to identical. The active drug and placebo were manufactured by the same pharmaceutical company. Intensity of malaria transmission during the course of both studies was comparable, not only in the comparable EIR, but also in the rates of malaria episodes in the placebo groups during the first year of life Table . At the The overall incidence of severe anaemia up to 1 year of age was not significantly affected by the SP intervention in Manhiça . ProphylThe administration of the IPTi regimen differed slightly in the two trials because of the different EPI immunization schedules. Consequently, in Ifakara the first two doses were given at 2 and 3 months, while in Manhiça they were administered at 3 and 4 months of age. It is unlikely that this variation in the intervention regimen would explain the difference found in the efficacy; particularly since the time between administration of the second and third dose was shorter in Mozambique. The risk of malaria is greater between age 5 and 9 months than during the first 3 months of life, and thus implies a theoretical advantage to the infants in the Manhiça study, which was not observed .HIV infection during pregnancy has been associated with increased susceptibility to malaria and lower efficacy of IPT with SP . In contThe analysis presented in this paper shows that, as expected, the risk of malaria was greatly reduced for 1 month in children who received a stat dose of a moderately effective antimalarial. However, the different protection levels between the two trials is mainly explained by the difference in risk of malaria outside the window of time when the drug is efficacious. There are no obvious reasons that explain these findings. In a previous report we have hypothesized that this reduced risk is a function of the accelerated acquisition of immunity in children receiving IPTi in the first year of life . The queThis comparative analysis provides an example of how existing data sources can be used to fine-tune malaria control strategies in the immediate future. Contrary to earlier speculation , the resCM. I declare that I have participated in the conception, design and conduction of the studies, and writing up of the manuscript, and have seen and approved the final version of the manuscript.DS. I declare that I have participated in the conception, design and conduction of the study (Ifakara), and writing up of the manuscript, and have seen and approved the final version of the manuscript.EM. I declare that I have participated in the conduction and analysis of the study (Manhiça), and writing up of the manuscript, and have seen and approved the final version of the manuscript.PA. I declare that I have participated in the conduction of the study (Manhiça), and writing up of the manuscript, and have seen and approved the final version of the manuscript.EK. I declare that I have participated in the conduction of the study (Ifakara), and writing up of the manuscript, and have seen and approved the final version of the manuscript.SS. I declare that I have participated in the analysis of the study (Manhiça), and writing up of the manuscript, and have seen and approved the final version of the manuscript.JJA. I declare that I have participated in the conception, design, and analysis of the studies, and writing up of the manuscript, and have seen and approved the final version of the manuscript.JS. I declare that I have participated in the conduction of the study (Manhiça), and writing up of the manuscript, and have seen and approved the final version of the manuscript.HM. I declare that I have participated in the conception, and design of the study (Ifakara), and writing up of the manuscript, and have seen and approved the final version of the manuscript.MT. I declare that I have participated in the conception, and design of the study (Ifakara), and writing up of the manuscript, and have seen and approved the final version of the manuscript.PLA. I declare that I have participated in the conception, and design of the studies, and writing up of the manuscript, and have seen and approved the final version of the manuscript


Dear Sir:In their short report on the utility of a point-of-care malaria rapid diagnostic test (RDT) in HIV-infected adults, Mills and others conclude that “the rapid diagnostic test accurately ruled “in or out” malaria.”At the “rule out” side, there is a moderate sensitivity of 85.7%, again with a disappointing lower confidence limit of 57.2%, which is caused by the low number of malaria cases. The high negative predictive value is probably caused by low pre-test probability  in the study population . Thus, the probability of not having malaria before the test was already 94.4%, and the diagnostic gain offered by the RDT is limited to 4.7%.Malaria is a life-threatening disease that requires prompt and adequate treatment. Two of 14 cases were missed; the confidence limits indicate that it might have been six. Rapid diagnostic tests can miss not only low parasite densities such as in the present study,3A cautious conclusion of this study might be that RDTs might be potentially useful to rule out malaria, but that studies on larger samples and patient populations with higher malaria prevalence are necessary to confirm this finding.an den EndeJ. VJan JacobsInstitute of Tropical MedicineAntwerp, BelgiumE-mail: jvdende@itg.beZeno BisoffiCenter for Tropical DiseasesSacro Cuore HospitalNegrar, Verona, Italy


Early diagnosis and prompt treatment including appropriate home-based treatment of malaria is a major strategy for malaria control. A major determinant of clinical outcome in case management is compliance and adherence to effective antimalarial regimen. Home-based malaria treatment with inappropriate medicines is ineffective and there is insufficient evidence on how this contributes to the outcome of severe malaria. This study evaluated the effects of pre-hospital antimalarial drugs use on the presentation and outcome of severe malaria in children in Ibadan, Nigeria.Two hundred and sixty-eight children with a median age of 30 months comprising 114 children with cerebral malaria and 154 with severe malarial anaemia (as defined by WHO) were prospectively enrolled. Data on socio-demographic data, treatments given at home, clinical course and outcome of admission were collected and analysed.A total of 168 children had treatment with an antimalarial treatment at home before presenting at the hospital when there was no improvement. There were no significant differences in the haematocrit levels, parasite counts and nutritional status of the pre-hospital treated and untreated groups. The most commonly used antimalarial medicine was chloroquine. Treatment policy was revised to Artemesinin-based Combination Therapy (ACT) in 2005 as a response to unacceptable levels of therapeutic failures with chloroquine, however chloroquine use remains high. The risk of presenting as cerebral malaria was 1.63 times higher with pre-hospital use of chloroquine for treatment of malaria, with a four-fold increase in the risk of mortality. Controlling for other confounding factors including age and clinical severity, pre-hospital treatment with chloroquine was an independent predictor of mortality.This study showed that, home treatment with chloroquine significantly impacts on the outcome of severe malaria. This finding underscores the need for wide-scale monitoring to withdraw chloroquine from circulation in Nigeria and efforts intensified at promoting prompt treatment with effective medicines in the community. Malaria remains a significant cause of morbidity and mortality among children and creates enormous social, economic and disease burdens in endemic regions,2. CurreIn Africa, majority of malaria episodes occur in rural areas with considerable variation in the treatment seeking pattern with over 50% self treated at home -5. Self-Plasmodium falciparum may be promoted by the presence of sub-curative concentrations of the antimalarial medicine in the blood, that will only eliminate the most sensitive parasites allowing those that are less sensitive to survive [Drug resistance remains a major problem affecting progress on management and control of malaria ,14. Chlo survive , thus pr survive . Based o survive ,14.Many studies have evaluated the effects of different antimalarial chemoprophylaxis in Nigerian children, -21 whichThe study was carried out in Ibadan, south-west Nigeria, a region that is exposed to stable, malaria transmission with seasonal . A totalP. falciparum independently by two