Malaria is one of the most important infectious diseases in the world. Although most cases are found distributed in the tropical regions of Africa, Asia, Central and South Americas, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in particular due to the higher mobility in today's society.Plasmodium species was assessed using Nucleic Acid Sequence Based Amplification (NASBA) assays on clinical samples collected from 195 study cases with no clinical signs related to malaria and coming from sub-Saharan African regions to Southern Italy. In addition, base-line demographic, clinical and socio-economic information was collected from study participants who also underwent a full clinical examination.The prevalence of a possible asymptomatic infection with Plasmodium using a pan Plasmodium specific NASBA which can detect all four Plasmodium species causing human disease, based on the small subunit 18S rRNA gene (18S NASBA). Twenty-four samples (38%) of the 62 18S NASBA positive study cases were found positive with a Pfs25 mRNA NASBA, which is specific for the detection of gametocytes of Plasmodium falciparum. A statistically significant association was observed between 18S NASBA positivity and splenomegaly, hepatomegaly and leukopaenia and country of origin.Sixty-two study subjects (31.8%) were found positive for This study showed that a substantial proportion of people originating from malaria endemic countries harbor malaria parasites in their blood. If transmission conditions are available, they could potentially be a reservoir. Thefore, health authorities should pay special attention to the health of this potential risk group and aim to improve their health conditions. Plasmodium falciparum  of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale was performed on an IQ5 Real-Time analyzer (Bio-RAD)  was performed on a NucliSens EasyQ analyzer (bioMèrieux) using the Nuclisens BasicKit for amplification according to the manufacturer's manual at a KCl concentration of 80 mM and the same conditions of Real-Time 18S QT_NASBA.18S rRNA NASBA positive samples were also analysed for gametocyte specific Pfs25 mRNA QT-NASBA [The forward primer was 5'-GACTGTAAATAAACCATGTGGAGA-3' (nucleotides 204 to 227); the reverse primer was 5'-AATTCTAATACGA CTCACTATAGGGAGAAGG-CATTTACCGTTACCACAAGTTA-3' ; the PfS25 molecular beacon was 5'-Texas-Red-CGATCG-CCCGTTTCATACGCTTGTAA-CGATCG-DABSYL-3' (molecular beacon stem of six paired nucleotides and nucleotides 259–278). Time to positivity was calculated, i.e. the time point during amplification at which the fluorescence detecting target amplicons exceeded the mean fluorescence of two negative controls plus 20 standard deviations. Standard gametocytes dilution series were used as positive controls. The samples and the positive controls were performed in duplicate.2 value were calculated. A p value of < 0.05 was considered statistically significant.In order to value the statistical association between history of malaria and signs and symptoms  contingency double-entry tables were used and Odds Ratio and relative 95% Confidence Intervals and X2 value were calculated. The t-Student's method was employed for the comparative evaluation of quantitative variables (continuous variables- i.e: age). A p value of < 0.05 was considered statistically significant. In order to value the potential confounding effect of considered variables  on 18S NASBA positivity logistic regression model was performed.In order to evaluate the statistical association between 18S NASBA and the following variables: sex, regions of origin , previous episodes of clinical malaria as well as current clinical signs  contingency double-entry tables were used and Odds Ratio and relative 95% Confidence Intervals and X2 = 9.53, p < 0.05; hepatomegaly: OR = 3.01, 95%CI = 1.16–7.76; χ2 = 6.8, p < 0.05). However, an inverse relationship was found between spenomegaly and group B .All examined study cases did not present at the time of examination or in the previous two months, the malaria paroxysm- characterized by high fever, chills and rigor. On physical examination 98/195 (50.2%) cases were found to have splenomegaly and tender hepatomegaly; 50/98 (51.0%) were in group A, 27/98 (27.6%) in group B and 21/98 (21.4%) in group C. Splenomegaly as well as hepatomegaly was significantly associated with group A , scleral icterus 61/195 (31.3%), urticaria 37/195 (19.9%). Auscultation of the chest revealed scattered rales in about 25% of subjects. Cardiac examination was generally normal except for tachycardia in about 10% of patients.2 = 6.05, p < 0.05). Liver function tests revealed elevated transaminase levels in 32/195 (16.4%) of the cases and a mild to moderate increase in bilirubin (mostly indirect) in 86/195 (44.1%) of the study subjects; this variable resulted inversely associated with group C .Some abnormalities in routine laboratory tests were found in some study participants. Normocytic haemolytic anaemia presented in 61/195 (31.3%) of the cases; leukopaenia due to a decrease in granulocytes and lymphocytes was present in 96/195 (49.2%) and these cases also presented with eosinophilia in the presence of urticaria. Leukopaenia was significant associated with group A  belong to Group A, 28/93 (30.1%) belong to Group B and 16/44 (36.4%) belong to Group C. The distribution according to country of origin is presented in Table In total, 62 study participants 31.7%) were found positive for the presence of .7% were Microscopy confirmed the positive NASBA results in 14 cases and these cases were distributed as follows: six belonged to Group A, six belonged to Group B and two belonged to Group C. Quantification of parasitaemia was not available.Plasmodium falciparum was observed in 13 samples and one sample was identified as being P. ovale. Thirteen cases (21%) of this Plasmodium18S rRNA positive population were also found positive with a Pfs25 mRNA NASBA, which is specific for the detection of gametocytes of P. falciparum. Four cases positive for gametocytes were confirmed by microscopy.No significant association (p > 0.05) was found between 18S NASBA positivity and sex, age, history of malaria and region of origins.2 = 89.97, p < 0.001), hepatomegaly  and leukopenia : in all subjects who were found positive with the 18S NASBA, splenomegaly, hepatomegaly and leukopaenia were reported. The relative OR values were not computable. No other signs and symptoms was associated with 18S NASBA positivity.A statistically significant association was found between 18S NASBA positivity and splenomegaly . In particular, a high probability to be positive at 18S NASBA was observed in people coming from West Africa , Central Africa  and East Africa . By using the same regression model, a significant inverse association was found between increased transaminase levels and 18S NASBA positivity  so that people with increased transaminase levels had a lower probability to be positive at 18S NASBA. By the model, no significant associations (p < 0.05) were found between 18S NASBA positivity and sex, age, history of malaria, other signs and symptoms .Plasmodium parasites were found with the applied NASBA technology. Furthermore, clinical examination revealed a statistically significant association between splenomegaly and 18S NASBA positivity. Malarial splenomegaly is believed to be a consequence of an immunological dysfunction due to recurrent episodes of malaria [In many countries, the increase in imported malaria cases from different countries is due to the increasing number of people traveling to and from endemic areas, without taking appropriate prophylaxis or immigration from low income countries ,28. In t malaria ,31. It i malaria ,33. Pati malaria . A signiPlasmodium18S rRNA positive population was also found positive with a Pfs25 mRNA NASBA, which indicates that these cases harbour gametocytes and may thus contribute to possible malaria transmission, even if the number of gametocytes is very low.It was further noted in the present study that 21% of the In Italy, malaria was eradicated after a campaign launched in 1947 just at the end of World War II. The campaign was carried out by indoor treatment with DDT; houses, stables, shelters and all rural structures were treated into the mid-1950s and also later in some endemic regions, such as Sardinia. Due to the persistence of sporadic malaria cases in some areas ,36, the P. falciparum in 2003 [Plasmodium falciparum was confirmed to be the most prevalent species imported in southern Europe [P. vivax for 7.7%, P. ovale and P. malariae, 6% and 1.5%, respectively [Plasmodium infections have also been reported in Italy, with 58% of all these cases reported from a single town, Verona [Over the last two decades, with the exception of one autochthonous case registered in 1997 in a rural area of the province of Grosseto (Tuscany), which was followed by four more cases . Unt in 2003  in both  in 2003  and in 2 in 2003  in Chinen Europe ,28,37; iectively ,37. In a, Verona .Anopheles labranchiae, Anopheles superpictus and Anopheles sacharovi) are also under strict surveillance by Istituto Superiore di Sanità (ISS). Anopheles labranchiae is present in several areas of Central and Southern Italy and in some regions  high density have been registered [An. superpictus is lower and breeding sites are restricted to Calabria coasts and Sicily while although An. sacharovi has not registered since over 30 years, its presence in low density cannot be excluded in Apulia and northern Sardinia regions [The distribution and density of the three potential vectors of malaria in Italy . If transmission prerequisites  prevail they could potential be a reservoir. Therefore, health authorities should pay special attention to the health of this potential risk group and aim to improve their health conditions.This study showed that a substantial proportion of people originating from malaria endemic countries and not showing clinical signs of malaria harbor The authors declare that they have no competing interests.MM was involved in the laboratory work . RDT was involved in the fieldwork (blood sample collection). DM was involved in statistical analysis. VF and GA were involved in clinical consultancy. PFM performed daily supervision, analysis and technical assistance of the NASBA assays and substantial revision of the manuscript. HDFHS, conception of the study, critical reading and revision of the manuscript. AG and GS, scientific and financial coordinators of the project and writers of the paper.


