Background. School children living in the tropics are often concurrently infected with plasmodium and helminth parasites. It has been hypothesized that immune responses evoked by helminths may modify malaria-specific immune responses and increase the risk of malaria.Methods. We performed a randomized, open-label, equivalence trial among 2436 school children in western Kenya. Eligible children were randomized to receive either 4 repeated doses or a single dose of albendazole and were followed up during 13 months to assess the incidence of clinical malaria. Secondary outcomes were Plasmodium prevalence and density, assessed by repeat cross-sectional surveys over 15 months. Analysis was conducted on an intention-to-treat basis with a prespecified equivalence range of 20%.Results. During 13 months of follow-up, the incidence rate of malaria was 0.27 episodes/person-year in the repeated treatment group and 0.26 episodes/person-year in the annual treatment group . The prevalence and density of malaria parasitemia did not differ by treatment group at any of the cross-sectional surveys.Conclusions. Our findings suggest that repeated deworming does not alter risks of clinical malaria or malaria parasitemia among school children and that school-based deworming in Africa may have no adverse consequences for malaria.Clinical Trials Registration. NCT01658774. Plasmodium organisms are some of the most common parasites infecting humans worldwide [Helminths and orldwide , 2. Theiorldwide , 3. The orldwide , with scorldwide , 6. Helmorldwide , and it orldwide , 9. Howeorldwide –15.Plasmodium species. In Madagascar, bimonthly treatment with the anthelmintic levamisole had no effect on Plasmodium falciparum parasite density among children aged <5 years but, among children aged ≥15 years, resulted in a significant increase in parasitemia, compared with untreated controls [Plasmodium prevalence or density did not differ among those who received 4 monthly albendazole treatments, compared with children who received a placebo [A few randomized longitudinal studies have to date investigated interactions between worm and controls . A trial placebo . However placebo . This la placebo ; howeverWe present results from an individual randomized, open-label trial evaluating the impact of repeated (every 4 months) anthelmintic treatment with albendazole on clinical malaria and malaria parasitemia among school children in an area where only STH species are endemic. Our hypothesis was that, although helminths elicit strong immune responses, repeated anthelmintic treatment does not decrease or increase the risk of clinical malaria or malaria parasitemia, compared with annual treatment. Thus, we tested the hypothesis of no difference  between the 2 treatment groups.Supplementary Materials).Reporting of the current trial is in accordance with the checklist of the extension of the CONSORT statement for noninferiority and equivalence randomized trials  . Most malaria is caused by children , 23, butchildren . Recent hookworm . As partPlasmodium species infection, assessed through cross-sectional surveys conducted at 3, 7, 11, and 15 months.The study was designed as individually randomized, open-label trial to compare the impact of repeated (every 4 months) anthelmintic treatment versus annual treatment on the incidence of clinical malaria and the prevalence and density of malaria parasitemia among school children. A placebo-controlled trial was considered unethical because of the ongoing national school-based deworming program . AlthougWritten informed consent was obtained from a parent or guardian, and assent was sought from children before enrollment into the study. A questionnaire was administered to parents and guardians to collect information on household socioeconomic characteristics, children's use of malaria prevention measures, and recent history of deworming. The study was approved by the Kenya Medical Research Institute and National Ethics Review Committee (SSC No.2242), the London School of Hygiene and Tropical Medicine Ethics Committee (6210), and the Makerere School of Public Health Institutional Review Board (IRB00005876).A. lumbricoides, Trichuris trichiura, and/or hookworms into the main study (n = 1505). In vitro studies have shown inhibition of P. falciparum by benzimidazoles [Rural schools in Bumula District that were accessible year-round were purposively selected with the assistance of district officials. Initially, 30 schools were screened in January 2013 to identify schools with highest prevalence of STH infection. Subsequently, 23 schools with an STH prevalence >20% were included in the study. All children in participating schools for whom informed consent was provided were screened for STH infection, using the Kato-Katz method. To maximize the