Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens.Naturally acquired immunity to blood-stage 42) was determined by IFN-γ ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-119 invasion inhibition antibodies (IIA) to the E-TSR (3D7) allele and growth inhibitory activity (GIA). The haplotype of MSP-119 alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months.Blood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 E-TSR haplotype.MSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-γ responses and GIA. MSP-1P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.Variation in the stability of cellular and humoral immune responses to  Plasmodium falciparum is intense and stable develop naturally acquired immunity that is characterized by a high degree of protection against high-density parasitaemia and clinical illness. This immunity develops as a consequence of experiencing multiple episodes of blood stage infection throughout infancy and childhood, and may be lost, or markedly diminished, in the absence of periodic boosting by clinically asymptomatic blood stage infections during adulthood [Individuals living in areas where transmission of dulthood . Adaptivdulthood ,2. Appredulthood ,4.P. falciparum merozoite antigens and susceptibility to blood stage infection or mild malaria during childhood. Some, but not all, have reported a significant association of IgG antibody levels to the C-terminal region of MSP-1 with parasitological and clinical phenotypes such as parasitaemia and fever with parasitaemia [42 appear to persist for at least four months in residents of hypoendemic regions of the Peruvian Amazon [Many studies have examined the relationship between antibody responses to sitaemia -10. The sitaemia . On the n Amazon . There hn Amazon -16, and P. falciparum MSP-1 by adults who are clinically immune to malaria. Assays that measure immune responses to the 3D7 and FVO alleles of the C-terminal fragment of MSP-1, both of which have been included in recent clinical vaccine trials [in vitro, GIA responses to P. falciparum were quantified.The goal of this study was to advance understanding of the temporal stability of quantifiable immune responses to the C-terminal region of e trials -19, wereEthical approval was obtained from the Institutional Review Board for Human Investigation at University Hospitals Case Medical Center and the Ethical Review Committee at the Kenya Medical Research Institute. Twenty-four healthy asymptomatic adult female (n = 7) and male residents (n = 17) with respective median ages 34 and 44 years (age range 18–79 years) from Kanyawegi sub-location, Nyanza Province, Kenya participated in this study. A total of six blood samples from each individual were collected over a period of nine months. Three venous blood samples (20 ml) were collected at three-week intervals from August to October 2004 during a period of relatively low malaria transmission, and again  from April to May 2005 during a period of high malaria transmission. Data collected at each time point included the reported history of recent febrile illness, demographic information  and the level of asexual parasitaemia by blood smear. Data from the 16 participants who provided blood samples at all 6 time points are included in the analysis. All of these individuals remained asymptomatic and reported taking no malaria medication during the nine-month period observation.P. falciparum-infected erythrocytes. The density of parasitaemia was expressed as the number of asexual P. falciparum per μl blood assuming a leukocyte count of 8,000 per μl.Thick and thin blood smears were prepared, fixed in 100% methanol, stained with 5% Giemsa solution, and examined by light microscopy for 42 or FVO MSP-142  or 1 μg/ml PHA in microtiter plates coated with 5 μg/ml of primary anti-IFN-γ antibody . IFN-γ secreting cells were detected after removal of PBMCs and addition of 0.75 μg/ml of biotinylated secondary anti-IFN-γ antibody (Endogen M-700B) followed by horseradish-peroxidase conjugated strepavidin peroxidase (DAKO PO397) and 1% 3-amino-9-ethyl-carbazole substrate in -0.1 mol/L acetate buffer. Spot Forming units (SFU) were counted by Immunospot Satellite analyzer . An IFN-γ ELISPOT assay was considered positive if the frequency of SFU for PBMCs incubated with MSP-142 or PHA was significantly greater (p < 0.05) than culture medium alone using a chi-square Fisher's exact test. Only PBMC samples with strong positive IFN-γ responses to PHA were included in the analysis (n = 88).IFN-γ ELISPOT was performed as described previously ,16,20. BP. falciparum MSP-142 were quantified serologically by ELISA as described previously [42. Plasma from nine North American adults never exposed to malaria was used as a negative control. Plasma from four known malaria immune Kenyan adults was pooled to create a positive standard. A standard curve was performed for each plate tested. The value obtained with a 1:50 dilution of the positive pool was designated as 100 arbitrary units (AU), 1:100 dilution as 50 AU, 1:200 dilution as 25 AU, 1:500 dilution as 10 AU, 1:1,000 dilution 5 AU, and 1:2,000 dilution as 1 AU. A four-parameter standard fit curve was constructed from the positive control plasma pool and applied to sample values. Positive values were greater than the mean + 3 SD of the value of the individual negative control plasma samples.IgG antibodies to recombinant 3D7 (E-TSR) and FVO (Q-KNG) alleles of eviously . Briefly19-specific IIA were as described previously [19MAD20/3D7/E-TSR allele and an isogenic D10-PcMEGF parasite line in which the antigenically unrelated P. chabaudi ortholog replaces P. falciparum MSP-119 were tested in parallel. Ring-stage parasites were synchronized twice by sorbitol lysis and allowed to mature to late trophozoite/schizont stages. Purified parasites were adjusted to 4% haematocrit with 0.5% infected red cells, and 50 μl aliquots were placed in 96-well flat-bottom microtiter plates with an equal volume of 1:5 pre-diluted plasma in culture medium . The same batch of pre-diluted plasma was added to the two parasite lines in the same assay. The cultures were incubated for 26 hours to allow for schizont rupture and merozoite invasion. 25 μl of re-suspended cultures were removed, fixed with 0.25% gluteraldehyde in PBS for 45 min, and placed in 1 μg Hoechst 33342 (HO) stain  in 400 μl 1× PBS for >24 hours at 4°C [4 cells using Becton-Dickinson FACS Diva 5.01. Ring parasitaemia was calculated by quantifying singly infected erythrocytes plus multiply infected erythrocytes (quantified as having two rings) according to flow cytometry gating previously described [19 antibodies was calculated by subtracting the percent invasion of D10-PfM3' relative to non-immune controls from the percent invasion of D10-PcMEGF relative to non-immune controls. A positive response was defined as ≥ 5% inhibition attributable to MSP-119 IIA. Overall antibody-mediated GIA was determined from results of the MSP-119 IIA assay using growth of the D10-PfM3' line only. Growth inhibition based on duplicate wells was calculated with the following equation: 100 – (test plasma parasitaemia/non-immune plasma parasitaemia) × 100. Plasma from four malaria naïve US adults was pooled as the non-immune plasma controls. A positive response was defined as ≥ 15% D10 inhibition.Methods to quantify MSP-1eviously ,22. Bries at 4°C ,24. Stai19 haplotypes by PCR/ligase detection reaction fluorescent microsphere assay (PCR/LDR-FMA) were as described previously [DNA was extracted from 200 μl venous blood using QIAamp DNA blood mini kit . Methods to quantify the four major MSP-1eviously .Monthly rainfall data for the Kisumu area was purchased from the Kenyan Ministry of Transport, Kenya Meteorological Department. Rainfall from July 2004 to July 2005 was recorded as mm per month.Kappa analyses were conducted using SAS Version 8.2 .t test. Pearson's correlation was used to compare MSP-119-specific IIA results with serologically determined anti-MSP-142 IgG antibody and responses measured at each time point within each assay. Generalized Estimating Equation (GEE) was utilized to examine repeated immune correlates of protection dichotomous values over time from the same individual. Mixed linear model was utilized to examine repeated immune correlates of protection continuous values over time from the same individual.Quantitative differences between experimental groups were evaluated by paired 42 IgG ELISA , 46% for overall GIA, 41% for MSP-119-specific IIA, 67% for T cell 3D7 MSP-142 specific IFN-γ production, and 10% for T cell FVO MSP-142 specific IFN-γ production. T cell responses to FVO MSP-142 were disproportionately low in this population and not evaluated further. Thirty-three percent of the positive immune outcomes were observed coincidental with a P. falciparum-positive blood smear.Ninety-six determinations  were made for each of the four tests. The percent positive outcomes for each test were 70% for MSP-142 stimulated IFN-γ production indicate fair to moderate temporal stability over the time intervals examined. In contrast, kappa scores for serologically determined IgG antibody to FVO MSP-142 indicate essentially substantial stability for all time intervals tested. IgG antibody to 3D7 MSP-142 shows good stability over three- and six-week intervals, which decreased over the six-month interval. GIA appears to be moderately stable over time intervals as long as six months. 3D7 MSP-119-specific IIA demonstrates fair stability at three-week intervals that disappears at longer time intervals. In summary, IgG antibody to 3D7 and FVO MSP-142 measured serologically by ELISA were the most stable over time, followed by 3D7 MSP-142 IFN-γ responses and GIA. MSP-119 IIA was the least stable over time with essentially no consistent response lasting more than three weeks.The kappa test of agreement between paired samples was used to determine the stability of immune measurements over four time intervals: three weeks, six weeks, six months, and nine months. Immune assay results were expressed as positive or negative using criteria described in the methods section. Kappa test establishes the percent agreement of responses beyond chance such that consistent positive responses and/or consistent negative responses generate higher kappa scores. A kappa score of 0.81–1.00 was regarded as "almost perfect" and considered to reflect consistent positive or negative results between paired samples over a specified time interval. A score of 0.61–0.80 was regarded as "substantial agreement." A score of 0.41–0.60 was regarded as "moderate agreement," a score of 0.21–0.4 as "fair agreement", and a score <0.20 as "slight agreement ,27." Fig19 IIA and time intervals, allowing for estimation of stability of repeated observations for each individual. Only MSP-119 IIA showed a significant change from August–October 2004 versus April–May 2005, as illustrated in Figure 42 IgG antibody determined by serology or T cell IFN-γ responses were seen in these individuals over time using the GEE model.The GEE final model included categorical data for blood smear positivity, sex, IFN-γ response, GIA, MSP-142 IFN-γ, 3D7 and FVO MSP-142 IgG, and GIA responses did not change markedly with time, substantiating results found with GEE analysis. MSP-119 IIA was associated with time increasing 0.1548 units per week (p = 0.003). Results were not affected by sex or age. Little fluctuation over time of 3D7 or FVO MSP-142 IgG antibody was observed . However, MSP-119-specific IIA levels increased significantly  in the six-month interval between October 2004 and April 2005 . Transgenic P. falciparum in which FVO MSP-119 has been replaced by the P. chabaudi ortholog have not been constructed. Additionally, there was no correlation between GIA and MSP-142 IgG . A modest positive correlation was observed between the functional MSP-119 IIA and GIA results (R2 = 0.1512). This is not surprising since MSP-119 IIA is a component of the overall anti-malarial antibody activity measured by GIA.No correlation was observed between 3D7 MSP-142 IFN-γ correlations ranged from R2 0.001 – 0.81 depending on the time interval examined. 