trained and experienced microscopists. Patients were managed on admission according to WHO guidelines and National Antimalarial Policy.Ethical approval was obtained from the joint University of Ibadan, University College Hospital, Ibadan Ethical Committee, the Oyo State Ministry of Health and the WHO ethical review committee. Written informed consent was obtained from the parents or guardian of the patients prior to enrollment. A semi-structured questionnaire was used to collect data on patients' demographic characteristics, history of febrile illness, antimalarial drug treatments given during current episode of clinical malaria prior to presentation at the hospital and findings from physical examination findings at the time of admission. All patients had and clinical history and examination performed, followed by blood films taken from finger pricks. Malaria parasitaemia was confirmed with thick blood smears stained with Giemsa examined for trophozoites of p-value of < 0.05 was taken as being statistically significant.Data was entered and analysed using SPSS 11.0 for Windows . Descriptive statistics  were computed for continuous variables while frequencies were computed for categorical variables. Comparisons of continuous variables were computed using Mann-Whitney U test because of their non-parametric nature while comparisons of categorical variables were done using the Fishers exact test because of the small numbers in some subcategories. The logistic linear regression technique was used to investigate the predictive value of pre-hospital use of antimalarial and outcome (died vs. survived) after inclusion of age and clinical severity in the models. A The study patients comprised 155 (57.8%) male and 113 (42.2%) female giving a male to female ratio of 1.4:1. The age of the patients ranged from five months to 10 years with a median age of 30 months. One hundred and sixty eight (62.7%) of the study population had pre-hospital antimalarial treatments. Table Of the 268 patients with severe malaria, 114 (42.5%) were cases of CM while 154 (57.5%) had SMA without coma  and 42 (27.3%) of CM and SMA, respectively (p = 0.009). Amodiaquine was given to 12 (10.5%) of CM and 8 (5.2%) of SMA groups. Sulphadoxine-pyrimethamine was given to 7 (6.1%) of CM patients and six (3.9%) of SMA patients. Of the 168 children who received an antimalarial drug before admission, 48.2% had CM compared with 33% in the group of patient who had no antimalarial treatment . Table .0% and 4Early treatment with effective antimalarial treatment has been demonstrated to decrease the morbidity and mortality of malaria in endemic countries. In most countries in the sub-Saharan Africa however, there has been little success in reducing both infant and child mortality even with the focus on better management of malaria. The present study has shown that pre-hospital antimalarial treatment of febrile children remains a significant practice among child care-givers in Nigeria as previously reported from the same area . The prePre-hospital administration of pre-packaged chloroquine was previously reported to have reduced the risk of developing severe malaria among children in some African communities at a time when CQ was still effective . Our datP. falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease [However, in a previous report from the same centre, CQ resistance of  disease . The cli disease .A recent study from Nigeria has shown that correct use of chloroquine increased from 2.6% to 52.3% among home care-givers after training, however as high as 47.7% still wrongly treat febrile children . This fiIn addition, there is some evidence to suggest that households of a high social class are more likely to use antimalarials to which there is less parasite resistance. As majority of our patients were from lower socioeconomic class, reducing the cost, improving the availability and access to quality pre-packaged artemisinin-based antimalarial may improve pre-hospital treatment of malaria and thus prevent resistance . White  observedIn conclusion, the use of ineffective antimalarial medicines for the home-based management of malaria could be a hindrance to prompt access to effective treatment, as the caregivers delay seeking treatment convinced they have initiated antimalarial treatment, albeit with compromised medicines. This study has demonstrated this practise to be deleterious on the outcome of malaria. To maximise the impact of chemotherapy on malaria morbidity and mortality, resources are urgently required to facilitate the use of ACTs for home-based treatment of malaria.The authors declare that they have no competing interests.AEO and OKA contributed equally in the conception, design, analysis of the data and drafted the manuscript. PEO participated in the interpretation of the data and helped in drafting and writing up the manuscript. OOO participated in the design of the study and contributed to drafting and writing up the manuscript. All the authors read and approved the final manuscript.


Information on the period during which infants lose their maternally derived antibodies to malaria and begin to acquire naturally their own immune responses against parasite antigens is crucial for understanding when malaria vaccines may be best administered. This study investigated the rates of decline and acquisition of serum antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA to Plasmodium falciparum antigens apical membrane antigen (AMA1), merozoite surface proteins  in a birth cohort of 53 children living in an urban area in the Gambia, followed over the first 3 years of life . Antigen-specific maternally transferred antibody isotypes of all IgG subclasses were detected at birth and were almost totally depleted by 4 months of age. Acquisition of specific antibody isotypes to the antigens began with IgM, followed by IgG1 and IgA. Against the MSP2 antigen, IgG1 but not IgG3 responses were observed in the children, in contrast with the maternally derived antibodies to this antigen that were mostly IgG3. This confirms that IgG subclass responses to MSP2 are strongly dependent on age or previous malaria experience, polarized towards IgG1 early in life and to IgG3 in older exposed individuals. Infants are protected from a number of infections by maternally transferred immunity. This immunity is not long-lasting, and children need to acquire immunity against common pathogens early in life. For malaria, a degree of resistance in endemic areas in the first 3–6 months of age has been attributed to a combination of protection by maternally transferred antibodies, foetal haemoglobin that cannot support parasite development, lack of p-aminobenzoic acid (PABA) in the infant breast milk diet which prevents parasite growth and limited exposure to vectors –7.Plasmodium falciparum antigens were found to be more susceptible to malarial attack, suggesting that maternally derived antibodies are an epidemiological marker for risk of infection in infants , apical membrane antigen (AMA1) and erythrocyte binding antigen (EBA175), have been shown to be associated with reduced incidence of malaria in prospective cohort studies and where the subclass has been investigated, it is cytophilic and complement fixing IgG1 and IgG3 subclasses that are most strongly associated with protection  inversely correlated with risk of infection and vice versa for IgG4  demonstrated the effect of antigen, age and exposure on IgG subclass responses . The plates were incubated at 4°C overnight and then washed four times with PBS-T (PBS with 0·05% v/v Tween 20). Unoccupied protein binding sites on the plate were blocked with 200 μL per well of blocking buffer  for 5 h at room temperature. Plates were washed prior to the addition of 100 μL of each plasma diluted with blocking buffer , dilutions chosen after optimization of assays for each isotype. After incubation at 4°C overnight, the plates were washed four times and 100 μL of horseradish peroxidase (HRP) conjugated sheep antibodies specific to detect each of the human IgG subclasses , and HRP conjugated rabbit anti-human IgM or IgA  at 1/5000 were added to each well and incubated at room temperature for 3 h. Plates were washed four times and antibody reactivities detected with 100 μL of substrate  in developing buffer . The reaction was stopped with 50 μL of 2 m H2SO4 and optical density (OD) of plates measured at 490 nm. The assays were run in duplicate wells with different time point samples from each individual run on the same plate. The OD reading was