potential immunological impact of worms among participants, we initially recruited children with detectable infection with idazoles , and a pidazoles  Thereforidazoles , age >15Enrolled children were randomly assigned to receive either a single dose of 400 mg of albendazole  every 4 months for 12 months or a single dose of 400 mg of albendazole at month 0 and a single 250 mg dose of vitamin C  at 4, 8, and 12 months. Allocation to treatment group was randomized using computer-generated randomization by an independent statistician. All drugs were administered under direct observation by study nurses who were not involved in other study activities. Albendazole and vitamin C tablets were received with water. In case of vomiting within the first 30 minutes, treatment was repeated; no vomiting occurred after the second administration.The study was designed to evaluate whether the rate of clinical malaria was equivalent among the treatment groups , and as such sample sizes were calculated on the basis of equivalence . TreatmeSupplementary Table 1). Third, sensitivity analysis conducted including only infected children revealed no change in the direction or magnitude of results (Supplementary Tables 3 and 4).Recruitment was delayed in 2012 and follow-up curtailed in 2013 by unpredictable nationwide teacher strikes. Therefore, faced with budget and time constraints, we conducted active case surveillance for only 13 months instead of the planned 18 months. Active case detection commenced at the beginning of the next school term after the teacher strike (September 2013) and was terminated at 13 months (October 2014). With this follow-up period, a sample size of 909 children in each treatment group would be required to maintain adequate power. To account for the shortened period of follow-up, we included both infected (n = 1484) and uninfected (n = 829) children in the final analyses. This was justified on the basis of 3 reasons. First, only 61 children (9.7%) uninfected at baseline remained uninfected throughout the study. Second, baseline characteristics were generally similar among both sets of children, with exception that infected children were more likely to be male and parasitemic  or who reported fever or other signs of malaria within the past 24 hours were asked to provide a blood sample by finger prick, which was used to perform a malaria rapid diagnostic test  and to prepare thick and thin blood smears. Any child with a diagnosis of uncomplicated clinical malaria (based on the rapid diagnostic test result) was treated with a 6-dose regimen of 20 mg artemether/120 mg lumefantrine in accordance with national guidelines.Plasmodium infection, were assessed by expert microscopy during cross-sectional surveys at 0, 3, 7, 11, and 15 months. Blood smears were stained with 2% Giemsa (pH 7.2) for 45 minutes. Parasite density was defined as the number of Plasmodium parasites per microliter of blood, counted against 200 leukocytes and with the assumption of a leukocyte count of 8000 leukocytes/µL. If <10 asexual parasites were detected in the first 200 leukocytes, counting was continued against 500 leukocytes. A blood smear finding was considered negative when the examination of 200 high-power fields failed to reveal asexual parasites. Thin smears were used for species identification. P. falciparum was the only species detected. The hemoglobin concentration was assessed using a HemoCue hemoglobin photometer . During the surveys conducted at 7, 11, and 15 months, children were asked to provide a stool sample, which was examined in duplicate for the presence and number of helminth ova by the Kato-Katz technique.Secondary outcomes, prevalence and density of Analysis was conducted on an intention-to-treat basis , with additional analysis conducted on a per-protocol basis . Data were analyzed using Stata, version 13 .z scores of height for age (HAZ), weight for age (WAZ), and body mass index for age (BMIZ)—were calculated using the AnthroPlus software for children aged 5–19 years [Summary statistics were calculated for all baseline data. Anthropometric indices—19 years , assumin19 years .Plasmodium species parasitemia (as determined by microscopy) and either an axillary temperature of >37.5°C or a reported history of fever or other signs of malaria during the preceding 24 hours. An alternative case definition that used a parasite density cutoff of >2500 parasites/µL was also used [Plasmodium infection, together with their 95% CIs, were calculated using exact binomial analysis. To allow for overdispersion of egg counts, arithmetic mean numbers of eggs per gram of feces, with their 95% CIs, were estimated using negative binomial regression, taking into account clustering by school. The differences in the prevalence of malaria parasitemia between