Plasmodium falciparum or Plasmodium vivax in a low transmission area on the north-western border of Thailand.Haematological changes associated with malaria in pregnancy are not well documented, and have focused predominantly on anaemia. Examined here is thrombocytopaenia in pregnant women infected with In this observational study we reviewed the platelet counts from routine complete blood count (CBC) in a cohort of healthy and malaria infected Karen pregnant women attending weekly antenatal clinics. A platelet count of 75,000/μL was the threshold at 2 standard deviations below the mean for healthy pregnant women used to indicate thrombocytopenia. Differences in platelet counts in non-pregnant and pregnant women were compared after matching for age, symptoms, malaria species and parasitaemia.Plasmodium falciparum and P. vivax caused a 34% (95% CI 24–47) and 22% (95% CI 8–36) reduction in platelet count, respectively. Pregnant compared to non pregnant women were at higher risk OR = 2.27 (95%CI 1.16–4.4) P = 0.017, for thrombocytopaenia. Platelets counts were higher in first compared with subsequent malaria infections within the same pregnancy. Malaria associated thrombocytopaenia had a median [range] time for recovery of 7  (N = 694) or vivax malaria 184,000  (N = 523) compared to healthy pregnant women 256,000  (N = 255), P < 0.05 for both comparisons. ery of 7  days whiPregnant women become more thrombocytopenic than non-pregnant women with acute uncomplicated malaria. Uncomplicated malaria associated thrombocytopaenia is seldom severe. Prompt antimalarial treatment resulted in normalization of platelet counts within a week. Plasmodium falciparum, can cause profound anaemia, thrombocytopaenia, activation of the coagulation cascade and rarely disseminated intravascular coagulopathy  25 years [15–45] years) with symptomatic uncomplicated falciparum malaria from the same population were analysed . These wBlood smears (thin and thick films) were prepared using Giemsa staining and were read for 200 fields before being declared negative. Parasite counts were reported per 500 white blood cells (WBC) and for counts above 1,000 parasites per 500 WBC by the percentage of infected red cells (RBC). All stages of the parasites were recorded .A 2 ml sample of venous blood was placed in an EDTA tube, refrigerated and transported on ice to SMRU Mae Sot laboratory, where the complete blood count was determined using a Sysmex pocH-100i automated haematology analyzer. Quality control of the Sysmex pocH-100i was determined on a daily basis by analysis of three different manufacturer-provided samples with known cell counts.Thrombocytopaenia in pregnancy was defined by a platelet count lower than 75,000/μL. This is the value  that falls two standard deviations below the mean platelet count in healthy Karen pregnant women  from a previously published cohort study from the same area . This vaP. falciparum or P. vivax on the microscopic examination of the peripheral blood in a pregnant women with no elevation of temperature , no history of fever or any of the following symptoms: headache, dizziness, joint pain, anorexia, nausea, spontaneous bleeding. Symptomatic malaria was defined as the presence of an elevated temperature , or a history of fever in the previous two days, with one or more of the above symptoms in a pregnant women together with asexual forms of Plasmodium on the microscopic examination of the peripheral blood. Uncomplicated malaria was defined by the absence of signs of severity  and uncEstimated gestational age (EGA) at the time of CBC was determined from ultrasound (US) dating of the pregnancy. As part of routine ANC pregnant women had a US scan at booking and at 18 weeks. Splenomegaly was recorded as present when the spleen was palpable below the costal margin and was measured in cm. Hepatomegaly was recorded as present when the liver was palpable below the costal margin in the mid-clavicular line.Continuous normally distributed data were described by the mean (standard deviation) and non-normally distributed data by the median [range]. Percentages were given for categorical data. Categorical data were compared using the Chi-square test or by Fisher's exact test, as appropriate. Student's t-test was used to analyze means, the Mann-Whitney test was used to analyze medians. The Wilcoxon rank sum test was performed on continuous data with a skewed distribution for paired analysis to determine whether there were significant differences of parasitaemia and haematological counts after or between episodes of malaria. Forward and backward stepwise logistic regression was used to assess the relationship with thrombocytopaenia, while controlling for demographic characteristics (age), and potential confounding factors significant on univariate analysis . Age, and log transformed parasitaemia were treated as a continuous variables and the others  as dichotomous variables. Data were analyzed using EpiInfo version 6, and SPSS version 14 for Windows (SPSS Inc).P. falciparum episodes, 523 P. vivax episodes, and 86 were repeat measurements following a report of thrombocytopaenia , 694 acute P. falciparum and P. vivax separately.Co-infections of falciparum with vivax malaria (n = 49) showed similar haematological changes to falciparum monoinfections but were excluded from analysis in order to focus on the effects of P. falciparum and first episode P. vivax) were comparable for age, gravidity and parity at the time of the first CBC   and 2.1%  were hyperparasitaemic episodes (≥ 4% RBC parasitized). The three groups  Figure . OverallAs eclampsia is typically associated with thrombocytopaenia we reviewed the 1.3% (13/964) of women with hypertension in pregnancy individually. As the CBC for malaria was measured earlier in pregnancy than the hypertension developed, particularly for women with a diagnosis of eclampsia (n = 3), and these women were not thrombocytopenic their CBC results were not excluded from the malaria analysis.P. falciparum  than for P. vivax  episodes, P = 0.001. , P = 0.001. Splenomegaly was associated with a 2.7 (95%CI: 1.5–4.7) fold risk of thrombocytopaenia. The number of women who had spontaneous bleeding at the time of the malaria episode such as nose bleeds or gum bleeding was very small, and not significantly associated with thrombocytopaenia . There was no difference in the proportion of reported antepartum haemorrhage (APH) in women who had any episode of thrombocytopaenia, 23% (5/22), compared to those who had none, 18% . The geometric mean parasitaemia was significantly lower in afebrile compared to febrile patients , and asymptomatic episodes compared to symptomatic episodes . Geometric mean parasitaemia of P. falciparum was higher than that of P. vivax: 2,176/μL and, 273/μL respectively, P = 0.001.On admission, falciparum cases had significantly higher parasitaemia, days of fever, proportion febrile or symptomatic, and higher rates of enlarged spleen and liver, compared to vivax cases Table . The proP. falciparum 134,000  and P. vivax 184,000 /μL, were significantly lower compared to healthy controls, 256,000 /μL, P < 0.05 for both comparisons. The platelet counts in falciparum cases were significantly lower than in vivax cases, P < 0.05. The proportion of women with thrombocytopaenia, were healthy 0.8% (2/255), falciparum 17.7% (123/694) and vivax 5.0% (26/523), P < 0.05 for both comparisons.Platelet counts  in episodes of malaria infection, P. falciparum infections.There were no cases of severe  thrombocytopaenia . In the remaining cases, there were 51.7% (78/151) counts from 10,000 to < 50,000/μL and 48.3% (73/151) count from 50,000 to < 75,000/μL. There was no association between species of infection and having a count of 10,000 to < 50,000/μL or 50,000 to < 75,000/μL . A parallel trend was observed between parasitaemia and thrombocytopaenia for falciparum and vivax cases . Platelet counts all decreased with malaria, the impact of P. falciparum being significantly greater than that of P. vivax  and then a subsequent CBC for a malaria episode , OR = 2.0 (95%CI 1.3–3.0), P = 0.001, and this was also observed for vivax malaria: 11% (30/279) versus 3% (6/238), respectively, OR = 2.9 (95%CI 1.2–7.4), P = 0.018.The proportion of women who had multiple episodes of malaria was 49% (381/781). A second episode occurred in 27% (214/781), a third in 11% (85/781), and 4 or more episodes in 10% (82/781). In falciparum malaria cases, the proportion of women with thrombocytopaenia was higher in the first episode 22%, (80/356), compared to later episodes of Data on women who had malaria and a repeat CBC due to thrombocytopaenia were limited to 56 women. The median [range] time for recovery from thrombocytopaenia was 7 -14 days versus 6,628 /μL, P = 0.012. A multivariate analysis controlling for independent factors such as the parasitaemia found that both pregnancy  and parasitaemia  were significant independent risk factors for thrombocytopaenia.CBC data from 71 non-pregnant women of child bearing age, median [range] 25 years [15–45] years) with uncomplicated falciparum malaria from the same population  were comvs 1.2% (6/501), P = 0.25. The two women with PPH and malaria both had P. vivax infection and both were placenta negative having started treatment 1 day and 4 days prior to delivery.Overall 76.0% (740/974) of women had documented deliveries, 5.7% (56/974) aborted and 18.3% (178/974) of women were lost to follow up before the pregnancy outcome was known mostly because they moved from the study area. Twins accounted for 0.7% (5/740) of deliveries. There were 1.5% (11/740) of women who had a caesarean section, all for obstetric indications and none as a result of thrombocytopenic haemorrhage. There were 69.5% (511/735) of singletons weighed in the first 3 days of life. The mean ± SD [range] birth weight was 2,868 ± 458  g and mean gestational age 39.5 ± 1.6 [28.0–44.4] weeks. There was no significant difference in mean birth weight or gestational age at delivery in women with malaria who had or did not have thrombocytopaenia reported during pregnancy (data not shown). The rate of post-partum haemorrhage (PPH), 2.0% (10/503), was low. PPH in women with malaria occurring less than 5 days before delivery compared to ≥ 5 days was 2.9%% (2/69) The observational data presented here demonstrates, as others have for non-pregnant patients, the reduction in platelet count associated with both falciparum and vivax malaria ,17-20. IReduced platelet counts during malaria infection result from platelet activation, splenic pooling, and a decreased platelet life-span to 2–3 days  ,17. The P. falciparum and one fifth in P. vivax infections. Another important finding was that thrombocytopaenia, although uncommon, was also seen in asymptomatic malaria infected women with P. falciparum. This has recently been reported in children in Nigeria [In this population of pregnant women with low premunition (similar to non-immune travelers), there was an approximate reduction in platelet count by one third in  Nigeria .Nearly 50% of women in this cohort had subsequent malaria infections. The risk of thrombocytopaenia was greatest in the first infection compared to later infections but one cannot determine whether this is a protective effect gained by a single infection or a by product of intensive weekly screening where parasitaemia may be detected earlier and before the woman becomes symptomatic.Does this level of thrombocytopaenia put women with malaria at the time of delivery at risk of post-partum haemorrhage? Spontaneous bleeding is uncommon unless the platelet count falls below 10,000/μL  when anyClearly treatment with antimalarials leads to platelet recovery but this takes closer to seven days than 48 hours. This implies it would probably be detrimental to try to postpone labour in women with treated uncomplicated malaria at term to allow for platelet recovery with treatment. This may well not apply to severe malaria where platelet counts are likely to be more severely affected, and there is a high risk of both fetal and maternal death[The women