3D7 and FVO MSP-142 IgG correlations ranged from R2 0.001–0.81 and 0.77 – 0.95, respectively. MSP-119 IIA and GIA correlations ranged from R2 0.001–0.55 and 0.2 – 0.69, respectively, depending on the time intervals examined.Additional materials . The average parasite density during both time intervals was <100 asexual parasites/μl blood. None of the participants experienced fever or other symptoms of malaria at any time during the 9-month observation period.Rainfall was used to estimate transmission intensity since this climatic variable has previously been found to track with of Kenya ,29. CumuP. falciparum MSP-1 has four predominant alleles based on point mutations resulting in four non-synonymous amino acid changes: E-TSR (3D7/PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (FVO/Wellcome type), and Q-TSR (Indo type). From August to October 2004, 41 of 48 samples were tested for MSP-119 haplotypes. By the MSP-119 PCR/LDR-FMA method, six positive samples had five E-KNG haplotypes and two Q-KNG haplotypes (one sample had both the E-KNG and Q-KNG haplotypes). From April to May 2005, 48 samples were tested. Nine positive samples were detected by MSP-119 PCR/LDR-FMA including three E-TSR, three E-KNG, and three Q-KNG haplotypes. All of those with the E-TSR (3D7) haplotype were found between April and May 2005 which coincided with increased MSP-119 IIA levels (E-TSR/3D7). In contrast, IgG antibody to the E-TSR/3D7 haplotype measured by serology did not increase.The 19 kDa C-terminal fragment of There was no correlation between T cell IFN-γ responses and antibody responses as determined by correlation coefficients, kappa test, and GEE at individual time points or over time.Plasmodium species. It is anchored to the outer membrane of the merozoite by glycophosphatidylinositol, and is considered to be essential to the cyclical process of erythrocyte invasion by malaria parasites. The msp-1 gene is conserved in the genomes of all Plasmodium species described to date, and the inability to delete the gene is consistent with the notion that the protein has an essential and non-redundant role in blood stage infection [P. falciparum MSP-1 have shown that induction of immunity to this single merozoite ligand can mediate protection against blood stage infection manifest as reduced or absent parasitaemia and reduced severity of anaemia [MSP-1 is among the most abundant merozoite surface proteins of nfection . Studies anaemia ,6,8,10.  anaemia ,31-33. T42 proteins measured by ELISA [19-specific IIA [42 are fair to moderately stable; 2) IgG antibodies to the FVO and 3D7 alleles of MSP-142 measured serologically by ELISA are most stable over time; and 3) functional measures of MSP-119 specific IIA is a transient but potentially sensitive indicator of antibody-mediated immunity that may reflect recent exposure to P. falciparum with the corresponding MSP-119 haplotype.Four immune assays were chosen for study based on previous observations suggesting that they may correlate with protection against parasitaemia and/or clinical morbidity: malaria antigen specific T cell IFN-γ measured by ELISPOT ,34-37, Iby ELISA ,6,8,10, ific IIA , and GIAific IIA ,38. BaseP. falciparum blood stage antigens in comparison to those living in low transmission regions or who experience few malaria episodes [42 were fair to moderately stable over the time intervals assessed is consistent with this observation. Similarly, in a repeat cross-sectional study of adults and children in western Kenya, IFN-γ T cell responses to LSA and TRAP peptides in adults were not stable over a nine-month time interval [per se may alter T cell responses since blood stage infection has been associated with reduced IFN-γ responses [P. falciparum by blood smear, although infection status had no correlation with IFN-γ ELISPOT results when analysed by GEE. In addition to extending the current study design to children and infants, a larger sample size may be needed to detect small but significant associations. Future studies that include flow cytometry to measure production of other cytokines in addition to IFN-γ, multifunctional T cells, and T cell memory subsets will be needed to define more clearly the contribution of merozoite antigen-specific T cells in mediating naturally acquired and vaccine-elicited immunity.Previous observations indicate that residents of high transmission regions have attenuated cellular responses to episodes ,40. The interval . The lacinterval . The paresponses . In the 19-specific IIA) can have arbitrary cut-off values to define a positive or negative response. Vaccine studies have often used a GIA cut-off of 15% for positive responders, as the case in this study. On the other hand, cut-off thresholds for MSP-119-specific IIA have not been well defined. In a previous analysis, high levels (upper quartile) of MSP-119 IIA were associated with protection in Kenyan adults living in a highland area where malaria transmission is low and unstable [19 IIA levels are frequently low or absent in Kenyans living in the nearby holoendemic area where the current study was performed (manuscript in preparation).The kappa test is an objective evaluation of agreement of an experimentally determined immune variable over defined time intervals. An inherent bias in interpreting the data described here  is the criteria used to determine a positive or negative immune response. Widely accepted criteria to differentiate positive and negative responses for ELISPOT and ELISA assays were used in this study. Functional antibody assays  allele but also other polymorphic merozoite proteins is warranted.The results of this study showing an increased level of MSP-1P. falciparum. Functional antibody measurements of MSP-119-specific IIA may be a transient indicator of recent haplotype-specific infection. Overcoming the temporal instability of immune responses generated against MSP-1 beyond those demonstrated by clinically immune adults may be necessary to develop a protective blood stage vaccine.These data underscore the lack of temporal stability of current assays that measure humoral and cellular immune responses to MSP-1 in adults with naturally acquired immunity to IFN: interferon; MSP: merozoite surface protein; IIA: invasion inhibition antibodies; GIA: growth inhibitory activity; PBMC: peripheral blood mononuclear cells; SFU: spot forming units; AU: arbitrary units; HO: Hoechst; PCR/LDR-FMA: PCR ligase detection reaction fluorescent microsphere assay; GEE: generalized estimating equation; ANOVA: analysis of variance.The authors declare that they have no competing interests.AED carried out antibody assays, data analysis and drafted the manuscript. KC carried out cellular assays. POS was responsible for participant recruitment and study coordination. MDS carried out cellular assays and participated in study design. BSC developed assays and manuscript preparation. AMM participated in study conception, study design, and manuscript preparation. DJT performed data analysis. JWK participated in study conception, study design, and helped to draft the manuscript. All authors read and approved the final manuscript.42 (3D7) stimulated INF-γQuantitative data for MSP-1. Correlation matrices and spaghetti plots for MSP-142 (3D7) stimulated INF-γ at all time pointsClick here for file42 (3D7) ELISA measured responsesQuantitative data for MSP-1. Correlation matrices and spaghetti plots for MSP-142 (3D7) ELISA measured responses at all time pointsClick here for file42 (FVO) ELISA measured responsesQuantitative data for MSP-1. Correlation matrices and spaghetti plots for MSP-142 (FVO) ELISA measured responses at all time pointsClick here for file19 IIA measured responsesQuantitative data for MSP-1. Correlation matrices and spaghetti plots for MSP-119 IIA measured responses at all time pointsClick here for fileQuantitative data for GIA measured responses. Correlation matrices and spaghetti plots for GIA measured responses at all time pointsClick here for file


Plasmodium falciparum malaria in pregnancy presents an enormous diagnostic challenge. The epidemiological and clinical relevance of the different types of malaria diagnosis as well as risk factors associated with malaria infection at delivery were investigated.In sub-Saharan Africa, P. falciparum in peripheral blood, placental blood and placental tissue sections by microscopy. Information relating to the use of intermittent preventive treatment in pregnancy with sulphadoxine/pyrimethamine, history of fever attack, infant birth weights and maternal anaemia were recorded.In a cross-sectional survey, 306 women reporting for delivery in the Mutenegene maternity clinic, Fako division, South West province, Cameroon were screened for P. falciparum infection was detected in 5.6%, 25.5% and 60.5% of the cases in peripheral blood, placental blood and placental histological sections respectively. Placental histology was more sensitive (97.4%) than placental blood film (41.5%) and peripheral blood (8.0%) microscopy. In multivariate analysis, age (≤ 20 years old) , history of fever attack  were significant risk factors associated with microscopically detected parasitaemia. The use of ≥ 2 SP doses  was associated with a significant reduction in the prevalence of microscopic parasitaemia at delivery. Age (>20 years)  was the only significant risk factor associated with parasitaemia diagnosed by histology only in univariate analysis. Microscopic parasitaemia  was a significant risk factor for maternal anaemia at delivery, but neither infection detected by histology only, nor past infection were associated with increased risk of anaemia.Among these women, Placenta histological examination was the most sensitive indicator of malaria infection at delivery. Microscopically detected parasitaemia was associated with increased risk of maternal anaemia at delivery, but not low-grade parasitaemia detected by placental histology only. Plasmodium falciparum infection during pregnancy is a serious public health problem in sub-Saharan Africa. One woman in four has evidence of placental infection at the time of delivery [Plasmodium falciparum erythrocyte membrane protein 1 (var2csa PfEMP1) is the principal chondroitin sulfate A (CSA) binding ligand mediating placental sequestration of IEs [Malaria due to delivery ,2. It isdelivery -5. Plasmn of IEs . Consequn of IEs , to highn of IEs . Moreoven of IEs .Placental malaria (PM) is one of the major features of malaria during pregnancy and has been widely used as a standard indicator to characterize malaria infection in epidemiologic investigations . A numbePlacental histology is considered the "gold standard" of malaria diagnosis in pregnancy for epidemiological or biological study purposes, because it can show signs of active (presence of IEs only in the IVS), active chronic (presence of IEs and pigment in monocytes) or past  infections ,22. HoweP. falciparum infections detected by blood smear microscopy, histology only and past infection, as well as risk factors associated with malaria infection at delivery.This study: i) determined and compared malaria infection at delivery in maternal peripheral blood, placental blood and placental histological sections; ii) assessed the epidemiological and clinical significance of The study was conducted in the Mutengene health area from March to October 2007. Mutengene is a semi-urban, road-junction town, located in the Mt Cameroon region, Fako Division of