corrected for nonspecific binding by subtracting the OD for antibody reactivity to the relevant fusion tags, MBP for MSP3 recombinant proteins and GST for MSP1 and MSP2 recombinant proteins. Antibody isotype reactivities of 20 nonimmune negative control individuals (European tourists in The Gambia) were also assayed.Enzyme-linked immunosorbent assays were used to detect the reactivity of antibody isotypes IgG1, IgG2, IgG3, IgG4, IgM and IgA in plasma to a panel of blood stage antigens AMA1, MSP1-19, MSP2-ch150/9, MSP2-Dd2, MSP3-3D7 and MSP3-K1. Briefly, wells of flat bottom 96-well plates  were coated with 50 ng of recombinant antigen  in 100 μL of coating buffer  and GraphPad Prism version 4·0  for analysis. An antibody isotype level was considered positive if the individual’s OD reading was above a cut-off value . Statistical comparisons of antibody isotype levels in cord blood among different calendar birth months were performed using Wilcoxon’s signed rank test.P = 0·04) or January 2004 (P = 0·02). For IgG3, levels were significantly lower in July 2003 than in August 2003 (P = 0·005), September 2003 (P = 0·006) or January 2004 (P = 0·02), and levels were also lower in August 2003 than in January 2004 (P = 0·04). Levels of IgG2 were very low generally, although even lower in September 2003 than in January 2004 (P = 0·01). For IgG4, there were no differences in levels between the birth months. Most of these differences were not significant after a Bonferroni correction for multiple comparisons, except for IgG3 levels that were significantly lower in July 2003  than that in other months.Of the 53 children analysed with all time point samples in the cohort, 5 (10%) were born in July 2003, 15 (28%) in August 2003, 17 (32%) in September 2003, 7 (13%) in January 2004 and 9 (17%) in February 2004. The mean levels of cord blood antibodies to the different antigens for each birth month are shown in The relative levels of different antibody isotypes in cord blood sera were observed to vary among antigens . PrevaleAntibody isotype reactivities to malaria antigens were analysed for samples collected at follow-up ages 4, 8 or 9, 18 and 36 months. A decline of all the IgG subclasses to all antigens was first observed in the children at 4 months of age. Detectable IgG1 to AMA1 persisted for longer because of high initial levels, as demonstrated by the proportion of children with positive levels at 4 months of age . After 9The primary antibody isotype IgM to AMA1 was detected in some children, with positive responses increasing with age, although only in a minority of individuals, 6% at 4 months, 15% at 18 months and 23% at 36 months . To eachMaternally transferred IgG subclass reactivity to MSP2 in the cord blood was dominated by IgG3. In contrast, acquisition of IgG1 to MSP2 occurred in individual children , whereasMaternally transferred antibody reactivities in cord blood to the panel of antigens followed the polarized pattern of IgG1 to AMA1 and MSP1-19, and IgG3 for MSP2-ch150/9 and MSP2-Dd2, previously described for endemic sera in The Gambia and elsewhere ,39,44,45The birth month of the children was associated with differences in the level of antibodies detected in the cord plasma. The Gambia experiences annual rainfall from June to October and thus seasonal malaria transmission occurs at this period, with a peak of incidence between August and November ,47. TherIt is important to note that although antibodies to MSP2 are predominantly IgG3 in the cord blood, children who made a response produced IgG1 against MSP2. This shows that IgG1 is the first IgG subclass synthesized against this malaria antigen after primary infections. This is consistent with the previous suggestion that there is an exposure-related shift from IgG1 to IgG3 to MSP2 antigens ,49. It aAn increase of antibody levels in these children was expected with age. However, it was observed that the proportions with any antibody responses from 9 months of age onwards were very low. A recent decline in the incidence of malaria in The Gambia, particularly since 2003 , account


Plasmodium falciparum infections is the gradual acquisition of clinical immunity resulting from repeated exposures to the parasite. While the molecular basis of protection against clinical malaria remains unresolved, its effects on epidemiological patterns are well recognized. Accumulating epidemiological data constitute a valuable resource that must be intensively explored and interpreted as to effectively inform control planning.A characteristic of Here we apply a mathematical model to clinical data from eight endemic regions in sub-Saharan Africa. The model provides a quantitative framework within which differences in age distribution of clinical disease are assessed in terms of the parameters underlying transmission. The shorter infectious periods estimated for clinical infections induce a regime of bistability of endemic and malaria-free states in regions of mesoendemic transmission. The two epidemiological states are separated by a threshold that provides a convenient measure for intervention design. Scenarios of eradication and resurgence are simulated.In regions that support mesoendemic transmission, intervention success depends critically on reducing prevalence below a threshold which separates endemic and malaria-free regimes. Plasmodium falciparum in 86 endemic countries, resulting in about 515 million clinical cases worldwide and over 1 million fatalities in Africa every year P. falciparum infections is the gradual acquisition of clinical immunity resulting from repeated exposures to the parasite Approximately 2.2 billion people are affected by Understanding the mechanism of acquisition of clinical immunity has become a classical problem in malaria research. Approaches to resolve this puzzle involve the development of comprehensive yet parsimonious mathematical models The model and methodologies developed here are not bound to the data, in the type and form presented here, and can be used with other data sets if desired.The model is represented diagrammatically in S) progress to a clinical form of malaria, requiring treatment, that we will henceforth call clinical malaria (I1). This group comprises both uncomplicated cases of clinical malaria which are treated without hospitalisation, and more severe cases requiring in-patient care. Based on the inference that immunity to severe malaria is acquired after one or two infections per se. Hence, upon reinfection, a recovered individual (R) develops a non-clinical form of malaria, which we will refer to as asymptomatic infection (I2). While in the R compartment, individuals may lose their immunity, return to S and acquire another clinical infection (I1). Each variable represents a proportion of the total population. At the population level, patterns of occurrence of the two types of infection, I1 and I2, are intertwined and our aim is to identify mechanisms consistent with reported prevalence of hospitalised clinical malaria. We specify rates of recovery from infection as τ1 and τ2, respectively, and assume that protection wanes at a rate, α, unless boosted by repeated exposures. The force of infection, λ(a), that varies between populations according to the value of λ0, and includes age dependence in agreement with previous studies We consider that infections in immunologically naïve individuals (λ0(1−c) (at age zero) and upper limit λ0. Parameter k determines how steeply the force of infection increases with age, and c controls the amplitude of the increase. A summary measure of transmission is obtained by integrating the force of infection over age asP(a) = μ eμa−, is the total population distributed over age and μ is the birth and death rate. Adopting standard assumptions, Λ is proportional to the frequency of infectious individuals, the proportionality constant being the transmission coefficient, β = Λ/(I1+φI2), where φ represents the relative infectivity of asymptomatic to clinical malaria infections.This function was derived from results of an entomological study, that correlates mosquito feeds with human body mass. The function is strictly increasing with age, with a minimum a = 0 are S = μ and R = I1 = I2 = 0.The boundary conditions for system (1) at age The datasets, from eight endemic sites, consist of retrospectively assembled data on paediatric admissions presenting from pre-defined catchment populations with clearly defined addresses to selected hospitals within 20km of their home, and where diagnosis was supported by detailed clinical