presented here reside in an area of low and unstable malaria transmission where acute symptomatic malaria can occur in women of all gravida  and placFalciparum and vivax malaria adversely affect platelet count in pregnancy and pregnant women are more susceptible to thrombocytopaenia than non-pregnant women. The effect of chronic malaria infection in pregnancy on thrombocytopaenia is unknown. Nevertheless treatment results in recovery from thrombocytopaenia and where efforts to prevent malaria fail, prompt detection and treatment, particularly in the last week before delivery should be provided.The authors declare that they have no competing interests.SOT, RM, EAA, MPJ, KS, KLT and YM participated in the clinical work and data compilation. RM, BE and FN conceived the need to review clinical records. SOT, KLT, YM and JZ performed the data management and JZ the statistical analysis. SOT, RM, JZ, EAA, BE, PS, NJW, and FN participated in drafting the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria. Because of their potential role in malaria pathogenesis, polymorphisms in these genes may be under selective pressure in populations where this infectious disease is endemic.Toll-like receptors (TLR) and related downstream signaling pathways of innate immunity have been implicated in the pathogenesis of TLR2, TLR4, TLR9, MyD88-Adaptor Like Protein (MAL) single nucleotide polymorphisms (SNPs), and TLR2 length polymorphisms in 170 residents of two regions of Kenya where malaria transmission is stable and high (holoendemic) or episodic and low, 346 residents of a malaria holoendemic region of Papua New Guinea, and 261 residents of North America of self-identified ethnicity.A post-PCR Ligation Detection Reaction-Fluorescent Microsphere Assay (LDR-FMA) was developed to determine the frequencies of glucose-6-phoshpate dehydrogenase (G6PD) and Hemoglobin S (HbS) in the holoendemic site compared to the episodic transmission site. However, this study detected no such difference in the TLR2, TLR4, TLR9, and MAL allele frequencies between the two study sites. All polymorphisms were in Hardy Weinberg Equilibrium in the Kenyan and Papua New Guinean populations. TLR9 SNPs and length polymorphisms within the TLR2 5' untranslated region were the only mutant alleles present at a frequency greater than 10% in all populations.The difference in historical malaria exposure between the two Kenyan sites has significantly increased the frequency of malaria protective alleles TLR2, TLR4, TLR9, and MAL genetic polymorphisms in populations with different histories of malaria exposure suggest that these innate immune pathways have not been under strong selective pressure by malaria. Genotype frequencies are consistent with Hardy-Weinberg Equilibrium and the Neutral Theory, suggesting that genetic drift has influenced allele frequencies to a greater extent than selective pressure from malaria or any other infectious agents in these populations.Similar frequencies of  Plasmodium falciparum [th century that such polymorphisms were under selective pressure, because they were protective against severe malaria infection and thereby increased the reproductive fitness of individuals living in malaria endemic areas [Every year 350–500 million cases of malaria occur worldwide, and up to 2.5 million of these individuals are estimated to die, mainly from neurologic complications, severe anaemia or respiratory distress due to lciparum . The burMycoplasma ligands [TLR2 gene is located on chromosome 4q32 and is comprised of 3 exons, which encode 784 amino acids [TLR2 Δ22) in the first untranslated exon was observed to be highly polymorphic in a Japanese population but was not associated with a disease phenotype [n) is present within the second intron, approximately 100 bp upstream of the translational start site. Repeats of varying length have been associated with susceptibility to tuberculosis, reversal reactions in leprosy, and colorectal cancer [in vitro [TLR2, Arg753Gln, is associated with susceptibility to tuberculosis [in vitro [Plasmodium falciparum GPI may weakly activate TLR4 [TLR4 SNPs (Asp299Gly and Thr399Ile) are in the extracellular domain, and commonly co-segregate in European Caucasian but not in African populations [in vitro in some studies, [TLR9 spans 5 kb on chromosome 3p21.3, consists of 2 exons, and encodes 1028 amino acids [TLR9 SNPs are present at approximately 15% or greater allele frequency in North American populations [TLR4 Asp299Gly and TLR9 -1486T/C were associated with low birth weight and maternal anaemia in a malaria endemic population [in vitro [Toll-like receptors are innate immune receptors that bind to conserved structural motifs expressed by microbial pathogens. TLR2, TLR4, TLR9, and downstream signaling pathways of these proteins have recently been implicated in human malaria pathogenesis . TLR2 is ligands -9. Malar ligands . The TLRno acids ,11. A 22henotype . A GT dil cancer -15. Shorin vitro . A non-srculosis , and prein vitro . TLR4 isin vitro ,19. Plasate TLR4 . Two nonulations ,22. Thesstudies,  but not studies, . TLR9 isstudies, ,26. Malastudies, ,28. TLR9ulations . Two of ulations -33. Bothpulation . MAL . Kenyan and Papua New Guinean study population characteristics in terms of age structure and malaria infection prevalence are presented in additional file Plasmodium spp. and P. falciparum asexual densities were obtained. All individuals were asymptomatic with no known episodes of severe malaria morbidity. Malaria transmission is perennial and stable in this area of Papua New Guinea. The entomologic inoculation rate is estimated to be 0.9 infectious bites per person per night, a transmission intensity similar to the holoendemic Kenya site [nya site . Mild maInformed consent, blood sample collection, and genotyping were performed under protocols approved by the Ethical and Institutional Review Boards for Human Investigation at the Kenya Medical Research Institute, the Medical Research Advisory Committee of the Papua New Guinea Department of Health, and University Hospitals of Cleveland/Case Western Reserve University.2, 125 nM primers, and 0.8 units Taq polymerase in a reaction volume of 25 μl. PCR primers and amplification conditions are listed in additional file TLR2 Δ 22 genotypes were assigned based on size discrimination of PCR products on a 4% agarose gel. TLR2 GTn genotypes were assigned based on size discrimination of PCR products run on a 6% polyacrylamide gel as described elsewhere [Genomic DNA was extracted from blood using the QIAamp 96 spin blood kit . PCR was performed using a master mix consisting of 1× PCR buffer, 125 μM dNTPs, 2.5 mM MgCllsewhere PCR amplification products from local donors were purified using the QIAquick PCR purification kit . Purified PCR products were sent to MWG Biotech, High Point, NC for sequencing. Sequences were analyzed using the Sequencher software . Sequenced DNA templates were subjected to PCR and used in the LDR-FMA as positive controls.PCR products were analyzed in a LDR-FMA divided into 3 steps: (1) ligation of oligonucleotides to the SNP, (2) FlexMAP microsphere hybridization, and (3) detection using the Bioplex suspension array system, which includes a fluorescence reader and the Bio-Plex Manager analytical software . This procedure is described in detail elsewhere . ConservTLR2, TLR9, and MAL SNPs. TLR4 SNPs were evaluated in a separate multiplexed LDR. Equal volumes of each PCR product were mixed, and 1 μl was added to the LDR. Conditions for the LDR step of this assay are described elsewhere [A multiplex assay was used for simultaneous detection of lsewhere .n, and allele B/A+B = Bn), where A and B are the 2 alleles of a SNP. To be homozygous for a particular allele, the ratio must be > 0.75. To be heterozygous, the ratio of the 2 alleles must be between 0.25 and 0.75. Consequently, an allele included in a ratio of < 0.25 is considered not present. Normalized values for An and Bn were divided, and the quotient was log-transformed. The mean and 95% confidence interval (CI) of the log-transformed quotients for the 777 individuals genotyped in this study are presented in additional file Mean Fluorescence Intensity (MFI) values were used to calculate the allelic ratio for each SNP by dividing the allele-specific MFI value by the sum of the MFI values for that SNP . A p-value < 0.05 was considered to be significant. Statistical comparisons of allele frequencies were made between the following groups; Kenya malaria holoendemic vs. Kenya episodic transmission: each of the Kenyan study populations vs. African American, Kenya holoendemic vs. Papua New Guinea holoendemic, and Papua New Guinea holoendemic vs. Asian Americans. Linkage disequilibrium analysis was performed using SHEsis [Allele (or haplotype) and genotype (diplotype) frequencies were calculated for the different polymorphisms, the Hardy-Weinberg exact test , and the Ewens-Watterson homozygosity test was performed for each population using Arlequin version 3.01 g SHEsis .TLR2, TLR4, TLR9, and MAL SNPs, TLR2 Δ22, and TLR2 GTn polymorphisms in the populations from Kenya, Papua New Guinea, and North America.Table TLR2 Δ22 polymorphism in the 5' untranslated region was present in all populations examined at an allele frequency >10%. The lowest value was in Papua New Guineans (13.1%), compared with 32.6% for the Kenyan episodic transmission site. Although the holoendemic Kenyan and Papua New Guinea sites have similar malaria transmission intensities, this allele was significantly less common in Papua New Guinea compared to the Kenya holoendemic site (p < 0.001). Allele frequencies were similar for Kenyan study participants from the holoendemic and episodic transmission sites.The n repeat in the 5' untranslated region of TLR2 was highly polymorphic in all populations examined. Consistent with how this genetic variation has been analyzed previously [The GTeviously ,49, the TLR2 SNP Arg753Gln is absent in Urat and Urim speakers from Papua New Guinea [A previous report showed that the w Guinea . Therefow Guinea .TLR4 SNPs examined in this study are absent in the Urat and Urim linguistic groups in Papua New Guinea [TLR9 SNPs were present at a frequency greater than 10%. Both -1486T/C and 1174G/A were more common in Papua New Guinea compared to the Kenya holoendemic site . There were no significant differences in TLR9 SNP frequencies associated with different malaria exposures in Kenya. The MAL 180 mutant allele was most common in Papua New Guineans (14.9%). In contrast, this allele was completely absent in the holoendemic Kenyan transmission site, and present in only 2.5% in the episodic transmission site.The two w Guinea  and therTLR2 Δ22, TLR4, TLR9, and the MAL polymorphisms. All three TLR2 Δ22 genotypes were found in all populations, and were in Hardy-Weinberg equilibrium. The TLR4, TLR9, and MAL SNPs were in Hardy-Weinberg equilibrium in both Kenya and Papua New Guinea. In contrast to Papua New Guineans where -1237C was absent, this allele was present in Asian Americans, for whom there were fewer heterozygotes than expected (p = 0.022). The MAL genotypes were in Hardy-Weinberg equilibrium in all populations except in African Americans, where there was a deficit of heterozygosity (p = 0.02).Table TLR2, TLR4, and TLR9 are described in Table TLR2 haplotype combinations, all were present in Papua New Guineans and North Americans. The deletion allele did not co-segregate with any of the GT repeat length alleles, as determined in linkage disequilibrium analysis where two polymorphisms were considered in linkage disequilibrium if the values for D' and r2 were >0.9 (data not shown). The Asp299Gly and Thr399Ile SNPs in the TLR4 gene commonly co-segregated in Caucasian Americans. Consistent with these data, the two polymorphisms were in linkage disequilibrium as measured by D' and r2 (data not shown). In Kenya, the TLR4 mutations co-segregated less frequently, and the Gly-Thr haplotype was present at 5–6% frequency in both the episodic transmission and holoendemic study sites. This haplotype was also found in African Americans and Asian