the South West Province of Cameroon. The Mutengene medical centre is the only government-owned institution that offers antenatal care, preventive and curative services at affordable cost in the health area. This town is located at about 220 m above sea level and has a heterogeneous population of approximately 40,000 inhabitants who originate mainly from neighbouring provinces in search of its fertile farmland and business opportunities ,23.The Mt Cameroon region has an equatorial climate made up of a long rainy season that starts in March and ends in October with maximum rainfall in August and September. The dry season starts in late October and ends in February. The mean value of the minimum temperature varies between 20.0°C in December and 18.0°C in August and the mean value of the maximum temperature ranges between 35.0°C in February and 30.0°C in March . MutengeP. falciparum being the predominant malaria parasite species [The Mt Cameroon region is hyperendemic for malaria, with  species . A previ species . In addi species . Intermi species . The stuPregnant women reporting for delivery at the Mutengene maternity health centre and who consented to participate in the study were enrolled. Expectant mothers with evidence of chronic illness and complicated pregnancy  were not eligible and cases of multiple pregnancies were excluded. All maternal and infant characteristics  were documented using a standard questionnaire. IPT/SP during pregnancy is administered during antenatal visits under the supervision of a midwife who records and signs in patients' antenatal case report book. SP dosage and compliance and history of fever attack were confirmed from the patient's medical record book and by personal interview. Human immunodeficiency virus (HIV) infection status was known for mothers recruited. Women in this setting are offered routine confidential HIV testing at first antenatal clinic (ANC) visit, but testing was done at delivery for women who had no antenatal follow-up or had enrolled in maternity clinics out of the study area with no ANC cards. We did not obtain informed consent to publish data on HIV status of the participants.3) excised from the centre of the placenta and used to prepare impression smears. A larger biopsy specimen of placental tissue (2 by 2 by 1 cm) was fixed in 10% neutral buffered formalin for histopathological studies.Maternal peripheral venous blood (2 ml) was collected by venopuncture within 24 hours of parturition and used to prepare thick blood films. Immediately following delivery, the placenta was obtained and a small piece of placental tissue . Fixed placental biopsies were transferred to the Anatomy and Pathology (ANAPATH) Laboratory, University of Yaoundé I Teaching Hospital (CHU), Yaoundé, Cameroon, where they were processed, embedded in paraffin wax and sectioned onto slides by standard techniques. Sections were later stained with haematoxylin-eosin stain for detection of active and past infections. Microscopic examination of blood smears was done under oil immersion for parasite detection and 200 high power fields were examined before the smear was considered negative. Parasites were counted against 200 leucocytes assuming an average leucocyte count of 8,000 per microlitre of blood . To detePlacental histological sections were first examined by the one of the investigators (JKA) and later confirmed by a more experienced histopathologist (BN) without knowledge of the blood film microscopy results. One thousand intervillous cells (IVS) were counted to determine the level of parasitaemia in placenta tissue sections. Past infection was defined as the presence of malaria pigment in fibrin or monocyte/macrophage without malaria parasites . HistoloHeparinized, microhaematocrit tubes were filled with a sample of peripheral blood and centrifuged using a microhaematocrit centrifuge. The haematocrit (packed cell volume (PCV) was read using haematocrit reader (Hawksley). Haemoglobin concentration was calculated from PCV values as described by Topley . Anaemia2) test, odds ratio (OR) test in univariate analyses and logistic regression in multivariate analyses. Differences in group means were assessed using ANOVA, Mann-Whiney U test or Kruskall Wallis test. Age and parity was categorized as follows: age  years and parity , use of SP , fever history . Statistical significance was set at p ≤ 0.05.All data were entered and analysed using SPSS version 11.5. Malaria infection at delivery was classified as microscopic parasitaemia detected by blood smear microscopy, parasitaemia by histology only, past infection and no malaria. Associations of the different types of malaria diagnosis with age group, parity, use and doses of IPTp-SP and history of fever attack were evaluated using Pearson Chi-Square  years, where primiparae (19.5 ± 2.4) were significantly younger (p < 0.001) compared with secundiparae (22.6 ± 3.0) and multiparae (28.2 ± 4.6). Among the women, 90.1% (272/302) reported taking IPTp-SP during pregnancy. For women with number of doses recorded, 47.8% (129/270) had taken one dose, 52.2% (141/270) took two or more doses and 9.9% (30/302) did not take SP during the entire pregnancy period. Peripheral blood was collected from 287. Reported history of fever was 43.6% (125/287). One hundred and seventy-six  women were anaemic at delivery and the prevalence of low birth weight was 5.6% (17/306). The prevalence of HIV infection among women recruited was 5.6%; (17/306).P. falciparum infection, defined as the presence of malaria parasites as detected in thick peripheral blood, placental impression blood films or placental tissue sections. Of the active infections, 44.2% (53) were detected by histology only and 55.8% (67) by blood smear microscopy and histology. Of the 82(26.8%) women with past infection on histology, 12 were positive for malaria parasites on blood smear microscopy  thus the overall prevalence of past infection was 22.9% (70). The cumulative prevalence of malaria infection at delivery .Sixteen women  had malaria parasite infection diagnosed by peripheral blood microscopy. Seventy-eight  had placental infection diagnosed by placental blood smear examination. Of the 25% diagnosed by placental blood smear, 19.6% (60/306) were with active infection while 5.9% (18/306) had past infection. Placental histological examination detected 185  placental malaria positive women. Of these, 33.7% (103) had active placental infections while 26.8% (82) had past infection. Overall, 39.2% (120/306) had active Placental histology detected all but three of the placental infections detected by placental blood smear examination, as well as all but two of the malaria infection detected by peripheral blood smear; giving a sensitivity of 97.4%. One hundred and ten women with histological evidence of placental infection were missed on placental blood smear, which had a sensitivity of 41.5%. One hundred and sixty-one women positive by placental histology were missed on peripheral blood examination, which had a sensitivity of 8%  in primiparous (31.6%) compared to secundiparous (21.1%) and multiparous (17.4%) women. The presence of infections detected by histology only (low-grade parasitaemia) was significantly associated with age (>20 years)  where infections detected by histology only were more common  in women > 20 years old (20.8%) than in those ≤ 20 years (8.5%). Parity, SP dosage, or fever history were not associated with the prevalence of low-grade infection.Microscopically detected parasitaemia was the only clinically relevant malaria infection at delivery and factors influencing its presence were analysed Table . In multThe mean birth weights of babies did not differ with malaria infection status, while mean haemoglobin concentrations were significantly lower in women with microscopically detected infection compared to uninfected women Table . SimilarThis study determined the prevalence of malaria infection and parasite density in Cameroonian parturient women using maternal peripheral blood microscopy, placental blood microscopy and placental histology. The epidemiological and clinical relevance of the different types of malaria diagnosis as well as some risk factors associated with malaria infection were investigated.P. falciparum infection during pregnancy. Using blood smear microscopy, 21.9% of the women were found to harbour malaria parasites at delivery similar to findings reported in Yaoundé [P. falciparum histidine-rich protein 2 (HRP-2) [P. falciparum-specific DNA for detection of low-level parasitemia or circulating genetic material [Plasmodium falciparum [Overall, pregnant women in this semi-urban area of Cameroon were frequently (62.1%) exposed to  Yaoundé ,19. Mala Yaoundé ,16,30 an (HRP-2) ,31 and Pmaterial  comparedlciparum .There was a significant positive correlation between parasitaemia levels determined by placental blood smear and tissue sections , the median placental parasitaemia levels being higher in placental blood smears (0.83%) compared to that detected in placental tissue sections (0.38%). Two concepts are suggested. Firstly, impression smears correlate quite well with histology. Secondly, infection detected only by histology is lower level and potentially less significant than infection found on impression smears.P. falciparum to low parasite densities in areas of high and stable transmission [It is well established that primigravidae are indisputably at greater risk of malaria infection during pregnancy in stable malaria regions . In the smission . The relsmission , but at It is generally assumed that in areas of stable transmission, parasitaemic pregnant women are rarely symptomatic, and that severe disease or death from malaria is extremely unusual . HoweverP. falciparum infection in both peripheral and placental blood and increased risk of anaemia. On the contrary, in Ghana, as reported by Mockenhaupt et al [Women with blood smear microscopically detectable malaria were likely to be anaemic and had a mean haemoglobin concentration of 0.98 g/dl lower than that of uninfected women. Similar to findings reported elsewhere ,19,31 blpt et al , submicrpt et al . Anaemiaet al [In Malawi, Rogerson et al  showed tet al . This maet al . There iIt is not surprising that women who reported to have received one or more doses of SP had a decreased risk of microscopic parasitaemia at delivery than those who had no SP during pregnancy. Furthermore, two or more doses of SP significantly reduce the prevalence of microscopic parasitaemia among pregnant women similar to findings of previous studies ,45. The In summary, malaria in pregnancy was most sensitively diagnosed by placental histology. Younger age and history of fever attack during pregnancy were independent significant risk factors associated with microscopic parasitaemia at delivery. Blood smear microscopy identified a subset of women with malaria who had a high risk of anaemia whereas low-grade parasitaemia detected by histology only and past infection were not associated with maternal anaemia at delivery. Improving IPTp-SP coverage and adherence will substantially reduce malaria parasitaemia and maternal anaemia in pregnant women in this area. Proper diagnosis as well as ensuring appropriate and timely treatment of symptomatic malaria in pregnancy is recommended.The authors declare that they have no competing interests.JKAK: Conception and design of study, data collection, analysis, interpretation and manuscript preparation. EAA: Supervision and critically reading of manuscript for important and intellectual content. NB: Contributed in placental pathohistological studies. ESE: Design of study and critically reading of manuscript for important and intellectual content; MTB: Supervision and revision of manuscript. All authors read and approved the manuscript.