examination and parasitology  and local . More specifically, we consider that rates of recovery and waning immunity are global and the force of infection is a local parameter β, are calculated by dividing the forces of infection integrated over age obtained from equation (3) by the sum, I1+φI2, in equilibrium conditions. We will represent the obtained equilibrium solutions as a function of transmission intensity, which is often represented in the form of the basic reproduction number.Equilibrium solutions are calculated by analytically solving the model without age structure using the global parameter values estimated previously. The transmission coefficients, R0, is the average number of secondary infections generated by the introduction of a single infectious person into an otherwise naïve population. This theoretical number is calculated by multiplying the transmission coefficient, β, by the average duration of a primary infection, 1/(τ1+μ), to obtain R0 = β/(τ1+μ). We stress that although R0 can be indirectly calculated by manipulating parameters that can be estimated from an endemic equilibrium, this number is only meaningful when malaria is newly emerging in an immunologically naïve population. When transmission is endemic, a proportion p of infections occurs in clinically immune individuals. These infections are likely to show no clinical symptoms, remain untreated and transmit for a possibly different period, 1/(τ2+μ). In this case, the average number of infections generated by an infectious individual is R0 = β((1−p)/(τ1+μ)+p/(τ2+μ)).The basic reproduction number, η = 1, the estimates obtained for the global parameters and the corresponding 95% confidence intervals are:Assuming, initially, the maximal hospitalisation rate, φ = 1, we estimate region-specific forces of infection as listed in From (4) we can see that, on average, individuals with clinical malaria are infectious for a shorter time period (about 25 days) than those with an asymptomatic infection (circa 165 days). The duration of clinical immunity lasts for around 1 year and the age-dependent force of infection rises from 1% to 76% of its upper limit between ages 0 and 10 years. Under the initial assumption that clinical cases are as infectious as mild infections, η = 1, φ = 1), whose robustness is challenged in I1 (from the model) ranked by the estimated forces of infection. We infer that prevalence of clinical malaria peaks at low to intermediate transmission to gradually decline as transmission increases. This phenomenon has been termed as endemic stability R0<1. τ1>τ2). This framework provides an identification of regimes where malaria control is sustainable. Endemic and disease-free states are separated by an unstable equilibrium that represents the critical prevalence below which the system converges to the disease-free state. The datasets considered in this study include regions that are representative of the various scenarios produced by the model. We consider three regions for illustration purposes. In hyperendemic regions (e.g Kilifi) the endemic equilibrium is the only stable solution, while in hypoendemic regions (e.g. Bakau), the system shows no stable endemic equilibrium consistently with a scenario of unstable transmission. In regions of mesoendemic transmission , however, fixed parameter values can stably maintain a malaria-free state, or support transmission resulting in high levels of clinical disease. The outcome is determined by the initial conditions and can be manipulated by specially designed and carefully monitored interventions.The existence of two stable equilibria for the same parameter values is named bistability. This is generated by a positive feedback mechanism that enhances transmission when endemicities are established η. Reducing the hospitalisation rate systematically contracts the bistability region, which persists until η reaches the proximity of 0.1. While lacking a rigorous estimate for this parameter, we cannot exclude the possibility that η is so low as to compromise the bistable behaviour. The robustness of the bistable regime is, nevertheless, reassured when we explore the relative infectivity of asymptomatic to clinical infection, φ. φ,η) parameter space. For concreteness, the parameter combinations where bistability is predicted for Foni Kansala are also marked (in dark grey). Recent empirical studies φ>1. Coincidentally, the analysis demonstrates that increasing φ enhances the bistability phenomenon favouring the realism of our findings.In Confronting a mathematical model with observed age profiles of clinical malaria, we obtain estimates for the parameters underlying the dynamics of transmission. In particular, rates of recovery from infection are obtained indicating that, on average, individuals with clinical malaria are infectious for a shorter time period than those with an asymptomatic infection. We attribute this mainly to behavioural factors. A clinical infection is likely to lead to administration of anti-malarials and faster recovery. This trend is consistent with estimates obtained from epidemiological observations of populations under treatment, where the average duration of infection was brought down from 270 to 14 days by administration of drugs α. While Λ prevents the return to the susceptibility pool for clinical malaria, α has the opposite effect of generating more susceptibles. Since α is a global parameter, a peak in clinical disease is expected to occur as Λ varies across regions. This general model, applicable to malaria and other diseases  where patterns of clinical malaria incidence are consistent with perturbations of the malaria-free state rather than a stable endemic state.More relevant to malaria control, is the finding of bistable behaviour in regions of mesoendemic transmission, such as Foni Kansala and Sukuta. Bistability is generated by the establishment of longer infections in clinically immune individuals, in comparison to infections in those that are immunologically naïve. This asymmetry, which enhances transmission when endemicities are established, leads to the identification of conditions for sustainable malaria control. Interventions in the field have accumulated unsuccessful eradication attempts in regions of hyper to holoendemic transmission, such as the Garki district in Nigeria A practical tool for control planning comes from the observation that the two epidemiological states are separated by an unstable state (dashed branch) indicating a threshold for either eradication or resurgence. This threshold, expressed in terms of parasite prevalence, or other measures convenient to intervention design, is reduced if the typical duration of infection increases. Stability of the malaria-free state relies, therefore, on a system of early detection and response, whose purpose is to shorten the infectious period of parasite-positive individuals through antimalarial treatment. Shifts from disease-free to endemic states are usually triggered by a sufficiently intense migration from endemic regions, and can be prevented by early detection and treatment of malaria infections irrespective of symptoms The model adopted here is consistent with previous models in malaria epidemiology Mosquito population and infection dynamics is another classical topic in malaria research that has recently recovered attention from modellers R0 estimates in regions of holoendemic transmission by an order of magnitude R0 estimated directly in mesoendemic scenarios may appear higher than the number of secondary infections that would be generated by the introduction of a single infectious person into an otherwise naïve population. For concreteness, we calculate, from the endemic equilibrium solutions, the proportion, p, of infections which occurs in clinically immune individuals, and obtain values between 0.95 and 0.99 depending on the region considered. As for the recovery rate, τ2, this has been estimated as approximately 6.3 times lower than τ1. Replacing these values in the formula above given in R0≈6.3β/(τ1+μ), which is about 6 times higher than the “true” basic reproduction number. As a consequence, malaria eradication in those regions appears more viable than previously thought.Finally, the results presented here have important implications for the interpretation of measured indices of transmission. The basic reproduction number for malaria has recently been reformulated to consider heterogeneity in biting rates, resulting in an increase of Appendix S1(0.39 MB PDF)Click here for additional data file.