Americans. There was no linkage disequilibrium between Asp299Gly and Thr399Ile in the Kenyans, African Americans, or Asians Americans (data not shown). The most common TLR9 haplotypes were CTG and TTA, except in Kenyans and African Americans, where TCG and TTA were the most common. There were fewer TLR9 haplotypes present in Papua New Guineans compared to the other populations, largely due to the absence of the -1237 allele. There was linkage disequilibrium between all TLR9 SNP pairs present in Papua New Guineans (data not shown). This linkage disequilibrium was absent in the Kenyan and Asian American populations.Statistically inferred haplotypes for There is compelling evidence that evolution of the human genome has involved selective pressure exerted by microbial pathogens. Perhaps one of the best characterized examples of this natural selection process is historical exposure to falciparum malaria and its' association with the β-globin SNP underlying the haemoglobin S sickle cell trait. The frequency of this mutant allele (and other β-globin variants such as haemoglobin C) is greater than 25% in populations in Sub-Saharan Africa where it has been observed to mediate a strong protective effect against severe malaria . Data reTLR2, TLR4, TLR9 and MAL because these genes have been implicated in human malaria pathogenesis. Evidence for this includes in vitro experiments in which engagement of the immune receptor by putative malaria toxins and activation of downstream signaling pathways lead to production of pro-inflammatory cytokines [TLR or MAL polymorphisms examined have been under strong selective pressure by malaria. Although the protective haemoglobin S and G6PD polymorphisms are significantly more common in Kenyan residents of the malaria holoendemic area compared to residents of the nearby episodic low transmission area [TLR or MAL polymorphisms were not detected. A notable finding in the Papua New Guinea population is concerned with the MAL SNP that confers a Ser→Leu amino acid change. The mutation abrogates downstream signaling following ligand engagement of TLR2 and TLR4. The frequency of the mutant allele in the Papua New Guinea population was high . Other Africans , increasAfricans . BecauseTLR2 5' untranslated polymorphisms in Kenyans and Papua New Guineans from malaria endemic areas. These polymorphisms presumably affect transcription and/or translation. The Δ22 polymorphism is located in the first untranslated exon of TLR2, approximately 60 bp downstream of an NF-kB site critical for chromatin remodeling and binding of transcription factors [in vitro phenotypes of Δ22 and GTn need to be investigated more thoroughly to determine their effect on mRNA transcription and/or translation. Although these polymorphisms have not been studied in malaria case-control studies, TLR2 is potentially involved in the pathogenesis of malaria because of its putative role as a receptor for P. falciparum-derived GPI [This study is the first to describe the  factors . The in a priori knowledge of their biologic function and contribution to malaria pathogenesis [Future studies with larger sample sizes, additional SNPs, and other genetic loci are needed to determine more completely whether falciparum malaria has been involved in natural selection of genes that regulate innate immunity in humans. Advances in analytic and molecular tools such as tagged SNPs, such as those now available in the International HapMap project, also make it possible to identify signatures of natural selection in these and other genetic loci without ogenesis .TLR and TLR adaptor polymorphisms under selective pressure by malaria. The data suggest that history of malaria exposure of the two Kenyan populations has not been involved in selection of TLR2, 4, and 9 or MAL polymorphisms. Case-control studies comparing the frequencies of these polymorphisms among individuals with severe malaria with those with asymptomatic malaria infection need to be conducted to define more completely the potential role of these innate immune genes in human malaria pathogenesis.The goal of this study was to identify The authors declare that they have no competing interests.JG performed and analyzed genotyping assays and drafted manuscript. JK conceived of the study, participated in its design and helped draft the manuscript. PZ participated in the design of the genotyping strategy and assisted in the analysis of the genotyping data. AM, JV, and MB were involved in collection of samples and the design of population-based field studies and helped write the manuscript.Malariometric characteristics of Kenyan and Papua New Guinean study subjects. This file contains age, sex, and parasitaemia information for the Kenyan and Papua New Guinean study participants.Click here for filePCR primers and amplification conditions. This file contains sequences for the PCR primers used and amplification conditions.Click here for fileLDR-FMA primers for SNP genotyping. This file contains the oligonucleotide sequences for the LDR-FMA reaction, as well as the SNPs examined and their accession number.Click here for fileMean and 95% confidence interval (CI) values of allelic ratios to determine genotypes. This file contains the mean and 95% confidence intervals of the MFI values from the LDR-FMA reaction used to determine genotypes.Click here for file


Quinine can also be administered by the intra-rectal route and, over the last ten years, a series of studies have been conducted in children to determine the ideal dose and dilution in the African situation. The aim of the present study was to evaluate the feasibility and usefulness of a kit for an immediate administration of quinine alkaloids (Quinimax® (20 mg/ml) via the intra-rectal route before transfer. Patients were followed through complete cure and parasitological tests were carried out on Days 0, 3 and 7.A prospective, open, descriptive community intervention study conducted in northern Senegal at six village Health Units in children fewer than ten years of age with non-per-os malaria. Controls were given the routine care prior to transfer to a Health Center, and cases were in addition administered Quinimax16.8 hours versus 33.6 hours in the control group. Time-to cure was shorter in cases than in controls. Complete parasite clearance was obtained in all patients by Day 7. The kit was well accepted by all concerned and more than 80% of community health workers judged the kit easy to use.134 patients (79 cases/55 controls) were recruited between November 2003 and May 2004 or October and November 2004. The two groups were comparable at inclusion. In the case group, oral drugs could be administered after a mean of The emergency paediatric kit is a useful tool in the management of malaria in children who cannot be treated orally. It is feasible and easy to use for health workers in community-based Health Units where, according to the WHO, nearly 80% of malarial morbidity and mortality occurs. Malaria is the most serious parasitic disease throughout the tropics. According to the World Health Organization (WHO), more than two billion people are exposed, most of them living in Africa, the Americas and Southeast Asia. Malaria accounts for about 25% of all children's deaths in Africa . Every yThe recommended treatment for severe malaria includes the injection, ideally intravenous, of quinine salts as soon as the first signs of severe malaria or danger manifest (WHO). However, community health structures possess neither the equipment nor the expertise to perform this type of procedure. The situation has to be improved so that all children who develop severe malaria can be treated as quickly as possible, beginning close to home. Intra-rectal administration could provide a solution to these problems. When oral administration is impossible, community-based workers in remote village health care units could administer treatment via this route, prior to transferring the patient to a more sophisticated establishment for comprehensive management.A number of studies conducted in various countries in Africa  have shown that intra-rectal administration of diluted quinine is an effective, safe form of treatment in the management of malaria in children -6.In Senegal, the health care system in more remote areas is based on village-based health units run by community health workers with no formal medical or paramedical qualifications.non per os malaria).The aims of this study were (i) to study the feasibility of an emergency pediatric kit when used by community health workers; (ii)to evaluate the acceptance of this form of treatment; (iii) to evaluate the therapeutic benefit of immediate intra-rectal quinine administration in children with malaria who cannot be treated orally . The climate is Sahelian with a long dry season between November and June, and a short rainy season from July to October.Anopheles pharoensis and Anopheles gambiae s.l. [Malaria is unstable in this region with great variability from one spot to another. The main vectors are iae s.l. ,8. The Piae s.l. . AccordiA prospective, descriptive community intervention study was conducted in children consulting community health workers in six villages around the Savoigne Health Center Table . Most ofnon per os malaria were given conventional treatment before referral to the Health Center contains 3 villages. The second group of 3 villages named case group (or intervention group) in which the children received intra-rectal quinine before transfer. These 2 groups were made in order to describe the reference towards the health center whether or not a administration of quinine alkaloids was done to children with a non per os or severe malaria attacks in a same area at the same time. In order to investigate whether or not the supplementary treatment delayed referral, the research team did not get involved with the transfer of the sick children to the Health Center.The six villages were randomized into two groups of three. The first group named a control group in which the children suffering from severe or non per os malaria). Children in whom intra-rectal quinine administration was contra-indicated  were excluded from the case Group. Parental consent was obtained before enrolment.Included were all children of under 25 kilograms and/or 10 years of age presenting with a suspected malarial attack who could not be orally treated . In the case villages, the included children were also given an intra-rectal dose of 20 milligrams of quinine alkaloids per kilogram body weight (diluted in water to 30 mg/ml). The paediatric kit contains;• one phial of 4 ml wich contains 500 mg of quinine alkaloids composed by : 480 mg of quinine , 13,2 mg of quinidine , 3,4 mg of cinchonine  and 3,24 mg of cinchonidine . This quinine is to be diluted into• A bottle of 13,5 ml of purified water,• a syringe graduated according to children bodyweight and age wich to administrate by rectal route the reconstituted solution by rectal routeThe child was then monitored for thirty minutes. During this period, if any of the product was discharged, a further half-dose was administered before referral.On arrival at the Health Center, a blood smear was performed to confirm the diagnosis and, for those with demonstrated malaria, treatment was administered in accordance with current National Malaria Control Programme recommendations . As soon as possible, the switch was made to an oral regimen with the currently recommended combination (SP + amodiaquine). At the Health Center, the patient's clinical condition was monitored and parasitological tests were performed every day until oral drugs could be administered. The patients were followed up through at least Day 7.Two populations were analysed: Population 1 covering all eligible patients included at the village units who were referred to the Health Center; and Population 2 covering all eligible patients included at the village units who were found to have a confirmed parasitaemia on arrival at the Health Center.The statistical analysis was performed using SAS software Version 8.2 . Descriptive analyses were carried out for each treatment group. The statistical significance threshold was set at 5%. The analysis describe the time-to referral and clinical outcome in the intervention zone  versus the control group or non intervention zone.A total of 134 children (79 in case villages and 55 in control villages) were included in two phases, the first between November 2003 and May 2004, and then from October to November 2004.At inclusion, demographic characteristics were the same in the two groups Table The time elapsed since appearance of the first symptoms was the same (mean = 52 hours in both groups) and symptoms were similar – exceptions were the incidences of: hallucinations reported by 32.9% (n = 26) cases versus 45.5% (n = 25) controls; and agitation reported by 51.9% (n = 41) cases and 70.9% (n = 39) controls .After pre-calibrated dilution of the QuinimaxThe administered dose was considered correct if the dose administered corresponded exactly to the child's weight: it was found to be correct in 93.6% of cases Table .