Illumination was performed at 1, 2, 3, 4, 6 or 12 h after ALA administration with a 1-cm cylindrical diffuser placed in a balloon catheter . During illumination, fluorescence measurements and light dosimetry were performed. Animals were sacrificed at 48 h (n= 4) or 28 days (n= 4) after PDT. At day 28, an oesophagogram was performed. Largest PpIX fluorescence was found at 3 h after ALA administration. In vivo fluence rate was three times higher than the calculated incident fluence rate. At 48 h after PDT, major epithelial damage was found in all animals illuminated at 2 h, whereas less epithelial damage was found at 3–6 h and none at 1 and 12 h. In animals illuminated at 4, 6 and 12 h, but not at 2 h, oesophagograms showed severe dilatations and histology showed loss of Schwann cells. These results demonstrate that the choice of time interval between ALA administration and illumination is critical for achieving epithelial damage without oesophageal functional impairment. A short interval of 2–3 h seems to be most appropriate. © 1999 Cancer Research Campaign5-Aminolaevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. This study aimed to optimize the time of illumination after ALA administration. Six groups of eight rats received 200 mg kg


Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3−, CD45RO+ CD4+ T cells which coproduce IL-10 and IFN-γ. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation.Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4 P. falciparum malaria infections, suggesting that this component of the immunoregulatory arsenal may be rapidly overwhelmed by virulent infections. Further, we identified, for the first time in an acute human infection, a population of IL-10-producing Th-1 effector cells and found that IL-10-producing Th-1 cells were associated with development of uncomplicated as opposed to severe malaria, leading us to suggest that such “self-regulating” Th-1 cells may contribute to clearing malaria infections without inducing immune-mediated pathology. In addition, we found evidence that malaria-induced Tregs may limit the magnitude of malaria-specific memory responses detectable 28 days later, which may reduce the risk of immune-mediated pathology upon reinfection and may explain how immunity to severe disease can be gained after as little as one or two infections. We conclude that vaccines designed to induce cell-mediated responses should be assessed for their ability to induce IL-10 producing Th-1 cells and Tregs.While Tregs have been implicated in regulation of the immune response to chronic infections, their potential in determining disease outcome in acute infections is unclear. In this study we have found that Tregs are unable to control the florid inflammation during acute, severe  P. falciparum infection ranges from asymptomatic parasite carriage to a febrile disease that may develop into a severe, life-threatening illness. The factors that determine disease severity are not completely understood but are likely to include both parasite and host components The clinical spectrum of P. falciparum malaria While there is a growing consensus that killing of malaria parasites or malaria-infected red blood cells requires the synergistic action of antibodies and cell-mediated immune responses +CD25hiFOXP3+CD127−/lo regulatory T cells (Tregs) in determining the outcome of malaria infection. Induced and/or activated in response to malaria infection P. yoelii, ablation of Treg activity by depletion of CD25+ cells either allowed mice to control parasitaemia and survive + cells of BALB/c mice infected with either P. berghei NK65P berghei ANKA+ T cell-depleted BALB/c mice with P. chabaudi adami DS led to increased parasitaemia and more severe anaemia + T cell depletion around the time of parasite inoculation reduced the incidence of experimental cerebral malaria in C57BL/6 mice infected with P. berghei ANKA in two independent studies Given these associations between severe disease and exacerbated immune pathology, a number of studies have explored the role of CD4P. falciparum sporozoite infection showed an increase in FOXP3 mRNA expression and expansion of Tregs 10 days after infection; Treg induction correlated with high circulating levels of TGF-β, low levels of pro-inflammatory cytokines and rapid parasite growth Malaria naïve individuals undergoing experimental −, CD45RO+, CD4+ T cells which co-produce IL-10 and IFN-γ and which are more prevalent in children with uncomplicated malaria than in those with severe disease. We suggest that these IL-10 producing effector T cells may contribute to clearing malaria infection without-inducing immune-mediated pathology.To investigate the role of Tregs during clinical malaria infection, we have compared cellular immune responses of children with either severe or uncomplicated malaria. Interestingly, although we did not observe any significant differences in Treg numbers or function between severe and uncomplicated malaria cases, our data do indicate that malaria-induced Tregs may limit the magnitude of malaria-specific Th1 memory responses and thus moderate pro-inflammatory responses to subsequent infections, providing a possible explanation for the very rapid acquisition of immunity to severe malaria. Moreover, we have identified a population of FOXP3P. falciparum malaria were compared with those of 65 children with uncomplicated clinical malaria and with 20 healthy (control) children of similar age and recruited from the same study area at the same time ), and – as has been observed previously Immune responses of 59 Gambian children with severe ame time . On admid groups . ParasitA) and those suffering only from severe prostration (grouped as SB). Data were analysed using linear regression, with a random effect to allow for the within subject measurements over time, adjusting for age, sex, duration of prior symptoms and numbers of clones causing the infection. Due to the multiplicity of comparisons that were made within the model, resulting from multiple responses and multiple comparisons within response, hypotheses rejected with a probability of less than 0.012 have a false discovery rate of 5% For statistical analysis, patients were classified as uncomplicated or severe cases, with the latter being further subdivided into those patients suffering from cerebral malaria (CM), severe anaemia (SA) or severe respiratory distress (SRD)  and in healthy control children; on average, 2–3% of CD4+ T cells expressed the regulatory phenotype -driven lymphocyte proliferation. Anti CD25 treatment removed approximately half  of the CD4+ T cells that were FOXP3+CD127−/low and this was associated with a 1.76 fold and 1.57-fold (geometric means) increase in PfSE-induced lymphoproliferation in severe and uncomplicated cases, respectively, with no significant difference between the groups , no difference was observed between those with severe and those with uncomplicated malaria (data not shown).Next, since reduced expression of FOXP3 mRNA and protein expression Finally, since high concentrations of TNF-α have been reported to impair Treg activity  over Tregs among the various groups. As shown in T-BET/FOXP3 ratio was significantly higher during acute disease than during convalescence and a similar, albeit not significant, trend was observed for the ratio of T-effector/Tregs (data not shown). Moreover, since the absolute number of circulating T-effector cells was significantly higher in severe cases than in uncomplicated cases , the T-effector/Treg ratio tended to be higher among severe cases than uncomplicated cases on day 0 (p = 0.039) and a similar trend was seen for the T-BET/FOXP3 ratio (p = 0.058). The ratio of FOXP3 to GATA-3 (Th2 lineage factor) mRNA was similar for both time points in all groups (data not shown) but the Th1/Th2 ratio (T-BET/GATA-3 mRNA) was significantly higher during acute disease in children with CM, SA or SRD compared to those suffering from severe prostration (p = 0.0075), indicating that the expansion of the T-effector population is biased towards Th1 responses.Since the balance of T-effector to Treg responses is likely to be as important, or more important, than the absolute levels of either These data confirm previous studies indicating a shift towards a more inflammatory response during acute and severe malaria but, significantly, our data extend the previous observations by revealing that this inflammation is not balanced by a commensurate increase in Treg function. Indeed, our data strongly suggest that the potent inflammation induced during an acute malaria infection overwhelms the normal homeostatic capacity of the immune system and, in particular, that the Treg response in children with severe malaria is insufficient to balance a much stronger Th1 effector response.IL-10 and IFN-γ were also significantly elevated in all groups during acute disease, but there was no significant difference between severity groups , suggesting that IFN-γ production is being balanced by IL-10 production. Interestingly, IFN-γ mRNA levels on day 0 correlated with FOXP3 mRNA on day 0 for both severe (r: 0.39 p = 0.003) and uncomplicated cases (r: 0.44 p = 0.0001), suggesting that IFN-γ may also be driving FOXP3 expression.To investigate further the dynamics of pro-inflammatory/regulatory responses during clinical malaria, plasma concentrations and mRNA transcripts of inflammatory  and regulatory cytokines (IL-10) were assayed. In accordance with previous observations, plasma concentrations of IFN-γ, TNF-α and IL-10 were all significantly higher during acute disease than during convalescence, with significantly higher levels in severely ill children compared to uncomplicated cases . Levels y groups . For botIFN-γ to IL-10 mRNA were similar in all disease severity groups both on day 0 and day 28  producing both cytokines simultaneously  among PBMC collected from 34 of our convalescent malaria patients (19 severe and 15 uncomplicated) on Day 28.It has been reported that Tregs present at the time of infection FOXP3 mRNA levels measured on day 0, a linear by linear hyperbolic fit revealed that higher levels of FOXP3 mRNA on day 0 were highly significantly (p = 0.009) associated with lower malaria-specific IFN-γ memory responses on Day 28, suggesting that Tregs induced during the acute infection may