It is generally agreed that in high transmission areas, pregnant women have acquired a partial immunity to malaria and when infected they present few or no symptoms. However, longitudinal cohort studies investigating the clinical presentation of malaria infection in pregnant women in stable endemic areas are lacking, and the few studies exploring this issue are unconclusive.A prospective cohort of women followed monthly during pregnancy was conducted in three rural dispensaries in Benin from August 2008 to September 2010. The presence of symptoms suggestive of malaria infection in 982 women during antenatal visits (ANV), unscheduled visits and delivery were analysed. A multivariate logistic regression was used to determine the association between symptoms and a positive thick blood smear (TBS).During routine ANVs, headache was the only symptom associated with a higher risk of positive TBS . On the occasion of unscheduled visits, fever , headache  and shivering  were significantly associated with a malaria infection and almost 90% of infected women presented at least one of these symptoms. Two thirds of symptomatic malaria infections during unscheduled visits occurred in late pregnancy and long after the last intermittent preventive treatment dose (IPTp).The majority of pregnant women were symptomless during routine visits when infected with malaria in an endemic stable area. The only suggestive sign of malaria (fever) was associated with malaria only on the occasion of unscheduled visits. The prevention of malaria in pregnancy could be improved by reassessing the design of IPTp, i.e. by determining an optimal number of doses and time of administration of anti-malarial drugs. In sub-Saharan Africa, 30 million women become pregnant each year. ConsequTo prevent the consequences of MiP, the WHO recommends an intermittent preventive treatment during pregnancy (IPTp), an adequate management of clinical malaria, and the use of insecticide-treated nets. IPTp consists in the administration of two curative doses of sulphadoxine/pyrimethamine (SP), at least four weeks apart and beginning at the quickening, regardlLongitudinal cohort studies investigating the clinical presentation of malaria infection in pregnant women in stable endemic areas are lacking, and only a limited number of studies have focused on this issue-12. ConsThe STOPPAM project  consists in a prospective cohort of 1000 pregnant women in Benin. This longitudinal follow-up offered us the unique opportunity to assess the clinical presentation of infected women during the whole duration of pregnancy.The STOPPAM project has been conceived to study the underlying immunopathological processes causing poor outcomes in MiP, through the characterization and measurements of antibody and cellular immunological responses during pregnancy, at delivery and during the infant's first year of life.In this study, 1,000 pregnant women were followed-up in each of two sites: Benin and Tanzania. Specifically in Benin, 200 newborns to these mothers were followed-up during their first year of life to investigate the immunological responses in the infants.The study took place in the Mono province, located 70 km west of the economical capital of Benin, Cotonou. It is a high transmission area, with two peaks during the rainy seasons: from April to June, and from September to November. The entomological inoculation rate is 35-60 infective bites per person and per year . Three dThere are one district hospital , three government-run health dispensaries and 11 private clinics in the study area. Five nurses have been recruited and trained as "project assistants" to fill questionnaires and to collect blood samples from the study participants. Local midwives, nurses in charge of the health centre and the project assistants worked in close collaboration.Pregnant women meeting the following criteria: gestational age under 24 weeks, living for more than six months within 15 km from the dispensary and having planned to deliver at the hospital, were enrolled in the study after giving informed consent. At the initial visit  0), the assistants and the midwives collected information regarding the reproductive and the current pregnancy histories, medical history, socio-economic indicators, and the use of bed nets.At each monthly ANV, axillary temperature and blood pressure, weight, height and mid-upper arm circumference were measured. After clinical examination, rapid diagnostic tests (RDT) , thick and thin blood smears were systematically made to determine malaria infection. Any time during the follow-up, women were invited to attend health facilities in case of clinical symptoms to get care. The same clinical and biological information were collected as during ANVs. These visits were identified as "unscheduled visits".On all occasion during follow-up , symptoms were actively asked by the midwives in the private examination room following a standardized questionnaire. The symptoms included: headache, fever, stomach ache, nausea or vomiting, fever in the last 48 hours, shivering.Following the national guidelines, for IPTp two doses of SP (1500 mg sulphadoxine and 75 mg pyrimethamine) were administered at least one month apart during the second trimester of pregnancy under the supervision of midwives during antenatal visits. During the follow-up, a malaria infection was defined as a positive RDT and fever was defined as an axillary temperature above or equal to 37.5°. Following the national guidelines, all participants with malaria parasitaemia as detected by RDT received a treatment dose of quinine (24 mg/kg during 7 days), or SP if they were supposed to receive the scheduled dose on the same visit.Four ultrasound scans were performed from inclusion until delivery: the first to determine the exact term of the current pregnancy and the following to evaluate intrauterine growth and foetal morphology. At delivery, temperature, weight and blood pressure of the mother were measured. Venous blood samples for biological investigations, thick and thin blood smears and a RDT were obtained before delivery. The newborn child was fully examined.Thick blood smears (TBS) were stained with Giemsa and read by two experienced parasitology technicians. Smears were considered negative if no asexual-stage Plasmodium parasite was detected after 500 leucocytes had been counted. Malaria parasites were counted against 200 leukocytes and parasite densities were estimated using leukocyte count of the hemogram. If results were discrepant, the slides were read by a third microscopist.For statistical analyses, malaria infection was defined as parasitaemia detected by thick blood smear. Geometric means were used to calculate average parasitaemias.This study was approved by the ethics committees of the Research Institute for Development (IRD) in France and of the Science and Health Faculty  in Benin. Written informed consent was obtained from all participants.Data were double-entered, validated and cleaned using Access . Stata version 10 for Windows  was used for all statistical analyses. There were two sorts of missing data to deal with: missing symptoms for the first included women and missing malaria status at delivery, essentially because of public health worker's strike. First, the characteristics between the mothers with and without symptoms during first ANVs on one hand and the ones with and without malaria status at delivery on the other hand, were compared to ensure they were similar.Also, for the women whose symptoms were missing, the multiple imputation by chained equation method (MICE) based on the Gibbs algorithm was used and then the stability of the results was checked with and without MICE. Twenty imputed data sets were carried out with 15 iterations for each.For missing malaria status, a sensitivity analysis was performed to ensure that the results remained the same, and thus these missing data would not induce a bias. Differences in proportions and means were compared using the khi2 (or Fisher's exact test) and the Student t-test, respectively. A multivariate logistic regression was used to determine the association between signs and symptoms with positive malaria status at each visit. Covariates were included in the initial models only if the p-value was < 0.2 in the univariate analysis. In the final multivariate analysis, a p-value < 0.05 was considered significant.Figure Table The overall prevalence of malaria infections was 10.2% (602/5909). During the whole follow-up, primigravidae had more peripheral infections than multigravidae . At the initial ANV (prior to treatment and IPTp), the prevalence of malaria infections for primigravidae, secundigravidae and multigravidae were 30.7%, 18.7% and 11.7%, , respectively.Table As the STOPPAM project was not originally designed to assess malaria clinical presentation during pregnancy, the first 126 included women were not asked about symptoms. Symptoms during first ANVs were thus lacking for these women. To deal with these missing data, the MICE method based on the Gibbs algorithm was used.During ANVs (including enrolment), among the 982 women initially followed-up, 307 were infected at least once. Women under 25  and primigravidae  had a higher risk of peripheral malaria infections than older women and multigravidae, respectively.On a total of 4,357 visits, 397 malaria infections were found table . ConsideAmong the 524 women who consulted during unscheduled visits, 114 were infected at least once. Young age of the woman was a risk factor to develop a parasitaemia .On a total of 826 unscheduled visits, 130 malaria infections were found .th month.Based on the pharmacokinetic properties of SP, we analyAccording to what has been generally described in stable endemic areas, the majority (60%) of pregnant women who were followed-up in the STOPPAM project were asymptomatic when infected, although some of them displayed clinical signs. During ANVs, 5% of the infected women were febrile; but only headache, a non-specific symptom, was significantly associated with a higher risk of having a peripheral infection. However, its frequency was moderate . In contrast, during unscheduled visits, Almost 90% of the malaria episodes were symptomatic and fever , headache and shivering were strongly associated with malaria parasitaemia.In high transmission areas pregnant women still have an immune protection, and only exhibit limited symptoms when they get infected by malaria parasites-8. ThereA third study carried out in Ghana reported that MiP was often symptomatic when women were actively asked for clinical complaints. All womThe STOPPAM project was able to offer women both prospective routine ANVs and unscheduled visits in case of clinical complaints. Also, symptoms and signs were actively asked by midwives before TBS were performed. Therefore, it was not prone to selection biases unlike the abovementioned studies. Our results show that the majority of infected pregnant women in this stable malaria area only have few and mild clinical signs during routine visits. Unscheduled visits are frequently motivated by malaria-associated acute symptoms, particularly at distance from IPTp intakes. These women are important to identify and to treat, in order to avoid adverse consequences of MiP on the newborn child.In the statistical analysis, missing data related to symptoms for the first women included were an issue to deal with. First, gestity, age and education were shown not to be different between the women with missing data and the remaining women. Then, the MICE method for these missing data was used and all results remained similar using the MICE method or not. Therefore, missing data related to symptoms were unlikely to have induced a bias, and then to have altered the results.For the delivery, malaria status was missing for 140 women who were thus excluded from the analysis. Characteristics between excluded and remaining women were similar and the sensitivity analysis performed ensured that the results were similar. Moreover, the reason for excluding these women  could not possibly influence malaria status at delivery. Consequently, the mechanism of missing data could not have altered the results and induced a selection bias.Plasmodium isolate appears not to last over two months in non-pregnant women [According to the few pharmacokinetic studies available, the SP blood concentration effective against a sensitive nt women ,14, hencnt women . Even coTo avoid such late infections, different options could be considered to offer pregnant women a better protection. In sub-Saharan Africa, the attendance to ANVs is rather high: almost 70% of women attend at least once, 95% of them attend twice and more than half attend four times,16. The Another option to improve the coverage of IPTp is to give a third dose of SP. This strategy is currently recommended for HIV-positive pregnant women, but few studies have evaluated the benefits of increasing the number of SP administrations in HIV-negative women-19. BesiThis study found that pregnant women infected with Plasmodium only had symptoms related to malaria when infection was detected at unscheduled visits, generally at distance from the last dose of IPTp. A third dose of IPTp administered at the end of pregnancy might avoid such late infections. An alternative to SP-IPTp in endemic areas where malaria incidence is declining or SP resistance is high, could be an "intermittent screening and treatment", especially in areas with high seasonal transmission, which consists in screening all pregnant women during ANVs with RDTs and to treat only malaria infected women.The authors declare that they have no competing interests.BTH carried out the acquisition, the statistical analysis and the interpretation of data and drafted the manuscript. NF participated in the design and the coordination of the study, the acquisition of data and revised critically the manuscript. GB and SB participated in the acquisition of data. BGM and AM participated in the design of the study and revised critically the manuscript. PD coordinates the STOPPAM project, he participated in the design of this study and revised critically the manuscript. MC participated in the design and the coordination of the study and supervised the writing of the manuscript. All authors read and approved the final manuscript.