®.Administering the product was deemed easy in 77.2% of cases, and some of the difficulty of administration may be related the patients' reactions which were observed in 39% (n = 31), including agitation, crying out and tears (probably due to fear on introduction of the device). No parental reactions were reported. In 25% of cases, early discharge of the product within 30 minutes of the first dose necessitated the administration of a further half-dose of intra-rectal QuinimaxAll 134 patients were referred to the Savoigne Health Center. The mean time-to transfer in this rural area was 4.40 hours (± 11.07) in the case group, and 3.11 hours (± 5.76) in the control group for the parents to bring children from their villahe to the health post.This end point was evaluated by comparing the cases and the controls with a confirmed parasitaemia (n = 92) vis-à-vis fever and parasitaemia over time, as well as the time intervals to obtain cure, to stop vomiting and to the switch to oral drugs.On Day 1, fewer cases than controls had fever (71.4% versus 93.0%). This difference persisted through Day 2 but had disappeared by Day 3  and the switch to oral drugs was significantly shorter in the case group than in the control group (0.7 days versus 1.4 days) Table .Similarly, the interval between consultation and cure was shorter in the case group than in the control group .On Day 3, blood smear were carried out in the 92 patients with parasitaemia on arrival at the Health Center. Parasite clearance was recorded in 74.4% of cases and 69.2% of controls (NS)  collected at the Health Center were analysed for all included patients.On arrival at the Health Center, a difference was observed between the two groups in the mean number of feces in the preceding 24 hours: 1.2 in the case group versus 0.1 in the control group (p < 0.0001).Of the village children who passed a stool on arrival at the Health Center (78 including six controls), eight cases passed liquid stools compared with three controls (Table In the following days (Day 2 through Day 5), the number of faeces per day in the two groups became comparable Table .In the course of this Study, one control child died after having been referred from the Health Center to Saint Louis Regional Hospital  with severe dehydration and clinical anaemia.This study was conducted to evaluate the feasibility and safety of an emergency paediatric kit when used by unqualified, community-based personnel, and to assess acceptance of the kit by local people.The community-based health workers were observed to appreciate this novel therapeutic device.Malaria was incorrectly diagnosed by community health workers in 22% of cases in this Study. The mean percentage of mistaken diagnoses has been estimated at 45% in certain health care structures in Senegal , and at The administration of treatment prior to referral did not delay transfer of children to a more sophisticated health care facility for parenteral management.The emergency paediatric kit is an effective tool in the early management of malaria in children who cannot be treated orally. Its use significantly cut down the period of time before the switch could be made to oral drugs, and accelerated clinical cure.The percentage of children in whom early discharge of the administered product was observed was higher in this study 25%) than those reported by Barennes 12.9%)[5% than t.9%5% th. No partNo serious adverse reaction to the product was observed in the course of this study, and none had been reported in either Madagascar, where intrarectal quinine administration has been common since 1985  or in clThe paediatric kit used in this study provides a useful therapeutic option for early, community-based malaria management in children who cannot be treated orally, until they can be transferred to a more sophisticated health care establishment for definitive care. It is feasible at the community level and simple to use. Its generalization should be accompanied by the training of community health workers on how to use it, as well as its specific indications and contraindications.The author(s) declare that they have no competing interests.JLAN: designed the study, collected data, and prepared the manuscriptRCT: collected data, and prepared the manuscriptBF : contributed in the preparation of the manuscriptVL: designed the study and prepared the manuscriptELD and PAD: collected data and participated in the design of the studyOG: designed the study and prepared the manuscriptHDS: designed the study and prepared the manuscriptAll authors read and approved the final manuscript


Malaria represents a huge burden for the health care services across Africa. Describing malaria attending health services contributes to quantify the burden and describe the epidemiology and clinical presentation.Plasmodium falciparum parasitaemia of any density in the blood smear.Retrospective analysis of data collected through the Manhiça morbidity surveillance system (Mozambique) on all paediatric visits (<15 years) to the outpatient clinic from June 2003 to May 2005. Age-specific minimum community-based incidence rates (MCBIRs) of malaria were calculated using demographic surveillance system data. Malaria was defined as fever or history of fever in the preceding 24 hours with asexual A total of 94,941 outpatient visits were seen during the study period, of which 30.5% had malaria. Children younger than three years accounted for almost half of the total malaria cases and children aged ≥ 5 years represented 36.4% of the cases. Among children who presented with malaria, 56.7% had fever and among children who presented with fever or a history of fever only 37.2% had malaria. The geometric mean parasitaemia in malaria cases was 8582.2 parasites/μL, peaking in children aged two to three years. 13% of malaria cases had a PCV<25% and the mean PCV in malaria cases increased gradually with age, ranging from 27.8% in children aged 2–12 months to 34.4% in ≥ 5 years. The percentage of cases admitted or transferred showed a clear decreasing trend with age. MCBIRs of outpatient malaria per 1,000 child years at risk for the whole study period were of 394 in infants, 630 in children aged 1 to <5 years and 237 in children aged five years or more. A clustering of the cases was observed, whereby most children never had malaria, some had a few episodes and very few had many episodes.Preventive measures should be targeted at children younger than three years, as they carry the highest burden of malaria. Children aged 5–15 years represent around a third of the malaria cases and should also be included in control programmes. Concern should be raised about presumptive treatment of fever cases with artemisinin-combination therapies, as many children will, according to IMCI guidelines, receive treatment unnecessarily. Malaria continues to be the first single cause of mortality in children in sub-Saharan Africa  and a maIn Mozambique, as in most sub-Saharan African countries, the health management information system is weak . TherefoData are presented on the characteristics of malaria in children who attend the outpatient clinic of a rural hospital and minimum community-based incidence rates in a rural area of southern Mozambique. Data on children with malaria admitted to the same hospital are presented in a companion article .The study area is located in Manhiça, Maputo Province, southern Mozambique. The area has been described in detail elsewhere . There aPlasmodium falciparum, is perennial with substantial seasonality and of moderate intensity. The average entomological inoculation rate (EIR) was of 38 infective bites per person per year in 2002, being Anopheles funestus the main vector [Malaria transmission in the area, mainly caused by n vector ,11.Malaria control in the area is based on prompt treatment of cases, the coverage of insecticide-treated nets being <10% throughout the study period. The Manhiça District Hospital is the main health facility in the area, used for primary health care by the nearby population, and one of the two referral health centres for Manhiça District. It has a 110-bed inpatient ward, an outpatient clinic, a maternal and child health clinic with a small surgical room, and an emergency room . There aThe Manhiça Health Research Center  runs since 1996 a Demographic Surveillance System in the area . All chist of June 2003 to 31st of May 2005 were analysed.Retrospective analysis of data collected through the Manhiça morbidity surveillance system. All paediatric visits (<15 years) to the outpatient clinic from 1A passive case detection system was established in 1996 to cover all paediatric (children aged 0–<15 years) outpatient and inpatient visits to Manhiça District Hospital and all outpatient visits to the Maragra Health Post. A standardized questionnaire, which includes personal and demographic data (including the permanent identification number) and clinical signs and symptoms, is completed for each child seen at the outpatient clinic. The clinician on duty, usually a medical agent , records the physical signs found on examination and the symptoms and duration of those as referred by the child's guardian. The axillary temperature is taken with an electronic thermometer and a finger-prick blood sample is collected from children who present fever (axillary temperature ≥ 37.5°C) or report a history of fever in the preceding 24 hours. Blood is collected into heparinized capillaries to measure the packed cell volume (PCV) and thin and thick blood smears are prepared to determine parasitaemia. Final diagnosis/es are recorded on the questionnaire by the clinician upon discharge or admission to the ward, after review of all signs, symptoms and laboratory results.Children seen at the outpatient clinic of Manhiça District Hospital are either sent home after diagnosis and treatment, or, if they fulfill severity criteria, are referred to the day-care unit of the hospital. The day-care unit was created by CISM to operate as an intermediate step between the outpatient clinic and the paediatric ward. Children seen at the outpatient clinic who fulfill severity criteria or need close monitoring are referred to the day-care unit, where they are reassessed by a more senior clinician, stabilized, and they receive initial treatment. This unit only works during the day and before it closes all children are either discharged, after a final diagnosis and treatment, or admitted to the wards. The referral hospital for Manhiça is the Maputo Central Hospital, where some of the children are transferred, being the unavailability of blood for transfusion the main reason.