limit the magnitude of subsequent Th1 responses (As shown in esponses . For nei+ CD127−/lo FOXP3+) regulatory T cells. In partial support of these hypotheses, the number of cells expressing a Treg phenotype and FOXP3-mRNA levels were both significantly higher during convalescence than during the acute clinical episode and the ratio of the Th1 transcription factor T-BET to the Treg transcription factor FOXP3 was significantly higher during acute disease than during convalescence in both severe and uncomplicated cases, compatible with the notion that Tregs fail to sufficiently regulate pro-inflammatory responses which might contribute to the onset of symptomatic malaria infection.We hypothesized that the balance of inflammatory to regulatory immune responses would be biased towards a more inflammatory response in children with severe malaria than in children with uncomplicated malaria, that this balance would be restored during convalescence and – crucially – that this would be associated with differences in the proportion, absolute number or function of circulating classical  in severely ill children than in children with uncomplicated disease, suggesting that the classical FOXP3P. yoelii-infected mice + CD4+ T cells (that are CD25− and FOXP3−) and not classical Tregs are the only substantial source of IL-10 during acute malaria infection. This observation is reminiscent of that made by Nylen + T cells were simultaneously producing IFN-γ, identifying them as Th1 cells. Although IL-10 secreting Th1 cells have been described recently in two murine models of toxoplasmosis + T cell population was significantly higher in children with uncomplicated malaria than in children with severe malaria suggesting that in human P. falciparum infection, as in murine T.gondii infections IL-10 is well-established as a vital homeostatic regulator of malaria-induced inflammation that prevents immune-pathology in mice P. falciparum infections we did find evidence that they affect the magnitude of the malaria specific memory response induced by the current infection. A similar observation has been made in P. berghei ANKA-infected mice; animals that were depleted of CD25+ cells prior to infection and drug-cured on day 5 developed significantly stronger IFN-γ memory responses on day 14 than did intact infected/cured mice, and these mice also developed much more severe, and frequently fatal, clinical symptoms upon reinfection, despite more efficient parasite clearance Although Tregs may not seem to determine the outcome of current + Tregs are unable to control the florid inflammation that accompanies acute malaria infections and this component of the immunoregulatory arsenal is rapidly overwhelmed in children with either mild or severe malaria. Importantly however we have identified, for the first time in an acute human infection, a population of IL-10 producing Th1 effector cells which appear to be a major source of this key anti-inflammatory cytokine during acute malaria infection, and which are associated with development of uncomplicated as opposed to severe malaria. We propose that IL-10-producing Th1 cells may be the essential regulators of acute infection-induced inflammation and that such “self-regulating” Th1 cells may be essential for the infection to be cleared without inducing immune-mediated pathology. Moreover, we have found evidence in support of the hypothesis that Tregs limit the magnitude of the Th1 memory response raising the intriguing possibility that they may play an important role in the rapid evolution of clinical immunity to severe malaria.In summary, our data indicate that classical FOXP3A case-control study was conducted in Gambian children with severe or uncomplicated malaria, resident in a peri-urban area within a 40 km radius south of the capital, Banjul, with low levels of malaria transmission On admission (D0) and after 4 weeks (D28±3 days) one ml of blood was collected in RNA stabilizing agent  and a maximum of 4 mls of blood (mean: 3.2 mls CI 95%: 3.1–3.3 mls) were collected into heparinized vacutainers® (BD). All patients received standard care according to the Gambian Government Treatment Guidelines, provided by the health centre staff. The children's health was reviewed 7 days after admission. The study was reviewed and approved by the Joint Gambian Government/MRC Ethics Committee and the Ethics Committee of the London School of Hygiene & Tropical Medicine .P. falciparum parasites were identified by slide microscopy of 50 high power fields of a thick film. Full differential blood counts were obtained on days 0 and 28 using a Medonic™ instrument ; the presence of intestinal helminths was assessed by microscopy from stool samples collected into BioSepar ParasiTrap® diagnosis system, following the manufacturers' instructions. Sickle cell status was determined by metabisulfite test and confirmed on cellulose acetate electrophoresis ex-vivo, or cultured in RPMI 1640 containing 10% human AB+ serum, 100 µg/ml streptomycin, 100 U/ml penicillin , and 2 mM L-glutamine (Invitrogen Life Technologies), referred to as complete growth medium (GM).Blood samples were processed within 2 hours of collection. Plasma was removed, stored at −80°C and replaced by an equal volume of RPMI 1640 (Sigma-Aldrich). PBMC were isolated after density centrifugation over a 1.077 Nycoprep  gradient  and washed twice in RPM 1640. Cells were either stained for flow cytometry directly Fresh PBMC were stained using the following fluorochrome labeled mouse or rat anti-human antibodies: FITC anti-TNF-receptor II (R&D), PE anti-FOXP3 (clone PCH101), Pacific Blue anti CD3, APC-Alexa Fluor 750 anti-CD127 , APC anti-CD25, PerCP anti CD4 (BD systems), ECD anti-CD45R0 (Beckman-Coulter), and appropriate isotype controls.P. falciparum (D0) was assessed after 5 hours stimulation in GM containing PMA (50 ng/ml) and Ionomycin (1000 ng/ml) or GM alone.IL-10 and IFN-γ production by PBMC from 30 children with acute Cells were stained with FITC anti-IFN-γ, PE anti-FOXP3, PE-Cy7 anti-CD25, APC-AF750 anti-CD8, Pacific Blue anti-CD3 , PerCP anti-CD4, APC anti-IL-10 (both BD), and ECD anti-CD45R0 (Beckman-Coulter). To ascertain specificity of the intracellular cytokine staining, aliquots of some samples were incubated with saturating amounts of purified non-labelled antibody of the same clone prior to staining with the fluorochrome labeled ICS antibody. The FOXP3 staining buffer set (Ebioscience) was used following the manufacturer's protocol. Samples were acquired on a 3 laser/9 channel CyAn™ ADP flowcytometer using Summit 4.3 software (Dako). Analysis was performed using FlowJo (Tree Star Inc.). All flowcytometric analysis was performed at the MRC laboratories, The Gambia on freshly isolated cells.Plasma concentrations of IFN-γ, TNF-α and IL-10 were determined for each subject and time point on the Bio-Plex® 200 system, using X-Plex™ assays (both Bio-Rad Laboratories), according to the manufacturer's instructions. Data were analysed using the Bio-Plex® Manager software. The detection limit was defined as the concentration corresponding to a fluorescence value above the mean background fluorescence in control wells plus 3 SD, being 8.76 pg/ml for IFN-γ, 5 pg/ml for TNF-α and 0.57 pg/ml for IL-10. Values below this threshold were set to these levels.P. falciparum parasites (3D7 strain) were cultured in vitro as described hi cells or mock depleted using magnetic beads , at a bead to PBMC ratio of 7∶1, and cell proliferation was determined by [3H]-thymidine  incorporation after 6 days in culture with PfSE, uRBC , GM, or 2 days culture with PMA (10 ng/ml)+ Ionomycin (100 ng/ml), as described PBMC from 10 severe and 10 uncomplicated malaria cases collected on day 0 were depleted of CD255 cells seeded into duplicate wells onto Millipore MAIP S45 plates and restimulated overnight with PfSE, uRBC (concentrations as above), GM or PHA-L (5 µg/ml). IFN-γ ELISpot was performed using MabTech antibodies according to the manufacturer's instructions. Spot forming cell numbers were counted using an ELISPOT plate reader . Results are expressed as spot forming units (SFU) per million PBMC after subtraction of individual background values  being deducted. Assays were discounted if the positive control (PHA-L) was <50 SFU, or the negative control was >30 SFU.Cultured ELISPOTs were performed to assess malaria specific IFN-γ memory responses, adapting an established method FOXP3, IL-10, SOCS-2 and IFN-γ were measured by RT-PCR on a DNA Engine Opticon® (MJ Research) with QuantiTect SYBR Green PCR kits (Qiagen Ltd) using primers  previously described: IFN-γ, IL-10; FOXP3 designed by SOCS-2 designed by For quantitative reverse transcription-polymerase chain reaction (RT-PCR), total RNA was extracted from PAX tubes following the manufacturer's instructions and reverse transcribed into cDNA using TaqMan® reagents for reverse transcription (Applied Biosystems), following the manufacturer's protocol. Gene expression profiles for T-BET and GATA-3 gene expression was determined using the TaqMan® Probe kit using the primers  designed by msp1 gene to assess the number of clones infecting each patient Genomic DNA from each parasite isolate was genotyped by sequencing the highly polymorphic block 2 region of the A or SB), time (day 0 and day 28) and their interaction were adjusted for the possible confounding effects of age, sex, duration of prior symptoms and numbers of clones causing the infection. Where there was no significant malaria group and time interaction, p-values for the overall comparison of day 0 vs. day 28 are given. Within day 0, comparisons of severe vs. uncomplicated and the two groups of severely ill patients (SA vs SB) were adjusted for any malaria group and time interactions. To allow for the multiplicity of tests resulting from multiple responses and multiple comparisons within a response performed in the model, a false discovery rate (FDR) of 5% was assumed. Using the Benjamini and Hochberg approach Analysis was performed using linear regression, with a random effect to allow for the within subject measurements over time, where the response variables were log transformed to improve the normality and constant variance assumptions. Significance (measured at the 5% level) tests for the effects of malaria group Click here for additional data file.