treatment groups at 0, 3, 7, 12, and 15 months were determined using the binomial test for differences in proportions.Clinical malaria was defined as the presence of asexual lso used . ChildreEquivalence between treatment groups can be stated if the 95% CIs of the relative difference in malaria incidence and parasitemia lie within the predetermined margins of equivalence of −0.08 to 0.08 episodes/person-year for clinical malaria and −8.3% to 8.3% for prevalence of malaria parasitemia. Sensitivity analyses were performed to examine the intervention effect, excluding children who were uninfected at baseline.Between February and June 2013, 7075 children aged 5–18 years were screened; 1505 infected with at least 1 STH species were recruited into the trial. A further 841 randomly selected children without STH infection were also recruited. In total, 2346 children were included in the baseline survey and randomized to receive either repeated or annual treatment. Baseline characteristics were comparable between the 2 treatment groups  at 15 months of follow-up. In contrast, annual treatment only reduced the prevalence of A. lumbricoides to 14.7% and the prevalence of hookworm to 13.0% at 7 months of follow-up, and thereafter, at 11 and 15 months, infection levels rose slightly, to 24.8% and 26.4%, respectively. Of the 1505 children who were infected with any STH species at baseline, 1141 (75.8%) were followed up, and 257 (22.5%) remained infected at 15 months. Of the 841 children uninfected at baseline, 631 (75.0%) were followed up, and 61 (9.7%) remained uninfected at 15 months.Table A presents comparisons of incidence rates among treatment groups and shows that CIs included 0 and were within the predefined margin of equivalence  for intention-to-treat and per-protocol analyses. Similarly, by using a malaria case definition based on an increased parasite density cutoff, the results showed equivalence . Figure 3B and Table Supplementary Tables 2–4).During the 13 months of active case detection, 606 incident cases of malaria occurred, with 405 children having only 1 incident case and 93 having ≥2 cases. The incidence of malaria was 0.27 episodes/person-year in the repeated treatment group and 0.26 episodes/person-year in the annual treatment group, a rate difference of 0.01 , based on intention-to-treat analysis. Figure 3Plasmodium infection in the dry season, compared with findings in the placebo group, but this difference was found to be nonsignificant, partly because of the very low follow-up rate (26%) [Plasmodium among children, compared with findings associated with annual treatment [The results of our trial show that 4 rounds of albendazole at 4 monthly intervals did not increase or decrease the incidence of clinical malaria or malaria parasitemia, compared with a single round of treatment, based on our predefined margins of equivalence . Our study has a number of strengths, including a randomized design, a prespecified sample size, a specific case definition of malaria, and a high (85%) follow-up rate. A previous study in Nigeria found that anthelmintic treatment every 4 months for 14 months resulted in an increase in te (26%) . A placete (26%) . Howeverreatment . This stThere are a number of study limitations. The study was partially unblinded; participants were aware of treatment allocation, but assessors of main outcomes were blind. Second, our study reflects parasitemia based on expert microscopy findings, which underestimates infection, compared with molecular methods . Third, In areas of high malaria transmission, the main burden of malaria is among young children, but the risk of coinfection is low because of low levels of helminth infections in this age group . It woulAn estimated 81.6 million school-aged children living in sub-Saharan Africa benefitted from mass treatment with albendazole or mebendazole in 2013 . The resSupplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


The effect of CD4 count on malaria incidence in HIV infected adults on antiretroviral therapy (ART) was assessed in the context of a randomized controlled trial on the effect of stopping cotrimoxazole (CTX).This study presents a sub-analysis of the COSTOP trial (ISRCTN44723643) which was carried out among HIV-infected Ugandan adults stable on ART with CD4 counts ≥250 cells/µl. Participants were randomized (1:1) to continue CTX or stop CTX and receive matching placebo, and were followed up for a minimum of 1 year (median 2.5 years). CD4 counts were measured at baseline, 3 months and then every 6 months. Clinical malaria was defined as fever and a positive blood slide. First, the relationship between current CD4 count during follow-up and malaria among participants on placebo was examined; using random effects Poisson regression to account for