Both treatment and prevention strategies are recommended by the World HealthOrganization for the control of malaria during pregnancy in tropical areas.The aim of this study was to assess use of a rapid diagnostic test forprompt management of malaria in pregnancy in Bangui, Central AfricanRepublic.Pf® at each antenatal visit. The usefulness ofthe method was analysed by comparing the number of malaria episodesrequiring treatment in the cohort with the number of prescriptions receivedby another group of pregnant women followed-up in routine antenatalcare.A cohort of 76 pregnant women was screened systematically for malaria withParacheckPf®episodes during antenatal clinics visits was 13.8%, while episodes ofantimalarial prescriptions in the group which was followed-up routinely byantenatal personnel was estimated at 26.3%. Hence, the relative risk of thecohort for being prescribed an antimalarial drug was 0.53. Therefore, theattributable fraction of presumptive treatment avoided by systematicscreening with ParacheckPf® was 47%.In the cohort group, the proportion of positive ParacheckUse of a rapid diagnostic test is useful, affordable and easy for adequatetreatment of malaria in pregnant women. More powerful studies of theusefulness of introducing the test into antenatal care are needed in allheath centres in the country and in other tropical areas. Plasmodium isassociated with a 50% risk for low birth weight in areas where malaria is endemic[Malaria is a major public health problem, especially during pregnancy. In 2008,malaria affected more than 2 billion people, nearly 40% of the world population.More than 50 million women live in malaria-endemic areas, of whom 20% becomepregnant each year, and half of whom develop complications of pregnancy due tomalaria ,2. Place endemic. Clinica endemic-9.Sulfadoxine-pyrimethamine (SP) is a promising means of preventing the adverse effectsof malaria, as first suggested by studies conducted in East Africa . Hence, Lack of compliance of health care personnel with the new strategies could hamperimplementation of IPTsp in particular ,21. CertPlasmodium infection. [Plasmodium infectionand decrease the risk for low birth weight [P. falciparum to SP [Bardaji et al. in Mozambique  found thfection. . This prfection. . Although weight , unnecesum to SP ,28 jeopaum to SP , and useum to SP ,31.In public health care practice in the Central African Republic, malaria is diagnosedby analysing thick and thin blood smears; RDTs have not yet been introduced. The aimof this study was to assess the efficacy of systematic screening for malaria withRDTs during antenatal health care in reducing over-prescription of antimalarialdrugs to pregnant women. The number of episodes of malaria confirmed by a RDT in aprospective cohort of pregnant women receiving routine antenatal care was comparedwith the number of antimalarial prescriptions in a control group of pregnant womenat the same clinics.°C to 33 °C. The main malaria parasite isPlasmodium falciparum, and malaria transmission is endemic, withpeaks during the rainy season, although no data are available on the intensityof malaria transmission. Malaria accounts for more than 40% of morbidity in thecountry .This study was conducted in the two main public maternity clinics of Bangui,Central African Republic, the Castors Health Centre and the AmitiéHospital, between October 2009 and October 2011. In Bangui, the climate istropical, with rainfall peaks from April to November and temperatures rangingfrom 19 Plasmodium infection were compared with data collected from amatched control group followed-up routinely by the antenatal services staff.The objective of the National Malaria Programme is to reduce morbidity andmortality related to malaria in the general population, especially amongchildren under 5 years and pregnant women, to reach a coverage rate of at least80% with each WHO package . Thisstad hoc scientific committee of the University ofBangui in charge of validating scientific study protocols. The scientificcommittee of the ‘Ecole Doctorale des Sciences de la Vie et de laSanté de l’Université de la Méditerranée,Aix-Marseille’ also approved the study protocol and its amendments.Because there is no ethical committee in the country, this project was reviewedand approved by an We used 25% as a proxy for the number of malaria episodes during pregnancy, and 50%Antenatal services staff were informed about the study, and we collected data inthe context of usual non-malaria antenatal health care, including enrolment.During each working day, a maximum of two pregnant women from among thosepresenting at the clinics were randomly included in the cohort. The women wereof all parities with a gestational age less than 28 weeks, from whom writteninformed consent was obtained. Women who were temporary residents of Bangui, hadhad a prior dose of IPTsp, gave a history of sensitivity to SP, quinine or anartemisinin derivative, had an illness requiring hospital admission or declinedto join the study were excluded.Plasmodium infection with theParacheckPf® RDT . Werecorded sociodemographic data  and also gestational age, gravidity, parity and HIVserological status.At enrolment, all women were given an ITN , and a finger-prick blood sample was obtained for preparationof slides and for diagnosis of During follow-up, an IPTsp dose (1500 mg sulfadoxine and 75 mg pyrimethamine) wasadministered after 16 weeks of gestation and a second dose at least 1 monthlater. Smear slide analysis and the RDT were performed systematically duringeach scheduled antenatal visit or at any unscheduled visit for women whoreported malaria-like symptoms. Women with any symptom or clinical signsuggesting malaria  and withEach woman in our cohort was matched on gravidity (1 or ≥ 2) and age(± 5 years) to another women delivering at the same maternity clinic within1 week and from whom written informed consent was also obtained. Other criteriafor enrolment in the control group were: known last date of menstruation , at least one antenatal visit, known HIVserological status and sleeping under an ITN. The following data were collected:socio-demographic information , gestational age, gravidity, parity, HIV serological status, number ofantenatal visits and number of malaria treatment episodes (with or withoutlaboratory confirmation) during pregnancy.At each antenatal visit, the RDT and blood smears were performed for each womanin the cohort. At delivery, these tests were performed on both maternalperipheral blood and placental blood. The placental blood was collected from thematernal paracentric side of the placenta after incision, and thick blood filmswere prepared from a droplet collected by aspiration through a 21-gauge needleattached to a 5-ml syringe ,33. Newb× 100 oil immersion) todetect asexual forms of P. falciparum. The parasite density in maternalperipheral blood was determined from the number of parasites per 200 leukocyteson the assumption of an average leukocyte count of 8000/μl of blood. Formaternal blood films, a result was considered negative if no parasites weredetected per 200 leukocytes; for placental blood films, a result was considerednegative if no parasites were detected per 100 microscope fields. All the slideswere read independently by two microscopists. In case of a discrepancy, a thirdreading was done.The maternal and placental thick smears were air-dried, stained with 4% Giemsaand analysed under a light microscope (The RDT was performed according to the manufacturer’s guidelines and storedat room temperature (26–32 °C). Briefly, blood samples from thefinger-prick and placenta were blotted into the sample well of the test devicewith the loop provided in the kit. Then, six drops  ofclearing buffer were dispersed into the indicated well. The result was readafter exactly 15 min. It was considered negative if a pink band appeared only inthe control window and positive if, in addition to the control band, a distinctpink band also appeared in the test window. If no band appeared, the test wasconsidered invalid and was repeated with a new device.Data were double-entered into EpiInfo software version 3.5.3, and the databasewas checked and data entry errors corrected with the EpiInfo software‘data compare’ utility for finding differences between two tables.Statistical analysis was conducted with Stata 11.2 and MedCalc v11.6.1.χ2test or Fisher exact test, and quantitative variables were compared by theStudent t test (Mann–Whitney U test). The level ofsignificance (P) was fixed at 0.05 for all statistical tests.Categorical variables were compared by either the We used the attributable fraction calculation procedure  to estimDuring the study period, 874 pregnant women presented at their first antenatalvisit. At baseline, 412 women fulfilled the inclusion criteria. A cohort sampleof 83 pregnant women was randomly recruited from among women who fulfilled theinclusion criteria. The remaining 329 women were followed-up routinely by theantenatal services staff, providing an eligible population from which thecontrol group was recruited at delivery. In the cohort group, 76 women (91.6%)were followed-up until delivery. At this end-point, this cohort group wasmatched to the control group , and the mean graviditywas 2 range, 1–6), with no statistically significant differences betweenthe groups. The serological prevalence of HIV infection was 7.6%  and by thick blood smear analysis at 11.6% (43/369) of visits.Concordance was found for positive results in the RDT and in thick blood smearsbut not for negative results, as eight results were negative in the thick bloodsmear but positive with the RDT (15.7% discordance). Figure In the cohort, 40 pregnant women (52.6%) presented clinical signs at baselinesuggesting malaria. Of these women, 42.5% 17/40) had a positive RDT formalaria; none of the asymptomatic pregnant women had a positive result formalaria. During follow-up, 293 antenatal visits were made by the cohort group,and symptoms suggesting malaria were noted in 35.5% (104/293); however, positiveRDT were found for only 29.8% (31/104). Overall, positive results for 7/40 had Women positive results in the RDT were treated with eitherartemether-lumefantrine  or quinine . Duringfollow-up, three asymptomatic malaria episodes were seen, with positive resultsin both the RDT and thick blood smear analysis. Each of these women received onedose of SP, and thick blood smears analysed 8 days later and at the followingscheduled visit was negative.In the control group, 75 prescriptions for a possible malaria episode weredelivered by the antenatal services staff. Positive thick blood smears weredocumented on 35.0% of antenatal visit cards. These cards showed that 29.3%(22/75) prescriptions of antimalarial drugs were achieved after a documentedpositive laboratory result for malaria before treatment, while 36.0% (27/75)antimalarial drugs were achieved without laboratory analysis. For remainingprescriptions, laboratory