® was the second line treatment. Patients who were referred to the day-care unit and later to the wards received parenteral quinine for a minimum of six doses, followed by SP, or continued up to 21 doses if given as monotherapy.Children seen at the Maragra Health Post fulfilling severity criteria are transferred to the Manhiça District Hospital, where they follow the standard circuit. Malaria cases were treated according to the Mozambican national guidelines. The first line treatment for ambulatory patients was chloroquine until it was changed in 2003 to sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ). In 2006 it was changed again to SP plus artesunate. During the study period the first line treatment was SP+AQ  and Co-The PCV was measured using a microhaematocrit centrifuge and a Hawksley reader . Thin and thick blood films were read to quantify parasitaemia in the CISM laboratory according to standard procedures. Blood films were air-dried, Giemsa-stained, and examined using a light microscope fitted with a 10× oil immersion lens. Slides were declared negative only after 2000 leukocytes have been counted. Parasite numbers were converted to a count/μL by assuming a standard leukocyte count of 8000/μL.P. falciparum asexual parasitaemia of any density. No age cut-off for the level of parasitaemia was used to increase the specificity of the definition, as this is the definition used at the outpatient clinic for all ages to diagnose malaria and administer antimalarials.Malaria was defined as fever (axillary temperature of ≥ 37.5°C) or a history of fever in the preceding 24 hours plus a Mild anaemia was defined as a PCV between 25% and <33%, moderate anaemia as a PCV 15% to <25% and severe anaemia as a PCV<15%. Hyperpyrexia was defined as a measured axillary temperature of ≥ 39°C. The rainy season was defined as November to April and the dry season as May to October.Questionnaires were double entered into databases using a programme written in Fox Pro  at CISM. Statistical analyses were performed using Stata 9 . The DSS permanent identification number, which is recorded on the morbidity questionnaires when children attend the hospital, was used to classify children as residents/non-residents in the study area. Occasionally, the mother/accompanying person fails to show the card when attending the hospital and the number is not recorded, thus the number of outpatient visits of children from the study area are underestimates.Minimum community-based incidence rates (MCBIRs) were calculated as the age-specific yearly number of malaria cases visited at the outpatient clinic in children resident in the study area divided by the total child years at risk (CYAR) for that age group and year. CYAR were estimated from the DSS databases and children did not contribute to the numerator or denominator for a period of 28 days after each episode of malaria, when they were outside the study area or after death. Rate ratios and their p-values were estimated using the Mantel-Haenzel method to compare age-specific and year-specific rates.2 test or Fisher's exact test. Means of normally distributed variables were compared using the Student's t-test or ANOVA.Qualitative variables were compared using a χGeometric means of parasitaemia were compared using the Wilcoxon Rank sum test.Parasitaemia results were missing for 1.8% and PCV results for 1.5% of the outpatients who had fever or a history of fever. Age was missing in 0.2% of the outpatients.A total of 94,940 visits were seen at the outpatient clinic of Manhiça District Hospital and Maragra Health Post during the two year study period. Of these, 30.5%  had malaria, ranging from 34.1% in the first year of surveillance to 26.5% in the second year. Males accounted for 50.5% of the malaria cases.The age distribution of malaria cases is shown in Figure 2(1 d.f.) = 69.5, p < 0.0001).Figure Table The geometric mean parasitaemia (GMP) increased with age during the first three years of life, peaking in children aged two to three years and then decreased gradually with age. Figure 2(1 d.f.) = 3277.2, p < 0.0001), and 0.9% had severe anaemia, compared to 0.1% of the non-malaria cases (χ2(1 d.f.) = 242.1, p < 0.0001). The mean PCV was significantly lower in malaria cases  than in non-malaria cases . The mean PCV increased gradually with age both in malaria and non-malaria cases, although the increase was greater among malaria cases, in which the PCV ranged from 27.8% in infants aged 2 to 12 months to 34.4% in children aged 5 years or older (p < 0.0001 ANOVA and linear regression). The mean PCV difference between malaria and non-malaria cases was 4.9% (95% CI 4.7–5.1) in infants and 1.8% (95% CI 1.7–1.9) in children aged five years or above. The prevalence of moderate and severe anaemia presented no seasonality, being the same during the rainy and dry seasons both in malaria and total patients, although 63% and 60% of the total moderate and severe anaemia cases respectively occurred during the rainy season (p < 0.001).More than half of the malaria patients were anaemic, 11.9% had moderate anaemia, compared to 2.0% of the non-malaria cases  on average, while among children aged five years or older, this decrease was of 0.07 units .2(1 d.f.) = 344.7, p < 0.0001). Also, among malaria cases, children resident in the study area were transferred to the day-care unit and then admitted to the wards with a significantly lower frequency than children from outside the study area  = 371.7, p < 0.0001 and 45.4% vs. 62.0% of those transferred to the day-care unit, χ2(1 d.f.) = 225.1, p < 0.0001, respectively).Table Minimum community-based incidence rates of outpatient malaria per 1000 CYAR for the whole study period were of 394 in infants, 630 in children aged 1 to <5 years and 237 in children aged five years and older. Table The incidence of malaria in children aged 1 to 4 years was significantly higher than in infants , whereas the incidence in children aged 5 years or older was lower than in infants .The incidence in the first year of the study period was significantly higher than that during the second year , this difference being highest in infants .This paper describes the characteristics of children presenting with clinical malaria to the outpatient clinic of two health facilities in a rural area of southern Mozambique. Malaria represents a huge burden for the primary health care services, accounting for around 30% of the total visits. This implies a considerable health service utilization derived from one single disease, which is likely to translate into a significant health expenditure.The age distribution of the cases is typical of an area of moderate transmission, with most of the cases occurring in children younger than 5 years, with a peak in children aged 1 to 2 years . DespiteThe age distribution of outpatient malaria is more spread out than that of malaria requiring admission to the wards, as children younger than three years represent 45% of the cases among outpatient attendees, while children younger than two years account for 58% of the cases among admitted children , as the Infants, who suffer an important incidence of malaria, are transferred to the day-care unit and admitted to the wards with a significantly higher frequency than older children, reflecting either a greater severity of disease in infants, a high co-morbidity and/or a tendency of the medical staff to admit them more easily. Malaria control strategies should target children younger than three years, the age group most at risk, and particularly infants, who carry a disproportionate amount of severe disease.Presentation of malaria cases to the outpatient clinic showed marked seasonality, with a peak in December each year. This seasonality is related to the actual rainfall, but the peak in malaria cases does not always follow the peak in the rainfall, as shown in Figure According to the Integrated Management of Childhood Illness, children with a measured or reported fever in high malaria risk areas, and in the absence of facilities equipped with a microscope, should be classified as having malaria and receive presumptive antimalarial treatment . In thisOnly 57% of children with malaria presented with a measured fever, which indicates that if measured fever was used as the only criterion to collect a blood slide many cases would not be detected and treated.Parasite density was higher in admitted children, probably reflecting that high parasite levels on its own are very often a criterion for admission, especially when children are seen by a medical agent and not by a physician. Nevertheless, the level of parasitaemia – although it was associated with lower PCVs – was not found to be a risk factor for severity of malaria neither in admitted children in this area  nor elseThe decrease of parasitaemia density and the increase of the mean PCV with age reflect the development of naturally acquired immunity against malaria. The peak of the incidence of clinical malaria is in children aged one to three years, and coincides with the peak in parasitaemia density, that occurs in children aged two to three years. This argues against the widely accepted hypothesis that anti-disease immunity is different from anti-parasite immunity and supports the idea that actually these two immunities are the same and develop at the same time . In an aMore than half of malaria cases present with anaemia, being moderate or severe in 13% of the cases. The aetiology of anaemia in Manhiça and the relative contribution of malaria to its development have not been determined, but malaria appears to be an important contributor, as children with malaria have a significantly lower PCV than children without malaria. This difference in the mean PCV between malaria and non-malaria cases is greater in younger than in older children, suggesting that malaria is a greater contributor to anaemia in infants and young children . This isThe malaria MCBIR in children younger than five years from Manhiça, 575.7 per 1,000 CYAR, is within the interval of the estimated incidence rate for that age group in Africa in 2000 for rural areas with an EIR < 100 infective bites per person per year (414 – 1924 per 1000 CYAR) . The malThe MCBIRs show a 40% decrease from the first year to the second year of the study. This reduction in the incidence shifted the age distribution to the right, being this decrease highest for infants. The rate ratio of the incidence in children aged one to four years old versus infants was highest in the second year. This change also illustrates the interannual variability in malaria incidence, which can be due to multiple reasons, discussed more thoroughly in the second part of this article .More than 70% of the study area children have never presented to the health facilities with malaria, and among those who have, more than half had only one episode. These results are very similar to those found during the active case detection study, where 71% of children never had malaria and 50% of those who did, only had one episode, and to the results of the control group of the RTS, S malaria vaccine trial conducted in the area, where these percentages were even higher (80.5% and 70% respectively) . This suThis article presents a retrospective analysis of outpatient data on malaria in children. Despite the limitations of hospital-based data, these provide an indication of the burden and age pattern of the disease, that may guide policy makers. Children younger than three years carry the burden of malaria, and control strategies should be targeted at them. Children between five and 15 years also suffer an important proportion of the disease, although less severe, and should be included in control programmes. Moreover, this would also decrease the enormous toll paid by health facilities to manage this disease. Preventive measures together with effective treatment of correctly diagnosed cases should on the long term guarantee that malaria ceases to be the number one cause of childhood disease in Africa.The hospital surveillance system and the DSS in Manhiça were designed by JJA, CM and PLA. They were set up in 1996 and have received numerous contributions to its design and implementation since then. During the study period QB, EM, PA, BS, AB, JS and TN were involved in the diagnosis and management of malaria patients and collection of data. AN coordinates the DSS in Manhiça. CG and QB led the analysis, interpretation and write up of this data set, and received input from all authors. All authors read and approved the final manuscript.