repeated episodes. Second, the effect of CD4 count at enrolment, CD4 count at ART initiation, and CD4 count during follow-up on malaria, was assessed within each trial arm; to examine whether the effect of CD4 count differed by CTX use.2180 participants were enrolled into the COSTOP trial. The incidence of clinical malaria was approximately four episodes/100 person years in the CTX arm and 14 episodes/100 person years in the placebo arm. There was no evidence of an association of current CD4 and clinical malaria incidence (P = 0.56), or parasitaemia levels (P = 0.24), in the placebo arm. Malaria incidence did not differ by CD4 count at ART initiation, enrolment or during follow up, irrespective of CTX use. When compared with participants in the lowest CD4 stratum, rate ratios within each trial arm were all close to 1, and P values were all above P = 0.30.The immune status of HIV infected participants who are stable on ART as measured by CD4 count was not associated with malaria incidence and did not modify the effect of stopping CTX on malaria. The decision of whether to stop or continue CTX prophylaxis for malaria in HIV infected individuals who are stable on ART should not be based on CD4 counts alone.COSTOP trial registration number ISRCTN44723643The online version of this article (doi:10.1186/s12936-016-1426-z) contains supplementary material, which is available to authorized users. In many parts of sub-Saharan Africa, both malaria and HIV infection are highly endemic. HIV infection enhances malaria acquisition and severity; similarly malaria enhances HIV viral replication –5. The eDecreased CD4 counts have been associated with higher risk of acquiring malaria. In a study in rural Uganda, HIV infected ART-naïve adults with CD4 counts <200 cells/µl had a significantly higher risk of clinical malaria than those with CD4 counts ≥500 . A studyCTX is beneficial to HIV infected individuals as a prophylaxis against malaria and bacterial infections –13, evenTo address these research questions, we conducted a planned sub-group analysis to examine the relationship of CD4 count with malaria in the recently completed COSTOP trial in Uganda –21. COSTi.The effect of CD4 count on malaria incidenceii.Whether this effect differs in the presence and absence of CTX medicationThe study aimed to determine among HIV infected adults on ART with CD4 counts ≥250 cells/µl:This study used data gathered during the COSTOP trial conducted from 2011 to 2014 in Uganda (ISRCTN44723643). Trial methods have been described previously , 21. BriInformed consent for study procedures was obtained at screening and enrolment.At screening, data were documented on; disease history, duration of prior ART and CTX medication, and CD4 count at time of ART initiation. At enrollment, data were documented on socio-demographic characteristics and each participant was provided with an insecticide-treated bed net (ITN) and educated about the importance of using it. Participants were seen at scheduled follow-up visits every month for the first 3 months and 3-monthly thereafter, and were followed for 12 months to 3.5 years, depending on date of enrolment. At these visits, participants were asked about their health, symptoms suggestive of malaria, adherence to medication and bed net use. Blood samples were drawn; at enrollment, monthly for 3 months and 3 monthly thereafter for a malaria slide; at 3 months, 6 months and 6 monthly thereafter for CD4 count; and 3 monthly for the full blood count. Participants were asked to attend the study clinic at any time they felt unwell; if malaria was suspected, based on a history of malaria associated symptoms , a blood slide and other tests deemed necessary were done. Participants who reported having been treated for malaria elsewhere (for example during a journey) were asked to present documentary evidence of diagnoses and test results.A sample of blood was taken either from the fingertip using a lancet or from a peripheral vein using a syringe, and used to prepare thick and thin films on a glass slide. The specimens were processed using Leishman’s stain and examined by microscopy. Thick film specimens were used to record the number of parasites per 200 white blood cells and thin films to identify the plasmodium species. Venous blood samples were taken for CD4 cell counts and measured using a FACS-count system (Becton–Dickinson San Jose) at the MRC/UVRI laboratories in Entebbe and Masaka.Plasmodium falciparum asexual parasitaemia, no other obvious cause of symptoms and met any of the following criteria: convulsions, loss of consciousness, hypotension (systolic blood pressure <70 mmHg), admission to hospital due to malaria, laboratory evidence of liver or kidney damage, severe normocytic anaemia , or hyper parasitaemia on blood slide .Analyses