analysis was not recorded in antenatal visit cards.However, interviewed women were reported that smear analysis were positive.At delivery, the results of the two tests were concordant for both the maternalperipheral blood smear and the placental blood samples in the two groups. Fourplacental blood samples from the cohort and only two from the control group werepositive in the RDT. In the control group, five placental blood samples and twoblood slides were positive with the RDT Figure .During this study, none of the tests was invalid.U-test P values > 0.05)  of the pregnant women in our cohort needed antimalarialtreatment after a positive RDT, while 26.3% (75/285) of the control groupreceived such prescriptions. Antimalarial treatment was significantly morefrequent in the control group than in the cohort (P = 0.0001). The relative riskof the cohort for being prescribed an antimalarial drug was 0.53; therefore, 47%of prescriptions were avoided with use of the RDT. The distribution of malariatreatment according to gestational age was, however, similar in the two groups(Our findings highlight the value of using an RDT in managing malaria duringpregnancy. The usefulness of RDTs in the diagnosis of malaria in patients presentingwith symptoms or clinical signs is well established ,36, althThe ParacheckPf ® RDT was more sensitive than microscopic analysis of thickblood smears, as found in other studies -40. The We observed a significant reduction in the number of antimalarial drug prescriptionsin pregnant women when the ParacheckPf® RDT was used at each antenatal visit.This strategy therefore reduces unnecessary expenditure on drugs and also reducesunwarranted exposure of pregnant women to those drugs. Moreover, appropriate earlytreatment of malaria during pregnancy helps to eliminate parasites from bothmaternal blood and the placenta.It has been estimated that malaria parasites are present in the placenta in only 20%of cases ,45. InoOne potential limitation of our study is the small sample, which precluded furtheranalysis of birth weights; however, a study of three cohorts of a total of 3333women conducted in Ghana  led to tThis study shows that routine screening of pregnant women for malaria can avoidunnecessary prescription of drugs. As any use of medical products during pregnancyshould be avoided, the proportion of avoidable antimalarial drug exposure that weobserved in this study could be important if it were extrapolated to other primaryhealth care services.Ensuring clearance of malaria parasites is essential for the control of malariaduring pregnancy. Thus, the ease of use and relatively high sensitivity of theParacheckPf® RDT in comparison with microscopy could allow prompt, radicaltreatment of malaria. Its integration into primary health care services should,however, be accompanied by regular supervision of the activities of the personnelinvolved. More studies are needed in other areas of the Central African Republic andother countries to assess the introduction of RDTs in malaria control in pregnantwomen.The authors declare that they have no competing interests.AM, GS and JD conceived the study. AM organized and managed the study day-to-day andcontributed to writing the manuscript. DD managed the RDT kits. ES, LSH and NMparticipated in field data collection. All authors contributed to the preparation ofthe manuscript and have approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/482/prepub


Malaria is the commonest imported infection in the UK. Malaria requiring ICU admission has a reported mortality of up to 25%. The relationship between ethnicity, immunity, and risk of malaria is complex. The Malaria Score for Adults (MSA) and Coma Acidosis Malaria (CAM) score have recently been proposed to risk stratify patients with malaria.Retrospective study of patients with WHO severe falciparum malaria admitted to ICU at the Hospital for Tropical Diseases, London, UK. The relationship between clinical variables and risk of death or a prolonged ICU stay were examined with logistic regression. The predictive value of the MSA and CAM score were calculated.124 patients were included. Cerebral malaria and acute kidney injury occurred earlier (median day 1) than acute respiratory distress syndrome (median day 3). Six patients had community acquired bacterial co-infection. Eight patients were co-infected with HIV, five of whom were newly diagnosed. The positive predictive value of a CAM score ≥2 or an MSA ≥5 for death were 12% and 22% respectively. Five patients died. No variable was significantly associated with risk of death. There were no significant differences between individuals raised in endemic countries compared to non-endemic countries.per se. The clinical usefulness of the MSA and CAM score was limited. Co-infection with HIV was relatively common but compared to studies in children, bacteraemia was uncommon. The relationship between ethnicity and immunity to severe disease is complex.Mortality in patients managed in a specialist centre was low. Patients who died succumbed to complications associated with a prolonged stay on ICU rather than malaria  P. falciparum is both the most common and the most severe , impaired consciousness [seizures or GCS <11], multiple convulsions, shock [systolic BP <80 mmHg despite adequate filling], abnormal bleeding or coagulopathy , jaundice [bilirubin >50 μmol/L], hyperlactaemia [arterial lactate >5 mmol/L], acidosis [pH <7.35 or serum bicarbonate < 15 mmol/L], hypoglycaemia [blood glucose <2.2 mmol/L], severe anaemia [haemoglobin <5 g/dL]) [Plasmodia . Most were referred to HTD from other hospitals where they had already received treatment for malaria and, if there was no evidence of asexual parasitaemia at the time of admission to HTD/UCLH ICU, they were excluded. Of 165 patients admitted to ICU, 32 had notes which could not be found, eight no longer had asexual parasites on their blood film and one did not fulfil any of the WHO criteria for severe disease, leaving a total study population of 124.A retrospective study was conducted on all adult patients (>18 years) seen at HTD/UCLH between 1994 and 2010. During this period a total of 1616 patients with falciparum malaria were seen, of whom 165 were sufficiently unwell to require admission to intensive care. Patients were excluded if they did not meet at least one of the WHO criteria for severe disease  . The diaDemographic, travel, clinical, laboratory and modalities of treatment data were collected from clinical notes and laboratory databases using a standard case report form. Complications of malaria were defined according to WHO guidelines on severe malaria . Blood cAll patients were managed according to UK guidelines . Until 2Continuous variables were described with mean and standard deviation (SD) or with median and interquartile range (IQR) as appropriate. Categorical data were described with numbers and percent. “Severe ICU malaria” was defined as death or an ICU stay greater than seven days. The patients’ MSA and CAM scores on admission to hospital were calculated as previously described ,13. The The study was reviewed and approved by the Audit and Research Committee at HTD who granted ethical approval for the study and stated that individual patient consent was not required as this was a retrospective case note review.Demographic data are shown in Table Sixty-eight (55%) were classified as malaria-naïve. Forty-six (37%) were assumed to have “little” immunity to malaria as they had grown up in an endemic area. Seven were both born and usually resident in a malaria-endemic area at the time of admission and were assumed to have at least “some” degree of immunity.P. malariae, and one each with P. vivax and P. ovale.Median parasitaemia at admission, either to UCLH or the referring hospital, was 6.1% (IQR 2.6 - 18%). Ninety eight (79%) and 73 (59%) had a parasitaemia greater than 2% and 5% respectively. One hundred and eight (87%) and 82 (66%) patients had at least one parasite count greater than 2% and 5% respectively during admission. Twenty-two (18%) had schizonts at admission, and a further 53 (43%) had schizonts at some time during admission. Four had mixed infections, two with One hundred and twenty three (99%) received intravenous quinine as their initial treatment, 93 (76%) of whom received a loading dose. Twenty were subsequently switched to an artemisinin derivative, 18 of whom received artesunate. One was treated with an artemisinin derivative from the start.Thirty-three (27%), with a median parasitaemia of 25%, had an exchange transfusion performed. Forty six (37%) required ventilation at some time. Similarly 43 (35%) required renal replacement therapy and 43 (35%) required inotropic support. Median length of stay in ICU was 10 days (IQR 7–19) but several had a prolonged stay, the longest being 90 days.Patients met a median of three IQR 2–5) WHO criteria for severe disease  were acidotic at some stage. Hypoglycaemia (defined as blood glucose <2.2 mmol/L) was not seen at admission but five patients, all treated with intravenous quinine, developed this at some point. Shock occurred in 34 (27%). Significant bleeding or coagulopathy was less common, occurring in 13 (11%) patients most of whom had laboratory evidence of DIC. One patient, who ultimately made a complete recovery, sustained spontaneous rupture of their spleen. Another developed purpura fulminans, which necessitated bilateral below-knee amputations.Staphylococcus aureus and another Klebsiella pneumoniae. In one other, Mycobacterium tuberculosis was found in a sputum sample.Six had a community-acquired co-infection; three had clinical and/or radiographic evidence of pneumonia while one had a coincidental buttock abscess. Two patients had a positive blood culture at admission, one Staphylococcus aureus (n = 7), Haemophilus influenzae (n = 5) and Pseudomonas (n = 2). Sixteen developed pneumonia, two had proven fungal infection, one bacteraemia with Staphylococcus aureus and one an episode of Clostridium difficile colitis. Three patients, all African, were previously known to have HIV. Of 22 patients who were tested for HIV during admission, a further five were found to be positive, four of whom were African.Twenty (16%) developed nosocomial infection during their stay most commonly caused by One hundred and nineteen (96%) survived and all but one, the patient who developed purpura fulminans, ultimately made a complete recovery. Of the five patients who died, four were Caucasian; one was an African woman who had grown up in sub-Saharan Africa but left two years previously Table . Their mP. falciparum which responded to a further course of treatment.Twelve had persisting renal impairment at the time of discharge from ICU, but all of these ultimately recovered. After discharge from hospital, three needed readmission, one patient developed an acute confusional state which was diagnosed as a post malaria neurological syndrome and