Plasmodium ovale in a 34-year-old man. As in the majority other described cases of splenic infarction, the course was spontaneously favourable, suggesting that this complication was relatively benign compared to splenic rupture, which is life-threatening and usually necessitating surgery.The splenic complications of acute malaria include two different prognostic and treatment entities: splenic infarction and splenic rupture. This is the first case of splenic infarction during an acute malaria due to  Plasmodium falciparum or Plasmodium vivax. Splenic rupture is another, less well-known life-threatening malaria complication. A case of splenic infarction during acute malaria due to Plasmodium ovale is reported. This is the first case described in the literature with this species. This case supplements the splenic complication panel of acute malaria, with two different prognostic and treatment entities: splenic infarction and splenic rupture.Splenic infarction is a rare complication usually described during acute malaria with A 34-year-old man, a French professional soldier, with no remarkable medical history, had two previous exposures to endemic malaria zones . He stated having taken his anti-malarial chemoprophylaxis  more or less regularly during his stays, having omitted to take the medication on several occasions. He never had acute malaria previously.P. ovale parasitaemia. The species diagnosis was confirmed by molecular biology (polymerase chain reaction), without bi-parasitism .In 2008, he developed a 39,5°C fever with headache, asthenia, myalgias, with nausea and vomiting. After a few days of self-treatment with paracetamol, he presented to the Emergency Department at the Military Hospital Legouest, Metz, France. Physical examination showed a 14 cm painless splenomegaly, and a temperature of 39°C. All other physical findings were normal. Laboratory tests showed anaemia (Hb 11.8 g/dl), thrombocytopaenia (platelets 117×109/mm3), leukopaenia (3250/mm3), lymphocytopaenia (839/mm3) and a moderate increase in liver enzymes . The diagnosis of acute malaria was made by blood smear examination, with 0.001% The patient was admitted and treatment was started with intravenous quinine because of vomiting (8 mg/kg three times a day), and was well tolerated. Fever subsided gradually over three days, anaemia resolved without transfusion and signs of hepatic cytolysis disappeared within a few days.Six days after his admission, the patient complained of a sudden onset of left hypochondrial pain. Physical examination showed persistent, painful and tender splenomegaly. Abdominal CT-scan Figure  showed sHis haemodynamic status was stable and simple analgesics were effective, obviating the need for emergency surgery. Management consisted only of medical monitoring and analgesia (subcutaneous morphine).The splenic abnormalities regressed spontaneously over several months, during which the patient continued to tire easily. Haemoglobin was 10.6 g/dl one month after initial onset. Exploration of other causes of splenic infarction, including constitutional underlying coagulation disorder , sickle cell disease  and concomitant Salmonella infection, were negative.Malaria is one of the main infectious threats for the French army. Engaged in short-term missions or outside operations, 35,000 to 40,000 French soldiers stay every year in zones of endemic malaria disease. For 10 years, the rate of malaria incidence in the French army has been between 1,9 and 5,8 percent of men annually, and unfortunately, four deaths have been reported.P. vivax, 36% to P. falciparum, 1.2% to Plasmodium malariae and 6.3% to P.ovale, the frequency of which is on the increase.In 2008, 413 acute malaria cases were declared in the French army. 92,6% of which were uncomplicated clinical forms. 56.4% of the cases were due to et al [P. falciparum (except one case of co-infected with P. vivax and one case unknown), without anti-malarial prophylaxis . They all survived with medical treatment . No death due to splenic complications is mentioned.Splenic infarction is a rarely described, and probably under-diagnosed, complication of malaria. Bonnard et al  found onP. ovale or P. malariae infection. In a recent litterature analysis, Imbert et al [P. ovale. This is a very serious, life-threatening complication, even if the two cases described show that the issue was not always fatal.Rupture of the spleen is more common than splenic infarction during malaria and is a diagnostic emergency, usually necessitating surgery. Pathological rupture of the spleen has rarely been reported following rt et al  found onInfarction-induced haemorrhage does not consistently fit available pathological data. Haemorrhages have been observed in splenic tissue devoid of signs of infarction, and infarction was noted in only two of 55 spleen rupture cases reported over the last 50 years.P. vivax and in a P. falciparum mixed infection and in an adult suffering from P. vivax malaria) [In the 10 cases of splenic infarction reported over the last 30 years, only two cases were complicated by splenic rupture  . For thiWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.GC, FB and CR wrote the manuscript. DP, CSR, DA, and BG carried out the physical examination. CR and SV carried out the biological analysis. All authors read and approved the final manuscript.


The World Health Organization recommends insecticidal bednets and intermittent preventive treatment to reduce malaria in pregnancy. Longitudinal data of malaria prevalence and pregnancy outcomes are valuable in gauging the impact of these antimalarial interventions.We recruited 8,131 women delivering in a single Malawian hospital over 9 years. We recorded demographic data, antenatal prescription of intermittent preventive therapy during pregnancy with sulfadoxine-pyrimethamine and bed net use, and examined finger-prick blood for malaria parasites and hemoglobin concentration. In 4,712 women, we examined placental blood for malaria parasites and recorded the infant's birth weight. Peripheral and placental parasitemia prevalence declined from 23.5% to 5.0% and from 25.2% to 6.8% respectively. Smaller declines in prevalence of low birth weight and anemia were observed. Coverage of intermittent preventive treatment and bednets increased. Number of sulfadoxine-pyrimethamine doses received correlated inversely with placental parasitemia (Odds Ratio (95% CI): 0.79 ), maternal anemia ) and low birth weight from 1997–2001 ), but not from 2002–2006. Bednet use protected from peripheral parasitemia ) and placental parasitemia ) and low birth weight ) but not anemia throughout the study. Compared to women without nets who did not receive 2-dose sulfadoxine-pyrimethamine, women using nets and receiving 2-dose sulfadoxine-pyrimethamine were less likely to have parasitemia or low birth weight babies. Women receiving 2-dose sulfadoxine-pyrimethamine alone had little evidence of protection whereas bednets alone gave intermediate protection.Increased bednet coverage explains changes in parasitemia and birth weight among pregnant women better than sulfadoxine-pyrimethamine use. High bed net coverage, and sulfadoxine-pyrimethamine resistance, may be contributing to its apparent loss of effectiveness. Malaria in pregnancy is a major public health problem in sub-Saharan Africa, causing significant maternal and infant morbidity and mortality P. falciparumIn 1993 Malawi introduced intermittent preventive treatment during pregnancy (IPTp), comprising two treatment doses of sulfadoxine-pyrimethamine (SP) for all pregnant women at Antenatal Clinics. SP has been shown to protect pregnant women from malaria Using longitudinal data collected between 1997 and 2006 in Blantyre, Malawi, we have investigated the changes in malaria prevalence among pregnant women and their pregnancy outcomes in a single hospital, and how the changes in these outcomes are associated with SP IPTp and bed net coverage.Between 1997 and 2006, we recruited 8131 pregnant women. Of these women, 4031 were primigravid (49.6%), 4088 were multigravidae (50.3%) and gravidity was not recorded for 12 women (0.1%). The percentage of primigravid women varied between 39.5% in 1997 and 54.0% in 1999; 48.8% of women delivering in 1997–2001 and 50.0% of women delivering from 2002 onwards were primigravidae. Overall, 796 out of 7671 women (10.4%) had peripheral parasitemia, 667 out of 4831 women (13.8%) had placental parasitemia, 2278 out of 7964 women (28.6%) were anemic and 555 out of 4715 (11.8%) newborn infants were LBW.For women who did not deliver while the study team was present, we were not able to collect information on placental malaria infection or birth weight. However, the characteristics of these women were similar to those of women with delivery data available .Over the study period there were declines in prevalence of peripheral and placental parasitemia, maternal anemia and LBW . The preAmong malaria infected women, between 1997-8 and 2005-6 peripheral parasite densities among malaria infected women increased from a geometric mean of 568/µl to 1302/µl, and placental parasite density increased from 1091/µl to 1737/µl . Over thThere were no significant changes in rainfall patterns over the study period .After adjusting for gravidity, season and local rainfall, we observed a strong association between year of enrollment and prevalence of malaria and associated pregnancy outcomes . The oddThe percentage of women receiving at least one dose of SP IPTp (documented on their ANC cards) increased from 77.0% in 1997 to 95.3% in 2004. For the second documented dose, the coverage increased from 27.0% in 1997 to 77.8% in 2004. Coverage decreased in 2005 and 2006 . The numP. falciparum parasites resistant to SP have become highly prevalent in Malawi We previously observed an inverse relationship between number of doses of SP IPTp prescribed and prevalence of malaria infection, anemia and LBW Information on bed net coverage was not recorded prior to 1999, leaving data available for analyses from 6064 pregnancies. Bed net coverage increased substantially from 14.4% in 1999 to 60.5% in 2006 , and wasAmong women receiving at least two doses of SP IPTp and using a bed net, the prevalence of peripheral malaria, placental malaria and LBW was significantly decreased in comparison to women without bed nets and receiving one, or no, doses of SP IPTp. Hemoglobin levels were marginally higher in the women who had used bed nets and received optimal dosing of SP IPTp compared with women receiving sub-optimal SP IPTp and not using a bed net. Women with bed nets but with ≤1 dose of SP IPTp showed intermediate levels of protection, whereas women receiving ≥2 SP IPTp doses without nets were not significantly protected from malaria or its consequences .In this study, we demonstrate marked changes in prevalence of malaria parasitemia among pregnant Malawian women attending a tertiary hospital for delivery over a 10-year period. These changes were associated with concomitant decreases in prevalence of maternal anemia and LBW, important indicators of maternal malaria morbidity. This result is in keeping with declines in malaria illness in young children from East and West Africa, over similar time frames Together with the decreased prevalence of malaria infection, maternal anemia and LBW, we observed increased coverage with SP IPTp and with bed nets. In other studies