were carried out using Stata 13. Clinical malaria was defined as presence or history (during the previous 2 weeks) of fever and microscopically confirmed malaria parasites. Severe malaria (based on WHO guidelines) , 22 was The CD4 count at enrolment was calculated from the mean of the two most recent pre-enrolment (screening) CD4 counts. Person years at risk were calculated from enrolment until the date last seen or end of trial. After each malaria episode, participants were considered to be not at risk for another episode until the episode resolved, or for 28 days, if a resolution date was not available. Follow-up data were organized into intervals corresponding with the visit schedule. For time-varying variables during follow-up , the most recent value measured at the start of each interval was used. CD4 count values were carried forward for the visits where CD4 counts were not done, until the next recorded CD4 count.First, the effect of current (time of infection) CD4 count on clinical malaria incidence during follow up was assessed; using random effects Poisson regression to account for the clustering of multiple episodes within the same participant. Since the incidence of malaria in the COSTOP trial had previously been shown to be significantly lower in the CTX arm (21), this analysis was restricted to participants in the placebo arm in order to examine the effect of CD4 counts in the absence of the anti-malarial effects of CTX. The effect of CD4 adjusted for baseline covariates that were considered as potential confounders a priori  and baseline CD4 count) was examined, and then including time-varying variables . SES was measured by combining baseline data from all trial participants on housing construction and ownership of household items into an asset index score using principal component analysis . In ordeAmong placebo participants with clinical malaria, the effect of CD4 count at infection on parasitaemia during each malaria episode as the outcome was assessed, using random effects linear regression; parasitaemia levels were log transformed for analysis. The analysis was adjusted for baseline and time-varying potential confounders as described above. In addition, CD4 count at infection was assessed for an effect on severe malaria; since there were only 15 episodes of severe malaria  , no atteSecondly, the effect of CD4 count at baseline, CD4 count at ART initiation, or CD4 count at infection on clinical malaria was assessed by treatment arm (CTX or placebo), using random effects Poisson regression. Regression models contained fixed effects for CD4 count group, treatment arm, enrolment site and year since enrolment, and an interaction term between CD4 count group and treatment arm.2180 participants were enrolled into the COSTOP trial, 1002 (46 %) at the Entebbe site, and 1091 (50 %) were allocated to placebo (stopping CTX). Baseline characteristics were well balanced between trial arms . There was no evidence of an effect of CD4 count at infection on clinical malaria . Severe malaria rates decreased with increasing CD4 counts among participants with CD4 <400, then remained fairly similar in participants with higher CD4 counts. , outcomes  and a variety of potential confounders made it possible to investigate the research questions in great detail.This study had some limitations. In spite of the large sample size only a small number of severe malaria episodes occurred which limited the power to detect a potential effect of CD4 count. Also, the study was not a priori designed to address detailed research questions related to malaria, but rather was conducted as a sub-analysis of data gathered in the context of a randomized trial on the effect of stopping CTX. Although some potential confounders were adjusted for, residual confounding cannot be ruled out as a result of imperfectly measured covariates which were adjusted for (e.g. SES) or covariates which were not measured. Furthermore, viral load, which may be a better indicator of immune competence than CD4 count was not measured: it has been shown that effective viral suppression reduces the incidence of opportunistic infections  and a siIn this study of HIV-infected individuals on ART with baseline CD4 counts ≥250 cells/µl, the incidence of clinical malaria and the intensity of parasitaemia among patients with clinical malaria were not influenced by CD4 counts at ART initiation, enrollment into the study, or at the time of malaria infection. The finding of no association between malaria and CD4 count was similar among participants randomized to stop prophylactic CTX and those who continued CTX. The decision of whether to stop or continue CTX prophylaxis for malaria in HIV infected patients who are stable on ART should not be based on a patient’s CD4 cell count alone.