the patient made a complete recovery while two had a relapse of There was no demonstrable association between the amount of “immunity” to malaria  and the risk of severe ICU malaria , cerebral malaria , ARDS  or AKI . In logistic regression adjusting for the potential confounding factors outlined in the methods, no clinical factor was significantly associated with either death or severe ICU malaria.A CAM score and an MSA score could be calculated at admission for 94 (76%) and 124 (100%) respectively. The positive predictive value of a CAM score <2 and an MSA score <5 for survival were 100% and 97% respectively. The positive predictive value of a CAM score ≥2 or an MSA ≥5 for death were 12% and 22% respectively. A CAM score ≥2 was significantly associated with an increased risk of death  and of severe ICU malaria . An MSA score ≥5 was associated with an increased risk of death  but was not associated with a statistically significant increased risk of severe ICU malaria .This report shows that patients with severe malaria requiring admission to ICU who are managed at a specialist centre have a good outcome, with a mortality rate of only 4%. Consistent with previous reports, acute kidney injury, ARDS, and cerebral malaria were the most common complications. This report demonstrates that ARDS occurs later and at lower parasitaemia and emphasises the need for vigilance even after patients appear to be responding to treatment. The data reinforce the idea that there is a complex inter-relationship between ethnicity, “immunity” and the response to malaria. There were no significant differences between those individuals who had grown up in an endemic region and those patients who were malaria-naive. Co-infection with HIV was common, especially in patients who were African in origin. Finally, this report adds to the limited number of series reporting the efficacy and safety of parenteral artemisinins in severe malaria in a non-endemic setting -20. The In this series, the case-fatality rate was 4%. In contrast, a paper from Kilifi  showed tMost patients were young, Caucasian travellers to Africa but the second largest group were Africans visiting friends and relatives. For both groups, adherence to chemoprophylaxis was poor. The UK Health Protection Agency have previously reported an increased risk of acquiring malaria in individuals travelling to visit friends and relatives . BetweenCerebral involvement and/or AKI tended to be present on admission or develop within the first 48 hours. ARDS tended to develop later, often when there was little or no evidence of persisting malaria. Those caring for patients with severe malaria need to be aware that an initial response to treatment, manifest by a reduction in parasitaemia, does not necessarily mean a successful outcome.Before the introduction of artesunate, this hospital recommended a six-unit exchange transfusion for patients with a parasitaemia greater than 20% or those with a parasitaemia greater than 10% but with evidence of end-organ dysfunction. In total, 33 (26%) patients had an exchange transfusion, including three of the five who died. Since switching to parenteral artemisinins, we have seen evidence of a rapid and dramatic decline in parasitaemia compared to the response to treatment with quinine. As a result, we have not performed an exchange transfusion since 2008 and, in agreement with other UK specialist centres such as the Liverpool School of Tropical Medicine , we no longer recommend this as a modality of treatment.Children presenting to African hospitals with malaria often have positive cultures for gram-negative organisms, particularly non-typhoidal salmonellae. The rate of blood culture positivity can be as high as 12% ,26. Co-eThe extent of the interaction between HIV and falciparum malaria remains open to debate, but in sub-Saharan Africa, HIV positive pregnant women in particular appear to be at an increased risk of developing severe disease . In thisThis study shows that both a CAM score <2 and an MSA <5 identified patients who would survive. However, these scores had limited ability to predict mortality and it remains unclear what role, if any, they may play in clinical practice in areas of the world where malaria is not endemic. No clinical factor was associated with a poor outcome but given the low case fatality rate, the study was under-powered to detect such a difference.The main limitation of this study was its retrospective nature; as a result some data were missing or incomplete. Despite this, data capture was high, in particular data relating to ICU care, which were recorded systematically. Although the possibility of referral bias can not be excluded it is likely that our data are applicable to all patients with imported malaria requiring admission to ICU.per se. Africans who have left an endemic area seem to be at the same risk of severe disease as malaria-naive individuals. The increasing use of artesunate has been associated with a reduced need for exchange transfusion, even in patients with very high parasitaemia. In contrast to children, bacterial co-infections were uncommon. No single factor consistently identified individuals at risk of death. Both the MSA and CAM scores accurately predicted survival in low-risk patients but had limited power to identify those at high risk of death. All patients with malaria should be screened for HIV.This paper suggests that most adult patients with severe malaria who are managed in a specialist centre survive. Cerebral malaria and AKI tend to occur early in the disease, while ARDS develops later. Patients who die tend to do so because of complications associated with intensive care rather than malaria The authors declare that they have no competing interests.MM extracted the data, carried out the analysis and wrote the initial draft of the paper. MA designed the research database and helped extract the data. MMS and PLC advised on the collection and analysis of data on the diagnosis of malaria and helped draft these sections of the manuscript. SB and GB generated the initial ICU dataset and advised on the collection and interpretation of all data relating to intensive care stay of patients. CJMW designed and carried out the statistical analysis. JD conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/118/prepub


Although 80% of malaria occurs in children under five years of age, infants under six months of age are known to have low rates of infection and disease. It is not clear why this youngest age group is protected; possible factors include maternal antibodies, unique nutrition (breast milk), and the presence of foetal haemoglobin (HbF). This work aims to gain insight into possible mechanisms of protection, and suggest pathways for focused empirical work, by modelling a range of possible effects of foetal haemoglobin and other red blood cell (RBC) developmental changes on parasite dynamics in infants.A set of ordinary differential equations was created to investigate the leading hypotheses about the possible protective mechanisms of HbF-containing red blood cells, in particular whether HbF suppresses parasite population growth because parasite multiplication in individual RBCs is lower, slower or absent. The model also incorporated the intrinsic changes in blood volume and haematocrit that occur with age, and the possibility of parasite affinities for HbF-containing RBCs or reticulocytes.The model identified several sets of conditions in which the infant remained protected, or displayed a much slower growth of parasitaemia in the first few months of life, without any intervening immune response. The most protective of the hypothesized mechanisms would be the inhibition of schizont division in foetal RBCs so that fewer merozoites are produced. The model showed that a parasite preference for HbF-containing RBCs increases protective effects for the host, while a preference for reticulocytes has little effect.Plasmodium falciparum infection dynamics emphasize the likely importance of HbF and RBC number as an explanatory factor in paediatric malaria, and suggest a framework for organizing related empirical research.The results from this simple model of haematological changes in infants and their effects on  Malaria disproportionately affects children under five years of age . RepeatePlasmodium falciparum or Plasmodium vivax, the most common species – in about 48 hours bursts the RBC to release eight to 32 new merozoites, which invade new RBCs and continue the cycle [A human malaria infection begins when the parasite enters the bloodstream in the saliva of an infectious, biting mosquito. The parasite invades the liver, multiplies there for approximately 10 days, and then releases thousands of merozoite forms into the bloodstream. Each merozoite invades a RBC, multiplies, and – if he cycle . Virtual6 ml-1 of blood may be similar between infants and adults, the blood volume of an adult is 10–15 times that of an infant, so any given population of parasites in an adult host will represent a much lower total parasitaemia than in an infant host.Presumably, the basic biological process of parasite invasion and multiplication in RBCs is independent of the literal age of a host , and so, whatever the host’s age, cycles proceed roughly as sketched above, with regular numbers of merozoites released at each cycle, in cycles of regular length. The total blood volume of a host increases very significantly with age and size ,9. ThereAnother notable difference between infants and older children is that a much higher proportion of RBCs in infants contain foetal haemoglobin (HbF) rather than adult haemoglobin (HbA). HbF is produced only in humans, apes and Old World monkeys: it has a stronger affinity for oxygen than HbA, and so facilitates oxygen transfer across the placenta from mother to foetus during gestation . BeginniPlasmodium population because Plasmodium parasites do not survive as well in HbF-containing RBCs as in those containing HbA [P. falciparum may have a higher affinity for HbF-containing RBCs than HbA-containing RBCs. P. falciparum may also have (like P. vivax) a higher affinity for reticulocytes – the youngest age class of RBCs – which may be present in higher proportions in growing infants, and so may affect infection dynamics. In any case, the proportion of HbF-containing RBCs may be a factor in the protection of infants from severe clinical disease and high parasitaemia: by slowing parasite population growth, they may provide extra time for an effective response by the infant’s developing immune system or maternal antibodies.Infants under six months of age have a low rate of infection and incidence of severe disease compared to older children , despitening HbA -23, thouAlthough several possible factors underlying variable protection in infants have been suggested, including the effects of maternal antibodies, nutritional differences, and HbF, none has yet been confirmed. Foetal haemoglobin is one of a number of mechanisms, reviewed in an earlier paper , that coP. falciparum infections in infants [No mathematical model to date has investigated the effects of any developmental age difference on a malaria infection. Here previous models of RBC dynamics in malaria infections are extended to focus specifically on the effects of lower RBC counts and higher HbF proportions on the dynamics of  infants -26.Plasmodium parasites are involved in the pathology of malaria in an infant: (1) those in infected foetal erythrocytes, (2) those in infected adult erythrocytes, and (3) free merozoites in the blood.  