in subsets of participating women, we found that the proportion of bed nets that were ITNs increased from 69% in 1997–1999 to 89% in 2001–2006 . Increased coverage with IPTp and ITNs appears to be the likeliest explanation for the changes we have documented. While SP primarily protects the individual woman, ITNs may both protect the individual and (through their mosquito-killing activity) have community-wide effects.Earlier studies showed that SP use was associated with decreased prevalence of placental malaria, maternal anemia and LBW P. falciparum in Malawi dhps) gene and three dihydrofolate reductase (dhfr) mutations were highly prevalent in Blantyre over the study period dhps 581dhfr 164 The relationship between IPT use and parasitemia, anemia and low birth weight changed over time. Between 1997 and 2001 SP IPTp was associated with decreased placental malaria, maternal anemia and LBW, but these associations were lost from 2002–2006. We considered two possible explanations: increased parasite resistance to SP, and masking of a protective effect of SP by increased ITN coverage or by declining malaria prevalence and malaria-attributable morbidity, for other external reasons. Parasite resistance may compromise the efficacy of SP as treatment for malaria Increasing bed net coverage  might make SP appear less effective. We found that bed net use was constantly associated with protection against malaria infection and LBW, as described for ITNs As previously reported Our data collection ended in 2006, and in 2007 Malawi adopted artemether-lumefantrine as first line treatment for malaria in children Changes in rainfall may cause fluctuations in malaria transmission and in the prevalence of parasitemia, but these seemed not to explain the large changes in malaria observed. HIV increases susceptibility to malaria In areas of high malaria transmission, pregnancy-specific immunity develops over the first one or two pregnancies. In children, interruption of malaria transmission leads to decreased specific immunity and increased susceptibility to severe disease This study does have some potential limitations. Although we recruited more than 8,000 women, these women only count for less than 10% of the total deliveries during the study period. Moreover, the number of study participants included each year varied because of changing availability of staff to recruit study participants; although logistical issues associated with starting a separate study A recent paper critiqued facility-based surveys as tools to evaluate changing malaria epidemiology It is not clear how representative our data are of Malawi as a whole, or of malaria-endemic countries in sub-Saharan Africa. Most women in our study were urban residents, and may have self-selected to deliver in hospital. Local programmes to improve uptake of SP IPTp In conclusion, malaria infection declined significantly among pregnant women delivering in Blantyre between 1997 and 2006, and this was accompanied by declines in prevalence of anemia and LBW deliveries. Mean birth weights increased by 97 g, a change of similar magnitude to the reported benefits of effective malaria chemoprophylaxis or IPTp in pregnancy Importantly, our data suggest that, among women with good access to health facilities, high rates of coverage with IPTp and ITNs can be achieved, and that these may be associated with significant reductions in the burden of malaria in pregnancy. With an increasing focus on reducing the malaria burden, it will be of great value to discover whether similar reductions can be obtained in other settings, through scale up of these recommended interventions to WHO's target levels.Ethics approval for this study was given by the Malawi Health Sciences Research Committee and the Research Ethics Committee of the College of Medicine, University of Malawi.We recruited women delivering in the maternity unit of Queen Elizabeth Central Hospital in Blantyre, Malawi, an urban hospital hosting 15,000 deliveries annually. Malaria transmission in Blantyre is perennial with a seasonal peak during the rainy season. Primigravidae and at-risk pregnant women are specifically encouraged to deliver in the hospital.We recruited study participants between July 1997 and August 2006. Data from 1644 women delivering between July 1997 and November 1999 were included in a previous publication Participants were selected under the same criteria as previously described We recorded participants' demographic and clinical data and noted the number of doses of SP prescribed at Antenatal Clinics (ANC) as shown on ANC cards. We separately asked women the number of courses of SP they had received during pregnancy. We used finger prick blood samples to prepare thick blood films for immediate microscopy for maternal malaria parasitemia and to measure hemoglobin using a HemoCue hemoglobinometer . When women delivered in the presence of the study team, we pricked the cleaned maternal placental surface with a grooved stylet (which collected a droplet of blood in its groove) and used this droplet to make a thick blood film. All thick blood films were examined twice by trained microscopists, counting at least 200 white blood cells, and parasite counts were recorded as the average of the two readings. If results disagreed , a third independent reading was performed, and the two closest results were averaged. An assumed white cell count of 6000/µl was used to convert parasite counts to densities per µl. While study microscopists changed over the study period, the procedures were consistent throughout.We collected information on bed net use from 1999 to 2006 except for 2001 by asking whether the women were usually sleeping under a bed net. Information on whether the net was insecticide impregnated, or when, was not routinely available. From the Bureau of Meteorology, Malawi, we obtained monthly rainfall data for Chichiri, Blantyre between 1997 and 2006.nd and 3rd trimesters. Cumulative rainfall exposure was not normally distributed, and, therefore, the variable was log2 transformed before analysis.We classified participants as primigravidae (first pregnancy) or multigravidae (second and subsequent pregnancy). Doses of SP IPTp were classified as no SP treatment, 1 dose, 2 doses, or ≥3 doses. For some analyses, we divided year of enrollment into early (1997–2001) and late (2002–2006). Bed net usage was coded as whether or not the woman usually slept under a bed net. We defined anemia as hemoglobin concentration <11 g/dl and low birth weight (LBW) as infant's birth weight <2500 g. Seasons were classified as rainy (November to April) and non-rainy (May to October) based on the delivery date. Monthly rainfall data were summed over the 6 months prior to delivery to represent rainfall exposure during the 2st January 2002, the mid-point of study period) was fitted, and tested using the likelihood ratio test. To investigate the combined effect of SP IPTp and bed net use we included a variable with the following categories: ≥2 doses of SP IPTp and bed net use; ≥2 doses of SP IPTp only; sub-optimal (≤1 dose) SP IPTp and bed net use; and no bed net and sub-optimal SP IPTp during pregnancy. All statistical analyses were performed using Stata 9.2 .The exposure variables of interest were SP IPTp  and bed net use. Outcome variables were peripheral parasitemia , placental parasitemia , maternal hemoglobin concentration at delivery and anemia, infant's birth weight and LBW. Maternal gravidity, season of enrollment and rainfall were considered as confounding factors. We performed multivariable logistic regression, adjusting for the confounders described above, to investigate how the prevalence of maternal peripheral parasitemia, placental parasitemia, maternal anemia and LBW changed over time and how these outcomes varied with SP IPTp and bed net use. We also analyzed the impact of SP IPTp and bed net use on maternal hemoglobin level and birth weight using multiple linear regression. To explore whether the SP IPTp protection effect was modified over the study period, an interaction term between SP IPTp and year of enrolment (before versus after 1


Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting.Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared.During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (≥38°C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/μL. There was an age correlation for the proportion of parasite density below 100/μL for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy , indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections.A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay. Accurate and timely diagnosis of malaria infections is a critical part of case management in malaria control programmes aiming to reduce malaria morbidity and mortality. Sensitive and effective detection of infected individuals also plays a vital role in areas where transmission has been reduced markedly due to intensified control measures and elimination is being considered.Malaria diagnosis is traditionally achieved by microscopic examination of blood smears. Microscopy is able to detect parasite species and determine parasite densities. The limit of detection by thick film microscopy is in the range of 5 to 100 parasites/μL of blood . HoweverP. falciparum but often poorer for P. vivax  woul woulP. fP. vivax will eventually become the problem due to relapsing cases caused by the activation of hypnozoites in the liver 2-3 years after the initial infection. Level 1 microscopy seems to offer an advantage in detecting this species at very low parasite densities. Again, the incorporation of molecular based PCR detection method was found to detect more P. vivax infected subjects. However, the conventional PCR is difficult to perform in the field due to requirements for DNA extraction, sophisticated equipment and reagents. Therefore, a simple, cheap and easy-to-perform molecular assay is required. The deployment of such an assay combined with microscopy or RDT will provide the best chance of detecting the majority of infected individuals. The purpose of using this molecular assay would not be for treating clinical patients, but for finding asymptomatic infections where the individuals can be followed up for treatment on the next day or even longer.Elimination of Plasmodium infected individuals were asymptomatic with low and sub-microscopic parasite densities. With the proposed elimination of malaria from this Province these findings present a challenge for malaria diagnostics in mass screening and treatment programmes and thus for elimination. A combination of methods, or new diagnostics, may be required to detect infections in these asymptomatic parasite reservoirs.Results of a mass blood survey conducted in Temotu Province in Solomon Islands showed that a large proportion of The authors declare that they have no competing interests.IH, DS and QC conceived the study. IH carried out microscopy examination, QA, data analysis and drafted the manuscript. WWS performed microscopy QA, PCR and data analysis. LMB, KG and DK performed PCR and data analysis. AB, LB, AV and DS coordinated and planned the survey. KL performed microscopy and QA. MJ coordinated data collating. MLG contributed to data analysis and the writing of manuscript. QC supervised the PCR, analysed data and drafted the manuscript. All authors read and approved the final manuscript.