P. falciparum endemic areas. We assessed the impact of micronutrient deficiencies on malaria incidence and vice versa in the Brazilian state of Amazonas.There is a growing body of evidence linking micronutrient deficiencies and malaria incidence arising mostly from P. vivax malaria episode. We included 95 children median age 4.8 years (interquartile range [IQR]: 2.3–6.6), mostly males (60.0%) and with high maternal illiteracy (72.6%). Vitamin A deficiencies were found in 36% of children, beta-carotene deficiency in 63%, zinc deficiency in 61% and iron deficiency in 51%. Most children (80%) had at least one intestinal parasite. During follow-up, 16 cases of vivax malaria were diagnosed amongst 13 individuals. Micronutrient deficiencies were not associated with increased malaria incidence: vitamin A deficiency ; beta-carotene ; zinc  and iron ). Upon reevaluation, children with al least one episode of malaria did not present significant changes in micronutrient levels.We evaluated children <10 years old living in rural communities in the state of Amazonas, Brazil, from May 2010 to May 2011. All children were assessed for sociodemographic, anthropometric and laboratory parameters, including vitamin A, beta-carotene, zinc and iron serum levels at the beginning of the study (May 2010) and one year later (May 2011). Children were followed in between using passive surveillance for detection of symptomatic malaria. Those living in the study area at the completion of the observation period were reassessed for micronutrient levels. Univariate Cox-proportional Hazards models were used to assess whether micronutrient deficiencies had an impact on time to first P. vivax endemic areas are warranted in order to validate these results.Micronutrient serum levels were not associated with a higher malaria incidence nor the malaria episode influenced micronutrient levels. Future studies targeting larger populations to assess micronutrients levels in  Plasmodium falciparum in African children under five years of age  and categorical variables as frequencies and percentages. Univariate Cox proportional hazard models were used to assess the impact of baseline characteristics on time to malaria incidence over the one year follow up. Chi-2 or Fisher’s exact test were performed to assess the effects of malaria incidence on the delta of micronutrients serum levels  by the time of the baseline assessment (May 2010). As 33 children had moved out of the area, ninety-five children were included in the study . Median P. vivax and 1 by P. falciparum. Three patients had P. vivax malaria twice during the observation period. In the time to P. vivax incidence analyses, baseline vitamin A serum levels were not associated with the outcome , nor did beta-carotene , or zinc   . We founThe impact of having had malaria on micronutrient levels was assessed based on reevaluation conducted 1 year after the baseline assessment. Because 27 children had moved out of the study area by reevaluation time, the remaining 68 children were reassessed for micronutrient levels and these results were compared with baseline assessments to obtain the Δ for each micronutrient. Having had malaria was not associated with a Δ of any of the assessed micronutrients . AdjustmP. falciparum endemic areas.We evaluated the bidirectional association of vivax malaria and micronutrient deficiencies and found no association of deficiencies of vitamin A, beta-carotene, zinc, or iron with malaria incidence. Incident malaria evaluated throughout a year was not associated with worsening of micronutrient deficiencies either. We reviewed the extant literature to compare our results with previous findings, mostly from Vitamin A deficiency is found in 17.4% of children in Brazil , 51. In P. vivax malaria in the Brazilian Amazon. Previous studies, mostly in P. falciparum had shown an association of vitamin A levels with severity [P. falciparum malaria. One published study in an area where P. vivax is predominant assessed hemoglobin levels by day 30 and found no difference between vitamin A supplementation and the control group [Our results did not support an association of vitamin A levels at baseline and incidence of severity , 56 and severity , 58, proseverity , 60. Howol group , in agreP. falciparum malaria and results may differ in P. vivax endemic areas. Our results are similar to a study that found no association of zinc supplementation and P. vivax outcomes in Peru where P. vivax also predominates [We did not find an association between zinc deficiency and malaria incidence. Interestingly that studies have addressed the role of zinc supplementation in malaria outcomes have shown dissimilar results. Some studies found a protective effect of zinc supplementation on the number of malaria related clinic visits in Gambia  and rateominates .P. falciparum incidence [We found a slight trend towards an association of iron deficiency and malaria incidence and a similar trend for maternal illiteracy, having had malaria and the number of intestinal parasites detected during the baseline evaluation. These parameters