Thus, the dynamical model considered has five separate populations, describing the dynamics of uninfected and infected foetal (HbF) RBCs, uninfected and infected adult (HbA) RBCs, and free merozoites within an infant host. For conciseness, vector and matrix notation  was used to model the dynamics of circulating RBCs, including the effect of infant growth on total blood volume (via the rate of growth of erythropoietic tissue), the switch in production of HbF- to HbA-containing RBCs, and the change in haematocrit in the first few months of life. The dynamics of two RBC lines were modelled – the uninfected 1) foetal and (2) adult erythrocytes – then the dynamics of malaria infection were incorporated into the dynamics of the circulating RBCs. Three main populations of  foetal aN × 1 vector P, the components of which may evolve in time. Define T(P) to be the sum of the components of P. Let the following ordinary differential equation (ODE) system in time t determine the evolution of the components:Consider an S(t) is greater than or equal to zero for all t, Λ > 0 and dΛ = 0. The components of the vector δ(1) are zero except for the first one. Matrix D is sparse with all diagonal components = 1, and all components just below the diagonal = −1; see Additional file T (P)/dt from S at time t - Δt is approximately a Gaussian function of Δt with a mean D = N Λ-1 and standard deviation σ = D N-1/2[T(P) and source term S(t). If individuals age with an average lifespan of D with standard deviation σ, then knowing the tangible quantities D and σ sets the abstract quantities N and Λ.  Although the vector–matrix notation is introduced for brevity, P itself can be thought of as containing all the information about the “state” of the population possible in this model. The matrix Λ D operates on the state vector P, incorporating the contribution of the source S(t) into the time evolution of P. This formalism may seem complicated initially, but it incorporates the non-instantaneous propagation of changes in the source S(t) into the population. One could think of other formalisms that incorporate time delays and dispersion in aging, but this particular one allows the use of efficient ODE solvers.where  vector δ are zero= D N-1/2,27. The 6 RBCs per μl of blood. In neonates, the haematocrit dips within the first few months of life, and then rises again to adult levels [In uninfected individuals, RBCs containing HbA circulate for ~120 days, while RBCs containing HbF circulate for ~70 days, at which point the RBC is cleared by the spleen . In adult levels . PresumaP, then consider ρ = VB-1XP, where VB is the blood volume of the host.  The time evolution of ρ is given bySince RBC and parasite populations are assessed empirically as cell counts per μl of blood, the model dynamics were formulated to consider the population per unit volume of blood rather than the total population in the host. However, the volume growth of the child must be carefully incorporated. If the state of a population of a parasite or RBC line is given by whereandρ, T(ρ), is VB-1X T(P), the density of the population in the blood. So from the point of view of population density, the growth of blood volume contributes a decay factor ΛV: a growing volume tends to dilute the concentration of cells. Note that ΛV itself is time dependent, as the rate of volume growth slows as the child ages.The sum of the components of t) as the host ages, while the production of adult erythrocytes per kilogram of mass of the host is a sigmoid-like function of t that reaches a maximum some months after birth.  The timW(t) is the mass of the host at age t. The time evolution of W(t) and VB(t) is discussed in Additional file Pamx, tOSa, τCa, Pfmx, τCf and Pfmn are given in Table Here hc was tracked, calculated asAs a measure of the health of the host, the haematocrit Vf and Va are the mean volumes of foetal and adult erythrocytes. Their values are given in Table Rea and Ma are the population state vectors for the reticulocytes and mature RBCs for adult RBCs and Ref and Mf are the corresponding state vectors for the foetal RBCs; see next subsection. If the value of hc drops under 24%, it is assumed that the host dies.Here P. falciparum preferentially invades young RBCs – reticulocytes – regardless of haemoglobin content [P. falciparum preferentially invades HbF-containing RBCs [P. falciparum in fact prefers HbF-containing RBCs, or, given the shorter lifespan and changing production patterns in the first few months of life, the key factor is that HbF-containing RBCs have a younger age distribution, skewed toward reticulocytes. The ability to test these possibilities was incorporated into the model by creating a reticulocyte subgroup and a mature subgroup for both the foetal and the adult RBC populations. The model incorporates the effects of the switch from the production of foetal erythrocytes to adult erythrocytes, as well as the increase in blood volume as the host ages. The equations that describe the dynamics of the RBC populations are:Some evidence indicates that  content ,30, and ing RBCs . It remaI is the identity matrix and ESa(t) and ESf(t) are the source rates of production of adult and foetal reticulocytes, respectively, per unit volume of blood. These quantities change as the host ages, in a manner specified below. ζRea, ζMa, ζRef, and ζMf are the binding affinities of the merozoites to the respective RBCs; the values are defined in the next section. The state vector of the merozoites is μ. The rates ΛRea, ΛMa, ΛRef, ΛMf and the vector lengths were defined as in the subsection “Basic Structure of Dynamical Model” using the values specified in Table Here, Five morphologically distinct populations of asexual parasite cells were considered: (1) ring stage, 2) early trophozoites, (3) late trophozoites, (4) schizonts, and (5) merozoites. The possibility is allowed that the parasite can develop in either foetal or adult erythrocytes, but possibly at different rates of development and with different efficiencies of reproduction in each type. The list of populations as well as the average durations of individual residence in those populations, along with the standard deviations in the durations, is given in Table  early trPR = 24 hr with total of 105 merozoites released into the blood volume VB at a constant rate per unit volume κPR = 105 (24 h X VB)-1. The average number of merozoites subsequently released by bursting schizonts of P. falciparum in human blood culture is known from direct microscopy to be >16 [pa = 16. The possibility is allowed that the average number of merozoites released from a bursting schizont formed in a foetal erythrocyte, pf, may be different from pa. The binding affinity of merozoites to target RBCs is inferred from observations of parasite growth in neurosyphilis patients treated for malaria therapy [-6 – 10-5 μl hr-1. Here, ζMa, the binding affinity of a merozoite to the mature adult RBC, is taken to be 3.33333 × 10-6 μl hr-1. The possibility is allowed that the merozoite binding affinity to adult reticulocytes, ζRea, mature foetal RBCs, ζMf, and foetal reticulocytes, ζRef, may differ from ζMa. The value is only changed when directly stated.The primary release of merozoites from the liver apparently involves the release of thousands of merozoites in clusters . For simo be >16 , so in t therapy ,34, and  therapy , to be 1t be the time since the birth of the infant, and taking AgPR as the age of the infant when primary release starts, the ODE system for the parasite populations is:Using the information above, letting x) = 1 if x > 0, zero otherwise.). The rates Λμ, ΛRa, ΛEa, ΛLa, ΛSa, ΛRf, ΛEf, ΛLf, ΛSf and the vector lengths were defined as in the subsection “Basic Structure of Dynamical Model,” using the values specified in Table Note that since For further details on the Methods, see Additional file P. falciparum to develop normally within an HbF-containing RBC were modelled. First, the possibility was investigated that HbF-containing RBCs release 0, 2, or 16 merozoites , while HbA-containing RBCs always release 16 (pa = 16). Second, the rate at which infants produce HbF and HbA during the first few months of life was varied . Third, the possibility was examined that P. falciparum has no preferential affinity, an increased affinity for HbF-containing RBCs, or an increased affinity for reticulocytes . Last, the possibility that in HbF-containing RBCs the schizont stage is delayed 60 h (DSf =72), (so that the life cycle is 108 h instead of 48 h), was modelled. Figure f = 0, the infection increases more slowly – graphically, a lower slope) or diminishes this effect . Adding a delay is mildly protective for the host, as it slightly lowers the parasite population growth rate, which increases the time until host death. This change is seen in Figure f = 0, for instance, as in Figure Because little is known about the protective effects of HbF at the single-RBC level, several hypotheses about the failure of f = 0), the infant remains protected from a fatal outcome of infection through approximately the first 15 days of life, despite the complete absence of an immune response , the infant dies but the time until death increases, most significantly in the first two months of life. If the HbF-containing RBCs release 16 merozoites (pf = 16), the same as HbA-containing RBCs, there is no effect; i e, this serves as a control. Now, other parameter modifications will be introduced and compared to this set of parameters.With the parameters set to the values in Tables e Figure . If the Cf and τCa, such that the host reaches a HbF:HbA ratio of 1:1 at 65, 97, and 163 days of age. Figure Cf = 100, and τCa = 150. Figure Cf = 200 and τCa = 300 the infant is most protected from a fatal outcome.The rate of change of the ratio of HbF- to HbA-containing RBCs is known to vary dramatically from infant to infant. While some infants reach adult levels of HbA at three months of age, others do not reach adult levels until six months. Three different rates of change were modeled by modifying the values of τP. falciparum life cycle within a RBC is 48 hours. It is possible that the presence of HbF delays this life cycle. A delay of 60 hours in the schizont phase of HbF-containing RBCs, leading to a total 108-hour life cycle (DSf =72) was modelled. This delay