Zeno Bisoffi and colleagues discuss a new clinical trial in Zanzibar comparing symptom-based clinical diagnosis of malaria versus clinical diagnosis plus rapid diagnostic tests. PLoS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1000070Msellem MI, Mårtensson A, Rotllant G, Bhattarai A, Strömberg J, et al. (2009) Influence of rapid malaria diagnostic tests on treatment and health outcome in fever patients, Zanzibar—A crossover validation study. PLoS Med 6(5): e1000070. Anders Bjorkman and colleagues report results from a crossover trial evaluating rapid diagnostic testing for malaria diagnosis in Zanzibar.In recent years malaria treatment policies have shifted in most African countries to artemisinin-based combination therapies (ACT), which are highly effective but also much more expensive than previous regimens However, recent research has shown that clinicians are reluctant to refrain from treating for malaria after a negative test PLoS Medicine recently published a provocative debate on whether or not presumptive treatment in under-fives should be abandoned. Valérie D'Acremont and colleagues argued that it is high time to move to laboratory-confirmed  diagnosis for younger children PLoS Medicine, by Anders Bjorkman and colleagues, addresses these research priorities The divided opinion on this topic reflects a lack of evidence. There is not yet enough evidence for the safety of a test-based strategy for young children, and there is no evidence that satisfactory adherence to a test-based management strategy can be achieved by training. Operational research is needed to fill in these evidence gaps Bjorkman and colleagues conducted a cross-over clinical trial of symptom-based clinical diagnosis (CD) versus CD plus RDT in four primary health care facilities in Zanzibar. Patients of all ages with reported fever lasting 48 hours were eligible, and they were allocated on alternate weeks to CD alone or CD+RDT. Follow-up was 14 days, and ACT was prescribed to patients diagnosed with malaria in both groups. Malaria management was provided by registered nurses, all of whom had been trained in malaria case management. The RDT was a histidine-rich protein 2–based test called Paracheck.The use of RDT had a major impact on clinical decisions. In children, the prescription rate of antimalarial treatments in the CD+RDT group was half that observed in the CD group . In adults, the difference was even more striking (16% versus 87%). Although the authors do not give details about specific decisions taken according to RDT results, this difference suggests that the compliance was very high, as most RDT-negative patients were not treated for malaria. The prescription rate of antibiotics was higher in the CD+RDT group than in the CD group, suggesting that negative malaria RDT results led the nurses to consider and treat alternative causes of fever.No death was recorded in either group at 14 days of follow-up. The authors do not specify if they observed a difference in frequency of severe complications and/or of referral to a higher level of care; however, the patients in the CD+RDT group were less likely to re-attend the clinic during the follow-up period.Are these results generalisable—do they suggest that a test-based policy should be recommended even for young children? We will briefly examine the main issues: adherence, safety, and cost.also the prescribers, having to record their own prescribing behaviour, which may have influenced their decisions. The very encouraging adherence rates in this new study should be corroborated by further research on teaching and supervision strategies aimed at achieving satisfactory compliance in real-life situations.For the purpose of this study, nurses were trained for one day only. In two of the four health centres, RDTs were introduced for the first time. The excellent adherence to test-based management seen in the trial is surprising, and contrasts with previous studies As far as safety is concerned, Bjorkman and colleagues' study indicates that the RDT-based management strategy is safe for both adults and children. The study found no severe malaria case after a missed malaria treatment following a false negative result. Moreover, the lower rate of re-attendance in the CD+RDT group suggests a better clinical outcome. Nevertheless, definitive evidence on safety can only be found by a study that is adequately powered to detect differences in severe clinical outcomes, including deaths, and/or by pooling results obtained in different study settings Are RDTs cost-effective? Bjorkman and colleagues found that overall cost was the same with CD as with CD+RDT. However, the test-based strategy was associated with cost savings in adults , due to the much lower proportion of malaria treatment in this group. Since Bjorkman and colleagues conducted their trial, malaria incidence in Zanzibar has further declined This new study brings additional arguments, albeit no definite evidence, in favour of extending an RDT-based malaria management strategy to young children, in a situation of moderate- to low-intensity malaria transmission. This strategy would, however, be justified only if adherence to test results were optimal, which remains to be proved in real-life situations. More research is also needed in settings where malaria transmission intensity is higher and/or seasonal variation is greater.


Plasmodium falciparum infections >2 months after leaving malaria-endemic areas. We conducted a case–control study to identify factors associated with prolonged P. falciparum infection in immigrant travelers. Results suggest that P. falciparum infection should be systematically suspected, even months after travel, especially in pregnant women and first-arrival immigrants.Few immigrant travelers have  Plasmodium falciparum malaria was estimated to be 4,500 in 2004, with a median time of 10 days between departure from an area endemic for malaria and diagnosis  in Paris, without epidemiologic evidence of autochthonous, transfusion-transmitted, or occupational malaria. Case-patients had P. falciparum infections detected >59 days after their arrival in France. Controls had P. falciparum infections detected <30 days after their arrival. For each case-patient, 4 controls were matched by calendar year and hospital of diagnosis (70 cases and 280 controls). Data were collected from the CNRP database in which all cases are prospectively included and medical records are checked. We only considered immigrants , which resulted in 61 case-patients and 197 controls. We distinguished first-arrival immigrants  from visiting friends and immigrant relatives .A case–control study was conducted among patients with P. falciparum infection and estimate odds ratios (ORs) and 95% confidence intervals (CIs). For multivariate analysis, variables with p values <0.25 were introduced into the model and removed after a backward stepwise approach, which resulted in only values with p<0.05 in the final model (except for age groups). Statistical analysis was performed by using Stata software version 8.2 .Logistic regression was used to identify factors associated with prolonged P. falciparum malaria infections. The median diagnosis delay was 5 months (interquartile range 3–9 months). These infections included 10 patients  with clinical malaria >1 year after their arrival. Four of them, all pregnant women, had clinical malaria >3 years after their arrival. For the case–control study, 197 controls were compared with 61 case-patients  late infections occurred among 2,680 diagnosed patients . Table 1P. falciparum infection: pregnant women, first-arrival immigrants, and HIV-positive patients. A total of 27.9% (n = 17) of the patients were pregnant women, with a median (range) age of 22 (16–36) years. All were of African origin and had become pregnant in France; 10 (58.8%) were in their second trimester, 5 (29.4%) were in their third trimester. First-arrival immigrants were younger than other patients . All patients were of African origin except for 1 Indian man. HIV infection was associated with prolonged infection, but HIV status was not introduced into the final model because of missing data. Although chemoprophylaxis with chloroquine-proguanil was less common among case-patients than controls , the reverse was seen, although not significantly, with mefloquine use . Multivariate analysis , being a pregnant woman , and mefloquine prophylaxis .Among immigrant travelers, 3 groups had a higher risk for prolonged analysis  showed tP. falciparum malaria (impaired consciousness) and responded to treatment. The girl had traveled in Mali for 2 weeks and took regular chloroquine-proguanil prophylaxis. She was hospitalized 4 months after her return with uncomplicated P. falciparum malaria occurring concomitantly with a Salmonella spp. infection that had been treated 1 week earlier with ceftriaxone.We also observed cases of malaria in a 26-year-old Caucasian man and a 2-year-old African girl who were hospitalized with diagnosis delays of 221 days and 127 days, respectively. The man was a French expatriate who lived in Madagascar for 2 years and took regular chloroquine-proguanil prophylaxis. He was hospitalized 7 months after his return with severe P. falciparum infection: being a first-arrival immigrant, being a pregnant woman, and taking mefloquine prophylaxis. The first 2 factors most likely reflect partial control of parasitemia by acquired immunity. Persons living in areas with high transmission of malaria acquire this immunity during childhood. In these areas, P. falciparum infections in adults are mostly asymptomatic with transient low parasitemia levels  .Several immunologic mechanisms have been suggested to explain late manifestations of P. falciparum malaria in the United States during 1963–1999 . Current US guidelines recommend obtaining a thorough travel history and deferring blood donation if potential donors have emigrated from areas endemic for malaria in the preceding 3 years. However, this measure may not prevent transmission if P. falciparum is present for >3 years (as in 4 pregnant women in our study). In France, systematic serologic analysis for Plasmodium spp. in blood donors born in areas endemic for malaria was implemented in 2002 (This study also highlights the risk for blood transfusion–transmitted malaria, a rare but serious complication. Mungai et al. (P. falciparum infection in immigrant pregnant women and first-arrival immigrants even without recent travel to a country endemic for malaria. The prevalence of asymptomatic P. falciparum carriers in France or other northern countries is unknown but could be high with the increase in immigration. Public health authorities should be aware of the risk these persons represent for blood donations.Our findings suggest that physicians should consider the risk for prolonged