are likely interrelated and may serve as markers of a lower socio-economical status: living in areas with the poorest hygienic conditions may result in an increased risk of acquiring malaria. This trend, although not statistically significant, may go against previous studies that found a possible protective effect of lower iron levels on ncidence . An assoncidence . In a stncidence , the autncidence , 65. Thencidence , 66–68.P. falciparum endemic areas. Our study does not support the same effect by P. vivax.We also assessed whether patients having malaria during the 1 year follow up would have a downtrend in their micronutrient serum levels by comparing the measurements from 2010 and 2011. We did not find a statistically significant association of malaria incidence and Δ of any of the micronutrients. Previous studies have shown that malaria incidence may reduce the ingestion of micronutrients  and theiP. vivax malaria in several ways. We found a high prevalence of micronutrient deficiencies and intestinal parasitosis in the study area.Our findings should be interpreted in the context of some limitations. We report the experience of one municipality in the Brazilian Amazon and our findings may not be generalizable to other settings. Our sample size and, especially, the low number of events, could have prevented us from detecting existing associations a larger population would be able to. Unmeasured patient, socio-economical and administrative factors may affect malaria incidence and micronutrient levels. We were not able to assess dietary patterns, and although there is evidence of homogeneous habits within the study population , 65, theP. vivax endemic areas in larger populations are needed to validate our results.In conclusion, we were not able to observe an association between serum micronutrient levels and malaria incidence, although we found a trend towards an association between iron deficiency and malaria incidence. Iron deficiency may be a marker of poorer socio-economical status and higher malaria risk as a result. Further studies assessing micronutrient levels in


Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting.Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections.Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR  or dPCR-HRM . Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM . Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials.The coupled nPCR-HRM assay detected Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions.The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia  The World Health Organization (WHO) estimates that despite recent reductions in malaria incidence, malaria contributed to the morbidity and mortality of about 166 million and 564,000 people, respectively, in sub-Saharan Africa (SSA) in 2012 , since pPlasmodium parasitaemia, has been implicated in undermining considerations of such differential diagnoses  and Plasmodium ovale [GenBank:AJ001527] using Geneious 6.1.6 software [The extracted DNA was amplified using nested PCR, targeting 18S rRNA genes in software . The secRNA gene . Ten-folsoftware . Protocosoftware  were alsP. falciparum DNA, a standardized freeze-dried preparation of P. falciparum infected whole blood obtained from the National Institute for Biological Standards and Control . Following NIBC recommendations, the lyophilized material was suspended in 500 μL of sterile, nuclease-free water to a final concentration of 1 × 109 IU/mL, estimated to be 469,920 parasites/μL [To compare the detection limits of the three assays, we used the WHO International Standard for sites/μL . To estaMcNemar’s Chi-Square tests were used to compare the sensitivity of nPCR-HRM to nPCR and dPCR-HRM in detecting parasitaemia. Continuous/Gaussian data were compared using t-tests while Cohen’s Kappa was calculated to compare the agreement between the results obtained using the different methods. All statistical inferences were drawn on two-tailed distributions with α = 0.05. The data were analysed using R 3.0.1 software .While 5,672 and 1,412 patients tested positive for malaria at Sena and Tom Mboya Health Centres, respectively, during the study period, 197 subjects with undiagnosed febrile illness were enrolled into the study. Subjects were between 12 and 70 years of age with a mean and median age of 31.9 and 28.0 years, respectively. There were 114 females and 81 males, with the gender of two individuals not reported. About 53.3% (n = 105) of the subjects were recruited from Sena Health Centre between Oct 15, 2012 and Oct 15, 2013 and the rest (n = 92) from Tom Mboya Health Centre between May 28, 2012 and Feb 18, 2013. Although 7,553 and 2,378 patients tested negative for malaria at Sena and Tom Mboya Health Centres, respectively, only two smear/RDT-negative patients from each clinic were included in the study every week to minimize time-based selection bias. The mean age for the subjects recruited from Sena Health Centre was 29.1 years old. This population was significantly younger than that recruited from Tom Mboya Health Centre, which averaged 35.4 years old .The LoD of