Severe anaemia is a common, frequently fatal, condition in African children admitted to hospital, but its long term outcome is unknown. Early reports that survivors may be at risk of additional late morbidity and mortality may have significant implications for child survival in Africa. We assessed the short and long term outcome of severe anaemia in Malawian children and identified potential risk factors for death and further severe anaemia.For 18 months, we followed up children (6–60 months old) presenting to hospital with severe anaemia (haemoglobin ≤5g/dl) and their hospital and community controls with the aim to compare all cause mortality and severe anaemia recurrence rates between the groups, and to identify risk factors for these adverse outcomes. A total of 377 cases, 377 hospital controls and 380 community controls were recruited. Among cases, the in-hospital mortality was 6.4% and post-discharge all cause mortality was 12.6%, which was significantly greater than in hospital controls (2.9%) or community controls (1.4%) . The incidence of recurrence of severe anaemia among the cases was 0.102 per child-year , and was significantly higher than the 0.007 per child-year (95% CI 0.003–0.015) in the combined controls (p<0.0001). HIV was the most important risk factor both for post-discharge mortality  and for recurrence of severe anaemia .Severe anaemia carries a high ‘hidden’ morbidity and mortality occurring in the months after initial diagnosis and treatment. Because severe anaemia is very common, this is likely to contribute importantly to overall under-five mortality. If not adequately addressed, severe anaemia may be an obstacle to achievement of the Millennium development goal No.4 on child survival. Strategies to diagnose and properly treat HIV infected children early most likely will reduce the high post-discharge mortality in severe anaemia. Severe anaemia is a common condition causing significant morbidity and mortality in children in Africa. Of children admitted to hospital, 12–29% have severe anaemia requiring a blood transfusion and the in-hospital mortality is commonly very high, ranging between 4% and 10% Investigators in Kenya reported unexpectedly high post-discharge mortality and recurrent severe anaemia rates in children within two months of a severe anaemia episode In this study our first aim was to compare rates of survival and of recurrence of severe anaemia over 18 months between children admitted to hospital with severe anaemia, children admitted to hospital for other reasons and healthy children in the local community. Secondly we aimed to investigate risk factors for recurrent severe anaemia and all cause mortality during an 18-month follow-up period.This study was the longitudinal part of a case-control study investigating the aetiological factors for severe anaemia in children cases) were recruited if they had a blood haemoglobin concentration ([Hb]) of less than 5.0 g/dl, were aged 6–60 months and had not received a blood transfusion during the preceding four weeks. A child was not enrolled if the anaemia was known to be due to malignancy or trauma. For each case a Hospital Control (HC) was recruited the following day on presentation to the same out-patient department for a condition other than severe anaemia, and when the index case was discharged from hospital a Community Control (CC) was recruited from among apparently healthy children in the community. The guardian of the index case assisted in identifying the nearest child not part of the immediate family residing within 100–1000metres of the home of the index case. HC and CC had to be aged between 6 and 60 months, and not severely anaemic. Written informed consent was obtained from the guardians of the children and the study was approved by the ethics committees of the University of Malawi and the Liverpool School of Tropical Medicine, UK.Children were recruited from July 2002 to July 2004 and followed up until February 2006 from the hospital's Paediatric Accident and Emergency Unit (Blantyre) or Under-fives Clinic (Chikwawa). Each of these is a facility available to the public without charge, functioning in daylight hours for six days each week. Severely anaemic children (HCs) received 24 hour care in a specialised research ward while in hospital. An array of laboratory tests to determine the aetiology of the clinical condition (described below) were performed Cases received an urgent blood transfusion (20mls whole blood per kilogram body weight). Severe malaria was treated with parenteral quinine until the child was able to take oral medication, then sulfadoxine-pyrimethamine was given according to national policy at that time The process of recruitment included a detailed medical history and physical examination. Samples of venous blood, urine and stool were collected. Children requiring hospitalisation  were actively followed up at 1, 3, 6, 12 and 18 months from date of discharge. Guardians were additionally asked to return with the child to a study clinic whenever the child was sick. During each follow-up visit, a standard clinical form was completed and, if necessary, the child was treated by the attending clinician. At the time of this study, anti-retroviral therapy (ART) was not available to children in Malawi. Deaths and severe anaemia episodes were recorded, and if they occurred outside the study clinics, they were investigated as completely as possible using the patient's ‘Health Passport’ book, hospital records and home interviews with parents or guardians. Deaths occurring during the initial hospitalisation period were recorded as in-hospital mortality. Deaths occurring after discharge but during the study follow-up period were recorded as post-discharge all cause mortality and information surrounding the death was collected using a verbal autopsy All three study groups  Schistosoma haematobium using a semiquantative concentration method cases and HC according to a standard method using an automated BacT/Alert system  and cultured for 7 and 56 days for routine pathogens and mycobacteria respectively. Mixed growth or growth of Micrococci, Bacillus species or coagulase-negative staphylococci were considered contaminants. HIV testing was performed according to WHO guidelines using two rapid tests . Discordant results and reactive results in children less than 18 months were resolved by PCR IL10)  TNF)  All laboratory assays were done in all study groups, unless otherwise stated and were blinded to patient study group where possible. Haemoglobin concentration was measured on site using the HemoCue B-Haemoglobin analyser  and subsequently by Coulter counter analyser . Ferritin was measured using the electrochemiluminescence immunoassay  levels were measured using ELISA . Iron deficiency was defined as a sTfR/log ferritin index of 5.6 or more Data were analyzed using SPSS for Windows© version 12 and STATA© version 9. To compare the characteristics of the three groups of children, discrete data were analyzed by the Fisher exact test and continuous data by independent samples Student's t-tests. Survival times were recorded as the duration of follow-up from the date of recruitment or the date of discharge  until the date of a severe anaemia episode or post-discharge mortality. Severe anaemia was defined as [Hb] less than 5g/dl during the follow-up period and at least four weeks after a previous severe anaemia episode. Kaplan-Meier curves were plotted for the three groups of children and compared using the Log-rank test.cases. It was not possible to perform a similar analysis for HC and CC owing to the few deaths observed in these study groups. Among cases, we examined the association with all cause mortality of the following risk factors: residing in an urban/rural area, sex , age (in months), education of mothers of study participants (none/some), employment of parents (unemployed/employed), number of siblings (>2 siblings/≤2 siblings), history of previous blood transfusion (yes/no), history of jaundice (yes/no), blood in urine or stool (present/absent), wasting (<−2 Z-score weight-for-height/≥−2 Z-score weight-for-height), stunting (<−2 Z-score height-for-age/≥−2 Z-score height-for-age), undernutrition (<−2 Z-score weight-for-age/≥−2 Z-score weight-for-age), splenomegaly (present/absent), CRP (≤10 mmol/L/>10mmol/L), iron deficiency (≥5.6 sTfR/Log ferritin/<5.6 sTfR/Log ferritin), hookworm (present/absent), malaria , HIV infection (positive/negative), bacteraemia (present/absent), non-typhoidal salmonella bacteraemia (present/absent).We investigated the associations between risk factors determined at recruitment and post-discharge all cause mortality during the 18 month follow-up period among Cox proportional hazards models were used to examine the association between risk factors mentioned above and all cause mortality through the 18 months post-discharge follow-up period. Initially, each covariate (risk factor) was entered into a separate univariate model with time to post-discharge death entered as the dependent variable. All covariates with p≤0.10 in the univariate models and important confounders  were entered into a multivariable model. We also compared the proportion of children with HIV infection by study group using chi-squared test and determined the respective risk ratios.cases. This analysis was not possible for HC and CC owing to the few recurrences of severe anaemia in these study groups.We performed a similar analysis  to assess risk factors for the development of first recurrence of severe anaemia during the follow-up period among A total of 381 cases, 377 HC children and 380 CC children were recruited; 4 cases had to be excluded from the statistical analysis because outcome information was missing, reducing this cohort to 377 children. Of all children, 53.4% (606/1134) were from the urban site (Blantyre). Over the 18 months study period 17.8% (63/353) of cases, 19% (74/377) of HC and 15.3% (58/380) of CC were lost to follow-up, and the commonest reasons being emigration out of the study area and withdrawal of consent. There were no significant differences in baseline characteristics of the children lost to follow-up compared to those that completed the study follow-up period.Cases had a higher prevalence of stunting, wasting and malaria compared to controls. It was found that iron deficiency, determined in a subgroup of children only, was less common in cases  than in HC  or CC . Bacteraemia was more common among cases  than HC  with 72.1% (49/68) caused by non-typhoidal Salmonella. Blood cultures were not done on CC. HIV prevalence was significantly higher among the cases  than HC  or CC .There were a few differences in the baseline characteristics of the three groups . Cases hcases, 17.2% (65/377), than the combined control groups 2.0%  and they were ten times more likely to die than controls . Of these deaths among the cases, 36.9% (24/65) occurred in hospital  while the remaining 63.1% (41/65) occurred during the post-discharge18 month follow-up period. Most post-discharge deaths occurred in the community and hence their cause could not be exactly determined, however most deaths followed after a short history of illness. There were no deaths that could have been remotely related to anaemia e.g. trauma. The Kaplan-Meier plot (using censored data) of the three cohorts of children . Crude post-discharge death rates not using censored data are presented in cases occurred after a median time of 4.1 months (IQR 1.8–8.1), with 70.7% (29) occurring within the first six months.The overall all cause mortality rate  was significantly higher among the children  shows a cases HR 9.4, 95% CI 5.0–17.5; HC HR 12.7, 95% CI 3.2–50.8; CC HR 17.1, 95% CI 2.9–102.2).Nearly half the children who died, 45% (27/60) were infected with HIV . Among acases, risk factors for post-discharge all cause mortality are presented in For cases during the 18-month follow-up period, with a median time to event of 2.9 months (IQR 1.4–6.6)  during the follow-up period than HC (1.1%) or CC (0.6%). Crude estimates of rates of recurrence of first episode of severe anaemia are presented in cases was 0.102 per person-year (95% CI 0.075–0.138) compared to 0.007 per child-year (95% CI 0.003–0.015) for the combined controls (p<0.0001). All children with an episode of severe anaemia during the follow-up period were re-admitted to hospital and given a blood transfusion.Recurrence of first episode of severe anaemia was observed in 10.2% (Kaplan-Meier estimates) of 1.4–6.6) . Cases hcases who had had a previous transfusion or symptom of jaundice were more likely to develop severe anaemia during the follow-up period . Malaria on admission was associated with a protective effect, with malaria affected cases having an 80% reduced risk of recurrence of severe anaemia . HIV infection was associated with a five times increased risk of recurrent severe anaemia  among cases. Overall, among all study groups of children, univariate analysis showed that HIV infection was non-significantly associated with severe anaemia .Having more than 2 siblings, was strongly associated with an increased risk of severe anaemia (AHR 5.0 95% CI 1.9–13.3) . From thHC and CC respectively . During the follow-up period, a higher proportion of cases were re-admitted to hospital  as compared to HC  and CC . These proportions were not significantly different between the HC and CC (p = 0.8).There were 910, 1185 and 791 sick-clinic visits for cases ectively . Of thesThe in-hospital case-fatality rate of 6.4% in the present study is within the range of previously reported studies in Sub-Saharan Africa Of serious concern is the high post-discharge all cause mortality observed among the cases in this study, most deaths occurring within six months after admission for severe anaemia. The all cause mortality rate over the same period was significantly lower in other children attending the hospital  and in children in the community (community controls). Children with severe anaemia, even if treated in hospital, remain a highly vulnerable group after discharge. Outside of the context of a research study, such deaths may remain undetected by the health services, and if recorded, the link with a previous episode of severe anaemia may be unrecognized.Before this study the longest follow-up of children after an episode of severe anaemia was eight weeks, in an investigation in rural Kenya Several possible explanations for the high incidence of post-discharge all cause mortality and recurrent severe anaemia should be considered. Firstly, it is possible that the underlying cause of the initial episode of severe anaemia was neither diagnosed nor treated, and therefore haemoglobin levels may have deteriorated again after discharge. Considering the extensive diagnostic investigations Secondly, the aetiology of the severe anaemia episode may have been identified correctly, but treatment failure occurred as a result of multi-drug resistant malaria or bacterial infections. It has been shown that recrudescent and new malaria infections during the first six months can negate the initial haematological improvement attained from receiving blood transfusion cases may have been seen in the end-stage of a chronic underlying disease, with severe anaemia being merely a marker of disease severity. This hypothesis is supported by the fact that HIV infection was found to be the most important independent risk factor for post-discharge all cause mortality. Anaemia is not only a common paediatric manifestation of HIV, but has been shown to be strongly and consistently associated with the progression of HIV disease and death Thirdly, cases haemoglobin concentrations were still well below the normal range following transfusion–a situation that is likely to prevail in many health facilities in Africa–and a relatively minor additional haematological insult could have major consequences.Finally, inadequate blood transfusion may explain the high rates of post-discharge all cause mortality and recurrent severe anaemia, an explanation also suggested in the Kenyan study Iron status determination in populations were inflammatory conditions are common can be challenging. In this study sTfR-Log ferritin index was used after assessment with bone marrow iron aspirates found it to best predict bone marrow iron status S. typhimurium and S. enteritidis, are widely prevalent in Africa and are associated with malaria and HIV infection cases than HC, though a comparison with CC could not be made. This association may be due in part to the higher prevalence of malaria and HIV infection in cases than controls, malaria and HIV being known risk factors for bacteraemia. Bacteraemia was not an independent risk factor for post discharge all cause mortality or recurrence of severe anaemia. Whether routine treatment with antibiotics should be given to all children with severe anaemia remains a matter of debate. Our data suggest that this would be a reasonable policy where blood cultures cannot be carried out.The importance of bacteraemia has been shown in a previous study in Malawi, where it was associated with severe anaemia Our analysis included only risk factors that could be ascertained at the time of admission. Causes of a post-discharge death, if it occurred in the community, could only be investigated retrospectively by using the verbal autopsy method. Verbal autopsies are known to be neither very sensitive nor specific; therefore the true causes of post-discharge all cause mortality in the study population could not be clearly determined This study provides disturbing evidence of consequences of severe anaemia on child health and survival. It is commonly thought that most deaths due to severe anaemia occur in hospital