Malarial anaemia is an enormous public health problem in endemic areas and occurs predominantly in children in the first 3 years of life. Anaemia is due to both a great increase in clearance of uninfected cells and a failure of an adequate bone marrow response. Odhiambo, Stoute and colleagues show how the age distribution of malarial anaemia and the haemolysis of red blood cells may be linked by an age-dependent increase in the capacity of red blood cells to inactivate complement components absorbed or deposited directly on to the surface of the red blood cell. In this commentary, we discuss what has been established about the role of complement deposition on the surface of red blood cells in the pathology of malarial anaemia, how genetic polymorphisms of the complement control proteins influence the outcome of malaria infection and how the findings of Odhiambo, Stoute and colleagues and others shed light on the puzzling age distribution of different syndromes of severe malaria. In the accompanying article, Odhiambo, Stoute and colleagues show how the age distribution of malarial anaemia and the haemolysis of red blood cells (RBCs) may be linked by an age-dependent increase in the capacity of RBCs to inactivate complement components absorbed or deposited directly on to the surface of the RBC . The worMalaria remains an enormous problem in public health around the world . Over 2 Furthermore, there remain major unsolved problems about the fundamental pathophysiology of all syndromes of severe malaria. The rapid drop in haemoglobin during acute infection and the slower decline in chronic infection appear to be due to increased extravascular haemolysis of RBCs with a concomitant failure of the bone marrow to increase red cell production to compensate for these losses .The increased clearance of infected cells is readily explained by the rupture of cells after completion of the parasite's intra-erythrocytic life cycle and opsonisation and clearance of intact infected RBCs. Rather less obvious is why and how uninfected cells are also cleared. It has been estimated that approximately 10 uninfected cells are cleared from the circulation for every infected cell and so the clearance of uninfected cells is of crucial importance for the development of malarial anaemia .Why are uninfected RBCs cleared in such large numbers? Certainly the number and activation of splenic and other macrophages for phagocytosis of red cells is greatly increased during malarial infection -9. The iUninfected RBCs have a reduced deformability leading to enhanced clearance in the spleen and a severe reduction in red cell deformability is also a strong predictor for mortality measured on admission, both in adults and children with severe malaria ,11. SecoThe role of immunoglobulin and complement in marking uninfected RBCs for clearance by phagocytes was first studied by Facer and colleagues ,13 in ThThe story of how absorbed immune complexes may contribute to increased clearance of uninfected RBCs lay dormant for 20 years when Waitumbi, Stoute and colleagues based in Western Kenya began to study how immune complexes caused haemolysis . AppreciHere, a number of proteins are involved in the control of complement activation. Complement receptor 1 (CR1 or CD35), decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59) may enhance binding of C3b in immune complexes (CR1), enhance inactivation of C3 convertases (CR1 and CD55) and interfere with the assembly of the terminal components of complement that form the membrane attack complex (CD59) . Immune in vitro than RBCs from controls. Decline in CD35 or CR1 expression and increases in immune complexes bound on uninfected RBCs were associated with anaemia but these declines in CD35 (CR1) and CD55 expression were only transiently associated with malaria infection and levels returned to normal after infection had been cleared [In previous papers, Waitumbi, Stoute and colleagues have shown that the amount of red cell surface IgG is increased but red cell surface CR1 and CD55 reduced in children with severe malaria compared with asymptomatic and symptomatic controls . The dif cleared . These dHow does this relate to the age-dependent incidence of malarial anaemia? Population studies in Europe and Africa showed the CR1 expression was strongly age-dependent and increases of both CR1 and CD55 were seen after 4 years of age and low levels of CR1 and CD55 expression were seen in a cases of severe malarial anaemia compared with slightly older children with cerebral malaria . OdhimboHow do these findings help explain the age distribution of syndromes of severe malaria? If clearance of the immune complexes absorbed on to the surface of uninfected cells is reduced in younger children expressing lower levels of CD35 and CR1, then clearance of these cells would be increased, leading to more anaemia in these younger age groups.Genetic polymorphisms also affect the expression levels, sequence and domain  structure of CR1 in Africans and other populations ,20. MoreIt is possible therefore, that age-related and genetically determined reduction of levels of CR1 expressed on RBCs are associated with an increased susceptibility to anaemia but protection from other forms of severe malaria and may provide an example of how innate resistance to one syndrome of malaria may be at the expense of susceptibility to other pathophysiological pathways involved in malaria infection.These hypotheses should ideally be tested in longitudinal studies using genetically and phenotypically well-characterised children to ascertain the levels of these age-dependent, complement regulatory proteins prior to infection and determine their association with haemoglobin levels during acute infection and with the incidence of severe disease through childhood. Such studies can only be done in a few sites in Africa where large populations can be followed before and after infection and presentation to a health facility using a demographic surveillance system.In assessing the role of these age-related changes to the complement regulatory proteins in the differential presentation of anaemia and coma, one would also have to consider the recently described findings of increased levels of erythropoietin (EPO) seen in younger children with anaemia  and malaBMC Medicine provide us with a clearer understanding of the causes of anaemia in children with malaria. Translating this new understanding of pathology into fruitful avenues to investigate prevention and treatment of malaria is perhaps the greatest challenge of clinico-pathological studies in this and other disciplines.Finally, we would ask where these studies will lead in the quest for new methods to prevent or treat malarial infection. The findings of Odhiambo, Stoute and colleagues presented in the accompanying article in CR1: complement receptor 1; DAF: decay accelerating factor; DCT: Direct Coombs' Test; EPO: erythropoietin; MIRL: membrane inhibitor of reactive lysis; RBC: red blood cell.The pre-publication history for this paper can be accessed here:


Malaria is one of the major human infectious diseases in many endemic countries. For prevention of the spread of malaria, it is necessary to develop an early, sensitive, accurate and conventional diagnosis system.Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip.A cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured The potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10–100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes. Ltd.   [14]. ThP. falciparum strain 3D7 cells were cultured in RPMI 1640 medium  containing 50 µg/ml gentamycin  and 10% O+ human serum at a hematocrit of 5%, according to the established method of Trager and Jensen g for 10 min and then washed three times with culture medium to obtain human erythrocytes. The supernatant and buffy coat were carefully removed by aspiration after washing, and the leukocytes remaining among the washed erythrocytes were removed with a leukocyte isolation filter, LeukoLOCK™ , under gravitational force. It required less than 1 min for this isolation of leukocytes with LeukoLOCK™. The purified erythrocytes were employed for the malaria culture. The parasite concentration (parasitemia) was calculated by determining the number of malaria-infected erythrocytes among 30,000 erythrocytes and expressed as the ratio to non-malaria-infected erythrocytes. Erythrocytes exhibiting 0.1% parasitemia were added to each plate in 10 ml of culture medium to give a final hematocrit of 3%. The plates were incubated at 37°C under 5% CO2, 5% O2, and 90% N2 gas.Two microliters of a malaria culture was smeared so as to produce a thin film on a slide. Each slide was stained with 5% Giemsa  stain in phosphate-buffered saline (pH 7.2), and then examined under a light microscope , at a magnification of ×1,000, for the presence or absence of malaria parasites For tight confinement of erythrocytes in the microchambers, erythrocytes in RPMI 1640 medium at a hematocrit of 0.25%, 0.5%, 0.75%, or 1.0% were dispersed on a cell microarray chip, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers under gravitational force. The microchambers were then examined by light microscopy.2, and is fitted with filters with emission wavelengths of 535 and 585 nm. The fluorescence intensity of individual erythrocytes was determined with DNASIS Array version 2.1 software , and the erythrocytes that exhibited fluorescence intensities of two times above and ten times below that of uninfected erythrocytes were taken to be malaria-positive ones. The presence of malaria parasites in the fluorescence-positive erythorocytes was confirmed by Giemsa staining.For analysis of cultured malaria-infected erythrocytes on a cell microarray chip, an appropriate volume of a purified malaria-uninfected erythrocyte suspension in RPMI 1640 medium was added to a malaria culture (0.4 to 1.0% parasitemia) to give 0.0001%–0.01% parasitemia and a 0.75% hematocrit, this step being performed within 5 min. An aliquot of a 5 mM stock solution of SYTO 59 , which is a nuclear-specific fluorescence dye , was added to each erythrocyte suspension to give a final concentration of 1 µM g for 2 min, and the pellet was dispersed in 1.0 ml of RPMI 1640 medium to give an appropriate hematocrit. SYTO 59 was added to the erythrocyte suspension, and the mixture was subjected to analysis on a cell microarray chip as described above. The effect of leukocyte contamination on a cell microarray chip analysis was examined.For the preparation of erythrocytes from human whole blood without malaria infection, 20 µl of whole blood was mixed with 10 ml of RPMI 1640 medium, and the mixture was passed through LeukoLOCK™ under gravitational force. The isolation of leukocytes was performed with this isolation filter in less than 1 min. The eluted portion was centrifuged at 1,000×The number of fluorescence-positive erythrocytes was determined for each parasitemia sample, respectively. Data are expressed as the means ± standard error for five different experiments.To achieve the confinement of erythrocytes in the microchambers, the hydrophilicity on the microarray chip surface was optimized by means of reactive ion-etching exposure Malaria-infected erythrocytes in SYTO 59 for staining of malaria parasite nuclei were dispersed on a cell microarray chip and then scanning images were obtained . FluoresTo examine the possibility of contamination by leukocytes of purufied erythrocytes from whole blood, purified erythrocytes stained with SYTO 59 were analyzed on a cell microarray chip as described above. Similar accommodation of erythrocytes with tight confinement and monolayer formation in the microchambers using a malaria culture was observed on analysis of erythrocytes purified from whole blood , the numPlasmodium species of human is present in a positive sample with a cell microarray chip, high accuracy was obtained because only malaria-infected erythrocytes among mono-layered erythrocytes were targeted and detected with a confocal fluorescence laser scanning system. Erythrocytes for malaria cultures were prepared by centrifugation and a leukocyte isolation filter, and there is little possibility of contamination by means of leukocytes or cell debris of the sample for the cell microarray chip. The contamination risk is very low because we never found leukocytes or cell debris among over 100 fluorescence-positive erythrocytes on this cell microarray chip using a malaria culture. Using purified erythrocytes from human whole blood for cell microarray chip analysis, less than one leukocyte per 2,700,000 purified erythrocytes from whole blood was observed. If there is contamination by leukocytes in the erythrocyte suspension, it is relatively easy to distinguish malaria-infected erythrocytes and leukocytes by comparing their fluorescence intensities. The nuclei condensability in leukocytes is apparently greater than that in malaria parasites Our cell microarray chip exhibits 10–100 times higher sensitivity than that of light microscopy with Giemsa staining, and only 15 min is required for the detection of malaria parasites in purified erythrocytes. Although other more advanced methods based on flow cytometry or real-time PCR have been developed, the detection limit is only 0.003% for the flow cytometry measurement for parasitemia in malaria cultures with nuclear staining with YOYO-1 et al.P. falciparum is 11.9-fold per 48 hours. So, at least 2–4 days earlier detection of malaria is expected with our cell microarray chip than with conventional microscopic examination because of the 10 to 100 times higher sensitivity. Such diagnosis may lead to the detection of malaria before the appearance of some symptoms. Although the cost of a cell microarray chip and the dye is less than US$ 1.0, the cost is US$ 40 for the isolation of leukocytes with LeukoLOCK™. Furthermore, a confocal laser-based fluorescence microarray scanner is a precision machine and expensive, about US$ 100,000. A cell microarray chip is suitable at room temperature, but fluorescence dye SYTO 59 must be stored at −20°C until use. So, this method is not useful in a field setting. It is recommended that the treatment response should be assessed by a daily parasite count until clearance of all trophozoites is achieved Plasmodium falciparum infection cause serious difficulty in the identification and monitoring of malaria episodes Prompt and accurate diagnosis of malaria is a key factor for preventing contagion expansion. It has been reported that the median incubation period in nonimmune individuals (time from sporozoite incubation to development of symptoms) is 11 days