has little effect if the HbF-containing RBCs act as a “dead end”  for the parasite  in an infant infected at one day of age, the delay increases the time to host death by 400 hours, a gain which slowly decreases as the age at infection increases.In normal conditions, the e Figure  comparedP. falciparum has a greater affinity. This possibility was modelled by increasing the parasite’s affinity for HbF RBCs 10-fold (ζRef > ζRea and ζMf > ζMa). Compared to ζRef = ζRea and ζMf = ζMa, the stronger HbF affinity changes infection dynamics in favour of the host, most significantly when pf = 0 . An increased preference for reticulocytes slightly changes infection dynamics, although the maximum time to host death does not decrease dramatically , in an infant infected at one day of age, the time to host death increases by 400 hours, a gain which slowly decreases as the age at infection increases.It has been reported that ult RBCs . This poy Figure . IncorpoThe model presented here incorporates haematological changes that occur in infants under six months of age, and the results may offer some help toward explaining why neonates are relatively protected from severe malaria. The results are in accord with empirical studies which show that the youngest among the age group in question are protected, but the point at which the shift occurs varies based on transmission: in areas of high transmission, infants are protected only through the first three months of life, while in areas of low transmission, infants seem to be protected through six months . It is pf = 0, the host is protected for the first two months of life, despite the complete absence of an immune response. For the next few months of life, the rate at which parasitaemia increases in the course of an infection is slower than in an adult host, and this difference may be sufficient to allow for effective immune intervention: any host factor that slows down parasite population growth buys time for protective responses by maternal antibody or the developing, immature infant immune system. Whatever the mechanism by which HbF retards the expansion of a Plasmodium population – whether parasite multiplication in an individual RBC is lower, slower or absent – the presence of HbF would appear to be an unmitigated benefit to the malaria-exposed, of any age. Yet the sparse evidence suggests that HPFH is rare, in areas of high malaria endemicity as elsewhere. Sickle cell anaemia and β-thalassaemia are generally accompanied by elevated HbF levels, but the degree of elevation is highly variable [In the model, when the parameters are set as in Tables variable ,40. Becavariable : hydroxyvariable . The potvariable .It seems possible that there are opportunities for laboratory researchers to gain important insights from in vitro work. For instance, if sufficient quantities of HbF-containing RBCs could be obtained, and parasite cultures established in them, critical observations could be compared to those from the usual cultures of HbA-containing RBCs, on the number of merozoites released per infected HbF-containing RBC, and the time from when the parasite enters the HbF-containing RBC until it bursts.in vivo through the differing age distributions of HbA- and HbF-containing RBCs [The hypothesis that HbF-containing RBCs release fewer merozoites has a greater overall effect on infection dynamics than a delay in the parasite life cycle. Empirical research suggests that the parasite may have a reticulocyte preference, and that this may confound the seeming preference for HbF ing RBCs . AlthougIt has long been accepted that children under six months of age are protected from severe infection, and this is widely reflected in the literature on paediatric malaria. This topic has recently been analysed more carefully, as noted above, and it appears that the malaria burden in this age group – while still relatively low – may be higher than previously acknowledged . It may P. falciparum infection dynamics seem intuitively sound in a qualitative sense, and they emphasize the likely importance of HbF as an explanatory factor in paediatric malaria. The model provides a framework for examining hypotheses about the protective effects of foetal haemoglobin, organizing empirical observations and making critical quantitative comparisons among them.The results from this simple model of haematological changes in infants and their effects on P. falciparum: Plasmodium falciparum; HbF: Foetal haemoglobin; HbA: Adult haemoglobin; HPFH: Hereditary persistence of foetal haemoglobin; ODE: Ordinary differential equation.RBC: Red blood cell; The authors have declared no competing interests.EB conducted the model runs and compiled the output. All authors contributed to the development of the model and wrote, read and approved the final manuscript.Further Methods; Description: Further details on the methods.Click here for fileExplanation of Figure 2; Description: Further explanation of the interpretation of Figure2.Click here for file


Plasmodium falciparum and hookworm infections on the frequency of anaemia among pregnant women in two rural communities in Enugu, South East Nigeria.Malaria and hookworm infections are common in sub-Saharan Africa and they increase the prevalence of anaemia in pregnancy with resultant poor pregnancy outcomes. This study was carried out to assess the impact of A cross sectional descriptive study was carried out in a total of 226 women attending antenatal clinics at two rural Primary Health Centres (PHC) from April 2011 to July 2011(each PHC with 113 subjects). Socio-demographic data were collected through a structured questionnaire. Blood and stool samples were evaluated for haemoglobin estimation and malaria parasites, and stool samples examined for parasitic infection in all the women. Data was analyzed using STATA 10 software statistical analysis package. Student t-test was used for comparing mean values and chi square test for comparing categorical variables and level of significance set at p<0.05 and logistic regression was used to identify the risk factors associated with malaria in pregnancy.The mean age of the women was 27years with range 18 - 38years and SD of 5years. Most of the women were housewives and over 50% in their second trimester. 53% of them had malaria parasites while 27% had hookworm infection. About 40% of the women were anaemic (haemoglobin < 0.001). Similar association was found between hookworm infection and anaemia (p <0.001). Though both malaria and hookworm infections greatly increase the odds for anaemia  and  respectively, the odds for having anaemia in pregnancy was higher for malaria than hookworm infections.Plasmodium falciparum and hookworm infections have significant impact on the high frequency of anaemia in pregnancy in our rural communities. There is need to strengthen the control program that has been in place with an integrated intervention to combat these parasitic infections in our rural communities, with mass distribution of antihelminthics as one of the included relevant methods, among others. Plasmodium falciparum, the most common malaria species in Africa [Malaria infection during pregnancy is a major public health problem in tropical and sub-tropical regions throughout the world . This han Africa , 3.Malaria is a common cause of anaemia in pregnancy in both immune and non-immune individuals , 4 with Plasmodium falciparum and helminth infection in Africa [P. falciparum and hookworm co-infection has been shown to have additive impact on anaemia resulting in adverse pregnancy outcomes [Hookworm infections are also a common cause of iron deficiency anaemia in Nigeria , 12 and n Africa . P. falcoutcomes .P. falciparum and hookworm co-infection among pregnant women in rural communities where these parasites are endemic. This study was therefore aimed at assessing the impact of P. falciparum and hookworm infection on the frequency of anaemia among pregnant women in two rural communities in Enugu State in South East Nigeria.There have been few studies on the impact of The study was carried out in two Primary Health Centers (PHC) located in two different rural communities in Nkanu West Local Government Area (LGA) of Enugu state, Nigeria. These rural communities were randomly selected from the list of rural communities with PHC in the LGA.The communities were Ogonogoeji Ndiuno Akpugo and Eziokwe Amurri. Both are located over 10 kilometers away from the LGA capital (Agbani) which has a secondary health care facility. These communities share some form of homogeneity in both population and environmental factors. Each having average population of over 2000 inhabitants and surrounded by thick forest with no modern toilet and water drainage facilities which provide conducive environment for breeding mosquitoes.The people in both communities are mainly peasant farmers and the highest health facility is the PHCs located within the communities. The health centers provide among other primary health care services, antenatal and delivery services to pregnant women within and the surrounding communities.This cross sectional descriptive study was conducted among pregnant women at two rural Primary Health Centres (PHC) from April 2011 to July 2011 with each contributing 113 subjects. All the pregnant women coming for antenatal clinic (ANC) on daily basis were enrolled for the study. History, examination and investigations were carried out on all the women.Safety procedures were adopted in the collection of venous blood samples by swabbing the ante cubital fossae with 70% alcohol and 5mls of blood was drawn into EDTA bottle with sterile hypodermic needle. Thick and thin films were made on clean slides and labeled accordingly as recommended by WHO [The thin films were fixed with methanol and all films were stained with 10% Giemsa stain of pH 7.2 for 30 min as recommended by WHO . The thiFive milliliters of blood were collected inside EDTA bottle. Non-heparinized capillary tubes were filled with blood sample from the EDTA bottle. The tip of the capillary tubes were cleaned with cotton wool, sealed and they were arranged inside the haematocrit centrifuge. They were centrifuged for 5 minutes at 12, 000 revolutions per min. The Packed cell volume was determined by using haematocrit reader to read the level of the haemoglobin .All selected patients were given clean labeled stool containers. The patients were instructed to bring small quantities of their stool specimen the next morning within an hour of passing the stool to the primary health care centre. Wet mounts were prepared from the stool specimen using the direct smear method with normal saline and iodine preparation and the concentration procedure using formol/ether for the identification of helminth eggs as well as protozoan cysts .Approval to conduct the study was obtained from the Primary Health Care centre and the local government authorities of Nkanu West Local Government Area, Enugu