Plasmodium falciparum on B-cell homeostasis have not been well characterized. This study investigated whether an episode of acute malaria in young children results in changes in the peripheral B cell phenotype.The effects of Using flow-cytofluorimetric analysis, the B cell phenotypes found in the peripheral blood of children aged 2–5 years were characterized during an episode of acute uncomplicated clinical malaria and four weeks post-recovery and in healthy age-matched controls.+ B lymphocytes during acute malaria. Characterization of the CD19+ B cell subsets in the peripheral blood based on expression of IgD and CD38 revealed a significant decrease in the numbers of naive 1 CD38-IgD+ B cells while there was an increase in CD38+IgD- memory 3 B cells during acute malaria. Further analysis of the peripheral B cell phenotype also identified an expansion of transitional CD10+CD19+ B cells in children following an episode of acute malaria with up to 25% of total CD19+ B cell pool residing in this subset.There was a significant decrease in CD19Children experiencing an episode of acute uncomplicated clinical malaria experienced profound disturbances in B cell homeostasis. Plasmodium falciparum malaria annually [P. falciparum occurs mainly in children under five years of age living in the tropical and sub-tropical areas of the world, where malaria transmission is holoendemic [P. falciparum remains an important question to solve in order to achieve protective immunity by means of vaccination.There are over 500 million episodes of clinical annually . The prioendemic ,3. Malaroendemic . In addioendemic . The mecP. falciparum infection. Hypergammaglobulinaemia has been a well-described feature of Plasmodium infections [et al [P. falciparum-specific memory B cells as indicated by loss of P. falciparum antibodies, and Kassa et al [+ B cells. Polyclonal B cell activation induced by P. falciparum [P. falciparum erythrocyte membrane protein 1(PfEMP1) and merozoite antigens [A number of observations clearly indicate that B cells are affected by fections  and persfections . Dorfmans [et al  reportedsa et al  reportedlciparum  most likantigens .P. falciparum infection, chronic antigenic activation of B cells within the context of repeated P. falciparum infections may lead to cytogenic abnormalities or aberrations in B cell development and trafficking. This may be one possible explanation for the increased risk for Burkitt's lymphoma in children living in malaria endemic settings [In addition to disturbances in generation and maintenance of B cell immunity following settings .et al [+ B cells following acute P. falciparum and P. vivax infections, but no further phenotyping of B cell subsets was done. Recent development in B cell biology allow distinctions of peripheral B cells into distinct subsets i.e. naive (CD19+IgD+) and memory (CD19+IgD-) subsets based on IgD staining [et al [+CD19+ immature transitional B cells suggesting that the transitional B cells are potential marker of inflammation. Whether P. falciparum infection induces these cells is unknown.Although there is some data on the types and frequencies of mature B cells that populate the peripheral blood in healthy children and HIV infected children , few stuet al  found a staining . Furtherg [et al . They usg [et al ,17. Howeg [et al ,19. InfeP. falciparum infection modulates B cell homeostasis, flow cytofluorimetric (FCF) analysis was used to quantify the percentage of total B cells (CD19+) and to discriminate B cell subsets in the peripheral blood in children experiencing an acute clinical case of P. falciparum malaria and four weeks following recovery. In this study, it was shown that following acute clinical P. falciparum malaria there were significant increases in CD10+ B cells as well as a decline in CD19+IgD-CD38- population of memory B cells.To understand how This study was carried out at Chulaimbo Rural Training Center during the months of June and July 2003. This clinic serves a malaria holoendemic area  and is lP. falciparum parasitaemia ≥5,000 parasites/μl. Children were excluded from the study if they were severely immunosuppressed, malnourished, or had other concurrent infections that may cause fever such as lower respiratory tract infection; had haemoglobin (Hb) less than 5 g/dl; and parasitaemia with fever at the follow-up visit four weeks after enrollment in this study or relocation from study area. Follow-up was conducted four weeks post-recovery. Fifteen children met the inclusion criteria and were further analysed. Ten children were excluded from the study at follow-up based on the exclusion criteria . Eleven healthy-age matched children who were parasitaemic, but asymptomatic, and from the same geographic area were included as controls. The clinical and the demographic data of the study participants are displayed in Table All the protocols used in this study were reviewed and approved by the Kenya Medical Research Institute (KEMRI) Ethical Review Committee and Institutional Review Board of Human Studies at University Hospitals of Cleveland, Case Western Reserve University, USA. After informed consent was obtained from the parents or guardians, twenty-five children aged 2–6 years presenting with acute clinical malaria  were enrolled in the study. All malaria cases were treated according to the Kenyan national malaria treatment policy set in 1998 which using sulphadoxine-pyrimethamine (SP) as the first-line anti-malaria treatment. Inclusion criteria for children in the study included parental/guardians consent for blood samples to be drawn twice within four weeks, age two to six years, and admission with a diagnosis of acute malaria morbidity as defined by an axillary temperature ≥37.5°C and Plasmodium species and level of parasitaemia. Following microscopic confirmation of P. falciparum parasitaemia of sufficient density, 2–5 ml of blood whole blood was collected in EDTA vacutainer by venipuncture.After measurements of haemoglobin levels using a portable B-haemoglobin photometer , thick and thin blood films were prepared from finger prick samples and stained with 5% Giemsa to determine Blood was anticoagulated in EDTA and peripheral blood mononuclear cells (PBMC) were separated from whole blood by Ficoll-Hypaque density gradient centrifugation within two hours of venipuncture. Cell viability was determined using the trypan blue dye exclusion assay. Cells were resuspended in 1 ml of freezing media  and stored at -80°C for later batch analysis by flow cytometry.5 cells/100 μl flow buffer  were stained on ice with the following antibodies to lymphocyte surface receptors: CD3-FITC, CD8-FITC, IgD-FITC, CD38-FITC, CD19-PE, CD4-PE, CD38-PE, CD-10PE, CD23-PE, CD45-APC, CD3-APC, and CD19-APC . Isotype controls were IgG2a, K -FITC (mouse), IgG1, K -PE (mouse) and IgG1, K -FITC (mouse) (BD Pharmingen). After staining, cells were fixed with 1% paraformaldehyde for 15 minutes, and then resuspended in 300 μl flow buffer for analysis on a FACS Calibur flow cytometer . Data was analysed using CellQuestPro software (BD Immunocytometry Systems).Cryropreserved peripheral blood mononuclear cells were quickly thawed in a water bath at 37°C, washed in cold PBS with 3% BSA and 10 mM HEPES. PBMC were enumerated using a heamocytometer, and viability routinely assessed to be above 90%. A total of 3 × 10Total immunoglobulin  was measured using standard capture and detection sandwich enzyme linked immunosorbent assay (ELISA) . The plaPaired T-test was used to compare the differences in means of the same individuals at different time points . Independent-measures T-test was used to compare differences between acute clinical patients, post-recovery and an age-matched control group. Heterogeneity of variance assumptions was tested by the Levene statistic. In all cases P < 0.05 were considered statistically