Control measures which reduce individual exposure to malaria are expected to reduce disease, but also to eventually reduce immunity. Reassuringly, long term data following community wide ITN distribution show sustained benefits at a population level. However, the more common practice in Sub-Saharan Africa is to target ITN distribution on young children. There are few data on the long term outcomes of this practice.Episodes of febrile malaria were identified by active surveillance in 383 children over 18 months of follow up. In order to compare the short and long term outcomes of ITN use, we examined interactions between ITN use and age (12–42 months of age versus 42–80 months) in determining the risk of febrile malaria. ITN use and older age protected against the first or only episode of malaria . The interaction term between ITN use and older age was HR  = 2.91, 95%CI 1.02–8.3, p = 0.045, indicating that ITNs did not protect older children. When multiple episodes were included in analysis, ITN use and older age were again protective against malaria episodes (Incident Rate Ratio [IRR]  = 0.43 95%CI 0.27–0.7) and IRR  = 0.23, 95%CI 0.13–0.42, respectively) and the interaction term indicated that ITNs did not protect older children .These data on age interactions with ITN use suggest that larger scale studies on the long term individual outcomes should be undertaken if the policy of targeted ITN use for vulnerable groups is to continue. Malaria is a global public health problem. There were over 500 million episodes globally in 2002, and over a million deaths in Africa The relationship between malaria transmission and morbidity/mortality is not linear There are few data on the long term effects of individual ITN use. In order to examine the long term outcome of ITN use, we examine the interactions between ITN use and age of the children in observational data from Kilifi district, Kenya. We compare residence in villages at lower transmission intensity with the effect of ITN use.Ethical approval was obtained from the Kenyan Medical Research Institute National Ethics Committee, the Central Oxford Research Ethics Committee, and the London School of Hygiene and Tropical Medicine Ethics Committee. Parents of all children were approached for written informed consent before research began. The data presented here were generated during a randomised, controlled and double blind vaccine trial of an ineffective candidate vaccination to malaria. The details of study design are described elsewhere The study was carried out in Junju sublocation in Kilifi District, on the Kenyan coast. Junju contains a group of 5 closely related villages within the Chonyi area of Kilifi district. Kilifi is malaria-endemic, with all year round transmission and two high transmission seasons The participating children were aged 1–6 years old (inclusive) at enrolment, healthy, and resident in Junju sublocation, Kilifi District. They were recruited following public meetings to invite potential participants.Children were visited every week by fieldworkers and their axillary remperature was recorded. When the temperature was greater than 37.5 degrees, a blood film was made and a rapid near-patient test for malaria conducted. If the rapid test was positive, a full course of artmether/lumefantrine was given. Where the axillary temperature was less than 37.5 degrees, but the parents reported a fever, the fieldworkers returned three times over 24hours to take repeated measurements. If the child was febrile, a blood film and rapid test were taken. Parents could bring their children for assessment at any time, and the fieldworkers were resident in the study area. Cross-sectional blood films were performed on all children after 3 months of follow up.Bednet use was assessed by fieldworkers visiting the subject's home at the beginning of the study, at 4–5 monthly intervals and at the end of the study. They observed whether the bednet was hung over the child's sleeping space, asked whether it had been recently treated, and examined the number of holes that could admit a fingertip.Febrile malaria was defined as an axillary temperature greater than 37.5 degrees centigrade, with a Plasmodium falciparum parasitemia greater than 2,500 parasites per µl Subjects came from 5 villages. Transmission intensity for each village was determined by the rate of first reinfection occurring over three months monitoring following curative anti-malarial therapy (including both febrile and asymptomatic infection). Two villages formed the “high transmission” group (n = 207) and the remaining three villages formed the “low transmission” group (n = 176). Transmission was stratified according to re-infection rates measured during the three months following curative anti-malarials, which were 50% and 49% in the two villages in the “high transmission” group, and 39%, 36% and 27% among the three villages in the low transmission group. The parasite rates prior to curative anti-malarials were 78% and 69% in the high transmission group and 65%, 61% and 43% in the low transmission group Analysis of the 18 month follow up was done using survival models for the first or only episode of febrile malaria (Cox regression) and on the total number of episodes (Poisson regression). Analysis of ITN use was adjusted by village, analysis of transmission intensity was adjusted by ITN use, and analysis of the total number of episodes was adjusted by time at risk. The survival plots were not adjusted.Further analysis of the first 3 months of follow up was done using a categorisation of outcome to distinguish immunity from exposure, as described previously There were 355 children with data on ITN use. 102 children slept under an ITN, 253 did not. 18 children had nets with more than 3 holes, and 2 had nets with more than 6 holes. 74 children acquired an ITN during the 18 months of follow up, and 7 children had ITNs that developed more than 3 holes over the 18 months. 6 children lost the use of an ITN during the study. 236 episodes of febrile malaria were reported. Survival plots  and 2, iThe survival plots for time to first or only episode of febrile malaria  and 2 shOn Cox regression for risk of first event and Poisson regression for multiple events, the interaction terms for ITN use and older age, and for low transmission and older age, were significantly greater than 1. This indicates an antagonistic interaction .We examined how sensitive the analysis was to different definitions of ITN use. If we did not exclude children who acquired ITNs during the study or whose nets developed more than 6 holes during the study, the interaction term for survival analysis gave HR = 2.19 . If the definition of ITN use was a net with less than 3 holes or less than 6 holes the interaction terms were HR = 2.75  and HR = 2.52 , respectively. For Poisson analyses of multiple events, without the exclusions for a change in ITN status and for definitions of use based on 3 or 6 holes, the interaction terms were IRR = 3.02 , IRR = 3.28  and IRR = 2.83 .When age was used as a continuous variable, the interaction terms on survival analysis were HR = 1.25 per year  and 1.55  for the effect of ITN use and village respectively. The interaction terms on Poisson analysis for multiple events were IRR = 1.2 , IRR = 1.3 for ITN use and village, respectively. However, the goodness of fit by log likelihood was lower for age as a continuous variable compared with age as a categorical variable (log likelihood = −603.3 vs −601.6 for Cox regression and log likelihood = −401.3 vs −400.8 for Poisson regression). Furthermore, the residuals were unevenly distributed for age as a continuous variable (p = 0.001). Because of this non-linear effect of age on malaria risk, we preferred analyses that used age as a categorical variable (as given above).In order to examine the contributions of exposure and immunity to the altered effect of ITNs and transmission intensity, we examined odds ratios for febrile malaria versus asymptomatic parasitaemia . Age wasWe show that residence in a village at lower transmission intensity results in a relative increase in the risk of febrile malaria in older children. This is consistent with previous comparisons of different regions and countries within Sub-Saharan Africa However, our data should not be interpreted in isolation. Long term follow up following randomized trials of ITN use have consistently shown sustained reductions in morbidity and mortality Our study was observational, and competing explanations for the apparent reduction in efficacy of ITNs in older children should be considered: First, parents of older children might buy ITNs if they notice their child is at high risk of malaria, but parents of younger children have not yet noticed that their child is at high risk. Hence the child's risk determines ITN use in older children, but not in younger. This seems unlikely since there was no evidence that ITN use was significantly more common among older children in the higher transmission villages , and ITN use did not increase among older children (26% compared with 31% in younger children).Second, we have assumed that the older ITN users were long-term ITN users. Therefore, an increased risk of malaria can be explained by having not acquired immunity during a prolonged period of ITN use. In fact, some older children may have recently started using an ITN. This would not explain our results, however, since we would predict that older children who recently acquired an ITN would have the immunity of non-users, but the lowered exposure of an ITN user, and hence much lower than expected rates of malaria. Hence, this would increase the apparent protective efficacy of an ITN. Conversely, it is possible that some older ITN non-users were previous ITN users, but passed the ITNs on to younger siblings. We would predict that such children would have the lower immunity of ITN users, but the higher exposure of ITN non-users, and much higher rates of malaria. Hence, this would also increase the apparent protective efficacy of an ITN. As it happened, we found higher than expected rates of malaria among older children who were ITN users.Third, it is possible that the recently distributed ITNs were of better quality than those distributed several years previously. However, the state of repair of the ITNs was documented by fieldworker visits, and was generally good. Furthermore, the results were not sensitive to definitions of ITN use based on number of holes observed in the net. The pattern of HR and IRRs was similar with the varying definitions. Significance was variable on survival analysis, but consistent for multiple episodes.Anopheles dirus complex of South East Asia bites earlier in the evening than the common African vectors Fourth, it may be that ITNs are less effective in older children because of behavioural differences . Studies in Cambodia have suggested that older children are not protected when randomized to ITN use Fifth, use of ITNs by other homestead members is protective. If use by other homestead members was associated with the age of the ITN using child, this could explain our results. However, the majority of homesteads in our study (82%) contained both older and younger children, so this explanation is unlikely. Finally, it is unclear what effect clearing infection at the start of the study may have had. It is possible the chronic parasitaemia helps to maintain immunity (i.e. premunition), but there was no evidence of an early increase in malaria cases among older children in the survival plots. In any case, it is not obvious how the effect of clearing parasites could be different for older ITN users compared with younger ITN users.Other studies have demonstrated that ITNs protect against parasitaemia in all age groups Policy may move towards community wide ITN distribution programmes rather than continue targeting vulnerable populations, to ensure that reduced transmission offsets reductions in host immunity