State, Nigeria. Verbal consent from the study participants was also obtained before administering the questionnaire.2) was done to compare relationships between categorical variables. Factors that could cause anemia during pregnancy were identified and logistic regression done to factor these covariates. Anaemia in pregnancy was defined as those having haemoglobin concentration of less than 10g/dl, as this is the usual benchmark in most hospitals in Nigeria, and the reference work on it indicated under our discussion.Data was analyzed using STATA 10 software  statistical analysis package. A descriptive analysis was done on each variable and bi-variate analysis to assess relationships between the variables. Independent student t-test was used to assess significant mean difference of haemoglobin seen in malaria and hookworm infections, and one-way ANOVA was used to compare the level of anaemia seen in the three trimesters of pregnancy. Chi square . The highest form of education for most respondents was primary education (15%) . Those fP.falciparum parasitaemia found in our study is close to 60% reported by Fuseni et al in Ghana [Plasmodium parasitaemia in the communities are not surprising as the communities under study are surrounded with thick forest and have poor water drainage facilities which provide conducive environment for breeding mosquitoes. This situation is compounded by low use of insecticide treated nets among the rural dwellers. The prevalence of 27% for hookworm infection found in this study is similar to 30% reported by Ozumba et al in Enugu, Nigeria [Improving the understanding of the effect of malaria and hookworm infections  on the frequency of anaemia in pregnancy may help in the design of appropriate management strategies targeted at reducing adverse pregnancy outcomes often associated with these infections. The high prevalence of 53% for in Ghana . This isin Ghana , but lowin Ghana . The hig Nigeria , but hig Nigeria , 17% by  Nigeria , and 20. Nigeria . The praP.falciparum and hookworm infections. The significant relationship between Plasmodium parasite and hookworm infections on one side and anaemia on the other side is obvious in our findings in this study that 89% and over 50% of 153 pregnant women with malaria and hookworm infections respectively were anaemic. The frequency of anaemia is further aggravated by the additive effect of P.falciparum and hookworm co-infections in this study. This is similar to findings in the previous studies [P.falciparum is attribu to haemolysis of the infected red blood cells, decreased red blood cell production and other potential causes such as rupture of infected red blood cells with pathological effects of accompanying events, removal of uninfected red cells due to antibody sensitization, marrow hypoplasia seen in acute infections of malaria which may cause decreased production of red cells and consequent anaemia, among few other causes. Hookworm infections results in iron loss from the infested intestinal lumen. It has been estimated that a load of 1000 hookworm ova per gram of faeces is associated with loss of 1mg of iron per day [The 40% prevalence of anaemia in this study is consistent with 40.4% reported by Dim and Onah in Enugu , but hig studies . The pat per day Poor nutritional intake  common among the rural dwellers may also have contributed to the high prevalence of anaemia among these pregnant women. Interestingly, but as reported in other studies from Nigeria , 19–21, Plasmodium falciparum and hookworm infections have significant impact in the high prevalence. We suggest that an integrated interventional program to combat both malaria and helminthes infections in our communities should be strengthened to have significant health impact. Routine screening and treatment of both malaria and hookworm infections should be emphasized in our antenatal care. This will help prevent anaemia and reduce maternal and perinatal morbidity and mortalities in our communities.We conclude that the prevalence of anaemia in pregnancy is high in our rural communities.


Plasmodium vivax is responsible for the majority of malarial infection in the Indian subcontinent. This species of the parasite is generally believed to cause a relatively benign form of the disease. However, recent reports from different parts of the world indicate that vivax malaria can also have severe manifestation. Host response to the parasite invasion is thought to be an important factor in determining the severity of manifestation. In this paper, attempt was made to determine the host metabolic response associated with P. vivax infection by means of NMR spectroscopy-based metabonomic techniques in an attempt to better understand the disease pathology.P. vivax-infected patients, healthy individuals and non-malarial fever patients were carried out followed by multivariate statistical analysis. Two data analysis techniques were employed, namely, Principal Component Analysis [PCA] and Orthogonal Projection to Latent Structure Discriminant Analysis [OPLS-DA]. Several NMR signals from the urinary metabolites were further selected for univariate comparison among the classes.NMR spectroscopy of urine samples from P. vivax-infected patients were distinct from those of healthy individuals as well as of non-malarial fever patients. A highly predictive model was constructed from urine profile of malarial and non-malarial fever patients. Several metabolites were found to be varying significantly across these cohorts. Urinary ornithine seems to have the potential to be used as biomarkers of vivax malaria. An increasing trend in pipecolic acid was also observed. The results suggest impairment in the functioning of liver as well as impairment in urea cycle.The urine metabolic profiles of P. vivax using urine metabolic profile. Distinct variations in certain metabolites were recorded, and amongst these, ornithine may have the potential of being used as biomarker of malaria. Pipecolic acid also showed increasing trend in the malaria patient compared to the other groups.The results open up a possibility of non-invasive analysis and diagnosis of  Plasmodium. The five Plasmodium species that are responsible for human malaria are Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi [P. vivax is the predominant cause of clinical malaria [Malaria is caused by parasites of the genus knowlesi . Every yknowlesi . Sub-Sah malaria .Metabonomics is a comparatively recently developed technology defined as the global, dynamic response of living organism towards genetic and environmental perturbations . The tecP. falciparum infections [P. falciparum [Plasmodium berghei infection [P. vivax patients, although it has been observed recently that P. vivax can cause high levels of pathological complications [Malaria is an ancient infectious disease that has afflicted humans since pre-historic times. Severity in the clinical malarial disease occurs frequently, and has been well documented for fections ,11. The lciparum -14. Howelciparum . In anotnfection . No metaications ,18.P. vivax malaria patients and try to correlate the changes observed in them to the known and reported biochemical processes. The paper describes a study of global host metabolic responses towards P. vivax infection. The results ascertain differences in metabolic response in the urinary metabolic profile of the P. vivax infected persons with respect to that of healthy individuals and also with the patients of non-malarial fever. The differential metabolites are delineated, raising the possibility of a non-invasive diagnosis of malaria.In this report, a NMR based metabonomic approach is delinated to study the urine samples of This study was approved by and carried out according to the guidelines of the Local Institutional Ethics Committee. Informed consent was obtained prior to sample collection.P. vivax by blood smear microscopy and (3) Subjects able to consent through informed consent document. Certain exclusion criteria were also followed viz. (1) All females and males < 30 and > 50 years of age, (2) Patients with P. falciparum and mixed P. falciparum/P. vivax infections and (3) Patients with other chronic diseases and/or previous history of any type of malarial infection. In total 53 individuals  were recruited for the study: 21 P. vivax-infected patients, 21 healthy controls and 12 patients with non-malarial fever. Their age ranges were: malaria patients: 35 ± 3.95 years: healthy individuals: 44 ± 3.18 years: and non-malarial fever patients: 40.5 ± 5.33 years (median ± standard error). The samples were transported from the site of collection to the laboratory at 0°C and were immediately processed [Midstream urine samples (~10 ml) of malaria-infected individuals were collected from local pathological laboratories in sterile tubes containing a final concentration of 0.02% sodium azide. The inclusion criteria followed to recruit the subjects in the study were as follows- (1) Adult males (age ranging from 30 to 50 years), (2) Malaria cohort of those patients that tested positive for rocessed .2HPO4 and 0.2 M NaH2PO4; pH 7.4, made in deionized and 0.22 mm filtered water). This mixture was homogenized and left to stand for 5 min at ambient temperature following which it was centrifuged for 6 min at 6000 g to remove any suspended matter. The clear supernatant (600 μL) was transferred to 5 mm NMR tube  and 50 μL D2O, containing 2, 2-dimethyl-2-silapentane-5-sulfonic acid (DSS) at 0.01% final concentration, was added to it. D2O served as a field-frequency lock and DSS was the internal chemical shift reference. The samples were stored frozen at -20°C until required for NMR experiments.An aliquot (800 μL) of collected urine sample was mixed with 400 μL phosphate buffer (an 81:19 (v/v) mixture of 0.2 M Na1H. For the 1D experiments the spectral width was 12.01 ppm, an excitation pulse of 9 μs (~70°), and a relaxation delay of 1 s between consecutive pulses. This resulted in an acquisition time of 0.68 s and a cycle time of 1.68 s. A total of 64 transients were acquired into 8,192 data points. The water suppression routine involved excitation sculpting using gradients. For processing the FIDs were subjected to exponential multiplication leading to an additional line broadening of 0.20 Hz. A sine bell apodization was also used prior to Fourier transformation. The 1H NMR spectra were manually phase and baseline corrected. For identification of metabolites two dimensional (2D) experiments were performed on selected samples. 