significant. SPSS v16  was used for statistical analysis.P. falciparum parasite density/μl of 47,066 ± 32,105. Four weeks following treatment for malaria, the parasite density had dropped in all cases with a mean density of 3,464 parasites/μl. Some of the controls were asymptomatic, but parasitaemic and had a mean density of 1,820 parasites/μl. Children who had acute clinical malaria had the highest temperature (mean of 37.8°C), whereas during recovery they had comparable temperature to the control children (mean of 36.5°C). The haemoglobin levels were 8.6 g/dL, 10.3 g/dL, and 10.7 g/dL for acute clinical malaria, post-recovery and controls respectively.Twenty-five children presenting with acute clinical malaria were enrolled in this study. However, during follow-up conducted four weeks post recovery, only fifteen children met enrollment criteria and were included in the subsequent analysis. Clinical characteristics of the study participants are shown in Table + B cells relative to when they had an episode of acute clinical malaria (P = 0.02)  and more CD3+CD8+ T cells (P = 0.03) were measured in healthy age-matched controls relative to the children that had recovered from acute malaria.Since acute clinical malaria has been associated with perturbation of lymphocyte populations ,21, the -IgD+), naive 2 (CD38+IgD+), memory 3 (CD38+IgD-) and memory 4 (CD38-IgD-). To determine the frequencies of these B cell subsets in the study population, PBMC were live-gated on CD19+ cells to collect sufficient number of cells for analysis and then analysed for expression of IgD and CD38. A representative FCF analysis is shown in Figure -IgD+) B cell subsets compared to the same population following recovery (P = 0.03) and to age-matched healthy controls (P = 0.00). In contrast, elevation of the memory 3 (CD38+IgD-) B cell subset was observed in the acute clinical malaria compared to the same population following recovery (P = 0.00) and to age-matched healthy controls (P = 0.01). Interestingly, the memory 4 (CD38-IgD-) B cell subset was significantly lower in both acute clinical malaria and recovery compared to healthy controls (P = 0.01 and P = 0.00 respectively). This mirrors the increase in the naive 2 (CD38+IgD+) B cell subset during both acute clinical malaria and recovery compared to healthy controls (P = 0.04 and P = 0.01 respectively).Four major populations of B cells in the peripheral blood can be distinguished based on expression of IgD and CD38 : naive 1P. falciparum induces increased expression of CD10, phenotypic analysis of peripheral blood of children during acute clinical malaria, following recovery and in asymptomatic children was performed. The results illustrate that following recovery from malaria, children had significantly higher levels of CD10 expression than during acute clinical malaria (P = 0.04) and also compared to controls (P = .04)  Table . ImportaPrevious studies have demonstrated that acute clinical malaria is associated with hypergammaglobulinaemia . To deteP. falciparum malaria. In this study, a significant decline in the percentages of CD19+ B cells in the peripheral blood of children during an episode of acute clinical P. falciparum malaria compared to the same children post-recovery and to healthy age-matched children was observed. In addition, the children with acute malaria had an increase in a memory B cell subset and a concomitant decrease in a naive B cell subset in children as compared to the same children post-recovery and to healthy age-matched children. Thus, the data presented here suggests that P. falciparum infection results in perturbations of the B cell compartment and is consistent with accumulating evidence from a number of other studies showing dysregulation of B cell function following P. falciparum infection [Although extensive information is available on the types and frequencies of mature B cells that populate the peripheral blood, few studies have been done analysing how B-cell lymphopoiesis is altered in children infected with nfection ,21.+ B cells during acute malaria in this study. However, there were no significant differences in CD3+ T cells in this same population. Although the immunological pathways that orchestrate B lymphocyte depletion are unknown, P. falciparum malaria may interfere with B cell lymphopoiesis, induce lymphocyte apoptosis as shown following infection of macaques with P. coatneyi [P. falciparum infection, the possibility exists that P. falciparum infection, possibly through direct interactions of P. falciparum antigens with memory B cells, is driving memory B cells towards plasma cell differentiation and migration of these cells back to the bone marrow. Consistent with this, changes in B cell subsets were observed, with significantly higher memory 3 B cell subset observed in acute clinical malaria compared to recovery or controls, and a lower percent of memory 4 B cell subset in children both during acute and following recovery compared to healthy age-matched controls. Taken together, these observations have important implications in the maintenance of immunologic memory to vaccine antigens in populations chronically exposed to malaria infections, especially in children since they are most vulnerable to this parasitic infection.Lymphopaenia has long been recognized as a complication of acute malaria . Consistcoatneyi , or resucoatneyi . Lastly,coatneyi  have obs+CD38+ naive 2 equivalent of pre-germinal center B cells have been reported in the peripheral blood children with systemic lupus erythematosus [P. falciparum infection with B cell trafficking and/or differentiation, which could lead to cells exiting secondary lymphoid organs at early differentiation stages. A recent report found that the architecture of the germinal center was disrupted in a mouse model of malaria [P. falciparum malaria [P. facliparum infection resulting in alterations of lymphoid architecture, which could result in the changes in peripheral B cell subsets that were observed in this study.Elevated levels of the IgDematosus , but thi malaria . Further malaria . Togethe+CD19+ B cells was observed following recovery from acute clinical P. falciparum malaria, with up to 25% of total CD19+ B cell pool residing in this subset. The cells expressing CD10 cells were IgD+ and CD34-, suggesting that they were not pre-B cells that had aberrantly migrated to the periphery from the bone marrow [+ germinal center B cells [P. falciparum infection induces immature B cells to leave the bone marrow at an earlier differentiation stage [An expansion of CD10e marrow ,28. Stude marrow . Interes B cells . It is pon stage ,19, and + B cells as a percentage of CD45+ lymphocytes was 23.53% in healthy controls. This is within the range reported in healthy children and HIV infected children and is much higher than observed in healthy adults where the range is typically 12% of total lymphocytes [+CD19+ B cells than reported for one other study examining CD10+ levels in HIV infected children [The methods for determining the percentage of lymphocyte subsets in peripheral blood have over time, switched from PBMC isolation and then phenotyping to direct phenotyping from whole blood. The phenotype of peripheral blood lymphocytes was analysed following isolation of PBMC because of the difficulties in timing when samples were collected, the desire to do a batch analysis on all the samples to ensure consistency in our analysis, and the inherent challenges of the clinical setting in Kenya in which we were working. There are very few published studies of lymphocyte subsets in healthy children and most are based on children living in developed countries. The present study observed that the mean CD19phocytes . The prechildren .P. falciparum malaria in children, there were major perturbations in B cell subsets including a decline in memory B cells and an increase in transitional CD10+ cells. Understanding the mechanisms underlying the alterations in B cell lymphopoeisis are needed to aid in vaccine development.In summary, these results demonstrate that during acute uncomplicated episodes of The authors declare that they have no competing interests.ASA carried out the cell isolation, cell analysis and drafted the manuscript. AMM participated in the design and coordination of the study and draft of manuscript. CK participated in recruitment, FCF analysis and draft of manuscript. ZWN participated in study design. RPS carried out the statistical analysis. RR conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.