2D COSY spectra were acquired with 32 transients in the direct dimension for each of the 256 increments in the indirect dimension. The spectral width was 12.01 in both the dimensions. The pulse width used was 11.80 μs with a delay of 1.5 s. The processing parameters of the data included a QSINE window function, exponential multiplication of 0.20 and 0.30 Hz in the direct and indirect dimension respectively, followed by Fourier transformation in both the dimensions. The 2D TOCSY experiments were performed using similar acquisition parameters. The processing parameters were also identical with an extra Gaussian multiplication of 0.10 Hz prior to Fourier transformation in both the dimensions. The 2D spectra aided in assignment of the NMR resonances to the specific metabolites. In addition, comparison with reported experimental and calculated spectra was done using Human Metabolome Data Base (http://www.hmdb.ca/search/spectra).The NMR spectra were acquired on AVANCE 500 MHz Bruker NMR spectrometer equipped with a 5 mm broad-band inverse probe. The operating frequency on this machine was 500.13 MHz for 1H NMR spectra were manually phased and baseline corrected. For data reduction the individual spectrum (0.50 to 9.50 ppm) were divided into frequency bins of width 0.04 ppm each resulting in a total of 191 bins per spectrum. Each bin was integrated using MestReC 4.7.0. In order to avoid the artefacts of water suppression and the variable concentration of urea, the region 4.3 to 6.6 ppm was excluded from the binning procedure. The integrals were normalized to the total intensity of the spectrum to compensate for the inter-sample dilution effects. This was achieved by an algorithm developed in-house. The data was mean-centered  and Pareto scaled. The preceding operation resulted in similar weight being given to all the bins and the resultant multivariate models were not dominated by bins representing metabolites high in concentration. Next, the data so generated served as input for PCA and OPLS-DA, which were performed on the SIMCA-P + 12.0 platform .The frequency domain a priori. This helps in visualizing any pattern that is present in the data and also identifies the outliers. The class specific segregation can be refined using OPLS-DA. This is a supervised technique where class specificity is assigned towards the sample set a priori. This aided in the visualization of the data as scores plot. In such a plot each point represents a spectrum and the pattern and the segregation in the data can be visualized. This pattern is due to one or more spectral bin(s) which can be visualized using a loadings plot where each point represents a spectral bin. The OPLS-DA model is judged using two parameters. R2X signifies the total explained variation in the data whereas the Q2(cum) explains the extent of separation between the classes as well as the predictability of the model.PCA is an unsupervised pattern recognition technique where no class identity is assigned to the samples t-test to judge the significance in their difference across the classes.The spectral bins contributing to the variation seen in the OPLS-DA models  were assigned to specific metabolites as described in the previous section. For further quantification of these metabolites, univariate analysis of the NMR spectral peaks was performed in the following way: the peak of the metabolite contributing to the variation in the OPLS-DA model was assigned in the one-dimensional NMR spectral profiles of the individuals. These peaks were integrated, normalized to the whole region of the spectral profile and compared between the classes concerned using student's 1H NMR spectra of urine samples from patients and healthy individuals were recorded followed by multivariate statistical analyses. Representative 1H NMR spectra from individual belonging to the three groups are shown in Figure P. vivax, patients with non-malarial fever or healthy volunteers. For this analysis, only adult male samples were used. The individuals with infection had been referred to the pathology laboratories with malaria-like clinical symptoms  and their infection status was confirmed by standard peripheral blood smears.1H NMR spectra of urine samples. The first data set consisted of urine from P. vivax-infected individuals and healthy controls. The second data set consisted of patients with malaria and non-malarial fever. Earlier data analysis by PCA of the 1H NMR spectra of urine from the healthy individuals and P. vivax-infected persons showed an inherent segregation between the two classes [2X value was found to be 0.72 [P. vivax-infected patients and healthy controls or non-malarial fever patients. The OPLS-DA scores plot from the urinary NMR metabolic profile of P. vivax-infected individuals and healthy controls is shown in Figure 2X = 0.41 and Q2(cum) = 0.67. However, the spread is typical of human samples since there is enormous variation in genetic backgrounds as well as food habits. This is to say, the samples under consideration have factors other than malaria, which led to a larger scatter among them. To ensure that the effect of sex does not confound the analysis it was restricted to male patients only. In spite of all these factors, the Q2(cum) was found to be significantly high, therefore it implicated a considerable difference in the urinary metabolic profile of malarial patients and healthy individuals. OPLS-DA models made from NMR profile of urine samples of malaria patients and non malarial fever patients showed Q2(cum) = 0.89 and R2X = 0.67. This indicates a very good separation between the urinary profile of malaria patients and non-malarial fever patients, which is also evident from the PCA and OPLS-DA scores plot shown in Figures The multivariate analysis was performed on two data sets obtained from the  classes . The R2X be 0.72 . In the vs healthy and malaria vs non-malaria fever patients, respectively.In order to identify the metabolites corresponding to the spectral bins found significant two-dimensional NMR techniques such as COSY and TOCSY were employed, matching of the chemical shifts with values reported in metabolite databases such as Human Metabolome Database (HMDB). The compounds so identified are listed in Tables 1H NMR spectral peak(s) were integrated. The results are presented in Figures In order to compare the relative concentrations of the metabolites identified from OPLS-DA models, the corresponding Several metabolites also showed significant variation across malaria and non-malarial fever patients. Among them, glucose and ornithine were increased in the urine profile of the malaria patients significantly Figure , while ap < 0.05 for both) in the malaria patients. Along with this, pipecolic acid showed a trend towards increase in the malaria patients compared to non-malarial fever, although this was not statistically significant  to tyrosine. Alteration of this pathway leads to the formation of phenylpyruvate. Usually phenylpyruvate is a minor metabolite of phenylalanine. However, in patients with phenylketonuria, the phenylalanine-tyrosine pathway is blocked leading to excess formation of phenylpyruvate . A signichildren . HoweverOrnithine is a part of the urea cycle. In the first step of the cycle, ornithinecarbamoyltransferase (OCT) acts on ornithine and carbamoylphosphate to begin the natural detoxification of ammonia. Earlier reports suggested that elevation of serum value of OCT can be a potential marker of malaria and this was associated with the damage of the liver cells in the liver stage of the parasite ,28.P. vivax infection. In the light of results presented here, earlier reports of statistically significant decrease of ornithine in the plasma of patients with clinical malaria [Perturbation of OCT levels are likely to result in an impaired urea cycle. Urea cycle is the major pathway for ammonia detoxification in mammals. Earlier reports indicated ammonia toxicity during malaria in mice model . Observa malaria  may be iP. berghei ANKA infected mice [Pipecolic acid is a minor metabolite of lysine catabolism. In humans, pipecolic acid is found to be associated with chronic liver disease, pyridoxine dependent epilepsy and Zellweger syndrome -32. Receted mice . Hyperlyted mice . From thP. vivax infection. Febrile illnesses are often likely to be misdiagnosed as malaria. Lundqvist and co-workers showed that borreliosis can be misdiagnosed as malaria due to the similarity in the symptoms [P. vivax-infected individuals and n-butyrate and acetate which are decreased in these patients  which is equal to 0.89 from the OPLS-DA model made from the urinary metabolite profile of the non-malarial fever patients and P. vivax-infected individuals, urinary metabolic profiles have a potential to be of diagnostic use for detecting malaria and to differentiate malaria from other non-malarial fevers.The non-malarial fever category serves as a control for general fever related changes that are not specific to symptoms . Poorly symptoms ,36. In ts Figure , Table 4s Figure . As is eP. vivax-infected patients. Urinary metabolite profile of malaria patients were found to be distinct from both healthy individuals and those of non-malarial fever patients. Among several changes in metabolism, a disruption in the urea cycle in terms of ornithine excretion was noted, along with an elevated level of pipecolic acid indicating possible hepatic injury. Ornithine, therefore, promises to be candidate biomarker for malarial infection. Moreover, an extremely predictive model was constructed from urinary metabolite profile of the clinical malaria patients and individuals with non-malarial fever. This might help in the non-invasive diagnosis of malarial infection. Malaria-specific signatures were observed in the urine and showed that the urine profile of malaria patients cluster separately from different groups of control populations in spite of the high variability of urine metabolites across individuals. This study, therefore opens up the possibility of finding out malaria specific biomarkers in the urine, which might help in non invasive diagnosis of the infection in the long run. Many parameters, such as genetic make up, food habits and lifestyle, may influence urine metabolite patterns. In order to circumvent such confounding parameters, a longitudinal follow up and comparison of urine from cohorts of malaria patients and other control subjects will provide further specific information regarding disease related changes in patients of P. vivax malaria.This study reports on the understanding of the host metabolic changes in terms of the urinary metabolite profile of the The authors declare that they have no competing interests.AS, SG, SM, PJ were involved in collection and processing of the samples, NMR experiments, statistical analyses and model building and interpretation of the data. AS and SG, in addition, drafted the manuscript. SP, SS and HMS conceived the study, interpreted the data and were involved in writing the manuscript. All authors read and approved the final manuscript.The authors are grateful to the National Facility for High-field NMR, TIFR and Dr R.Y. Agarkar of TIFR Medical section for all their help. We thank Dr S. Bhayani and Dr A. Mehta for help in sample collection. AS thanks the Council of Scientific and Industrial Research, Govt. of India for the Shyama Prasad Mukherjee Fellowship.