the coupled nPCR-HRM technique was 236 parasites/mL, whereas the LODs of dPCR-HRM and nPCR were 1,490 parasites/mL and 4,700 parasites/mL, respectively.Plasmodium parasitaemia generated by our nPCR-HRM technique relative to those generated through nPCR or dPCR-HRM techniques are summarized in Figure Plasmodium infections than did either dPCR-HRM  or nPCR . More specifically, nPCR-HRM also detected significantly more cases of P. falciparum infections than did either dPCR-HRM  or nPCR .Prevalence estimates of Plasmodium parasites in their samples, of which 53  had P. falciparum parasites. While P. falciparum prevalence did not significantly differ between the two sampling sites during the study period (p = 0.137), the overall Plasmodium spp. prevalence was greater in samples collected at Tom Mboya Health Centre than those collected at Sena Health Centre (p = 0.013). Samples with Plasmodium collected at Tom Mboya Health Centre also had higher Plasmodium species diversity than those collected at Sena Health Centre. Samples collected at Tom Mboya Health Centre included 26 (72.2%) P. falciparum only infections, five (13.9%) P. malariae only infections, two (5.6%) P. ovale only infections, two (5.6%) double infections of P. falciparum and P. ovale, and one (2.8%) triple infection of P. falciparum, P. malariae, and P. ovale. Samples collected at Sena Health Centre had only two P. ovale infections with the remaining 24 infections being P. falciparum. The unique HRM profiles of the three Plasmodium parasites are represented in Figure Plasmodium parasites, among which 27  were P. falciparum infections. nPCR revealed malaria parasites in only 20  of the samples, among which 15  were P. falciparum infections. No mixed infections were detected by either nPCR or dPCR-HRM. To verify that the nested amplification approaches did not yield false positives due to cross-contamination, these assays were repeated with the same samples in three successive runs, all of which produced identical results for the individual samples.Based on nPCR-HRM analyses, 62  of the 197 subjects enrolled in the study had Plasmodium parasites were treated with anti-malarial drugs, 110 (81.5%) of the 135 patients without detectable Plasmodium parasites were incorrectly treated with anti-malarial drugs. However, these patterns differed between the two Health Centres, as represented in Figure Plasmodium parasites were treated with anti-malarial drugs, all 26 malaria patients with detectable parasites at Sena Health Centre were treated with anti-malarials. Yet, among the patients that did not have Plasmodium infection, more than half were treated with anti-malarial drugs at both Tom Mboya  and Sena Health Centres .Among the 197 patients presenting with febrile illness enrolled in this study, 152 (77.2%) were treated with anti-malarial drugs. While 42 (67.7%) of the 62 patients with detectable Plasmodium infection prevalence that includes low-parasitaemia infections can be synergistically enhanced to improve size estimates of its infectious human reservoir in regions where malaria elimination efforts are being scaled up. Indeed, nPCR-HRM was able to detect P. falciparum at orders of magnitude lower concentrations than either nPCR or dPCR-HRM. The LoD of the coupled nPCR-HRM assay (236 parasites/mL) is however in the same range as TaqMan Probe assays validated using the same P. falciparum DNA standard (313 parasites/mL) [Plasmodium parasites were detected by the novel nPCR-HRM technique, the well-established nPCR [Plasmodium parasites in only 20 and 39 samples, respectively. The same is evident for P. falciparum, where nPCR-HRM identified 26.9% prevalence among microscopically negative febrile illness patients, while nPCR and dPCR-HRM identified only 10.2% and 13.7% prevalence, respectively. These results are consistent with the lower sensitivity of nPCR previously reported in comparison to dPCR-HRM [Plasmodium parasites.By combining the high sensitivity of nested PCR with real-time species differentiating HRM analysis, the reporting of hed nPCR  and dPCRhed nPCR  moleculadPCR-HRM , yet furPlasmodium detection have been reported recently, they either failed to undercut the detection thresholds of microscopy on clinical specimen [Plasmodium species [Plasmodium infections, no previous studies have assessed drug administration among subjects with malaria parasitaemia below the detection limits of standard microscopy and RDT.Although other novel molecular methods for specimen  or were  species , 36. DuePlasmodium parasitaemia in rural clinics in malaria endemic regions of SSA, and that can differentially diagnose other non-malaria febrile illnesses, including bacterial, mycobacterial, fungal, and arboviral infections, is a big challenge [The lack of diagnostic tools sufficiently sensitive to detect low hallenge , 20–24. hallenge , clinicihallenge , 20–22.Plasmodium parasitaemia cases at Sena Health Centre were treated with anti-malarial drugs, only 44.4% of low-parasitaemia patients at Tom Mboya Health Centre were treated with anti-malarial drugs. However, in both Health Centres, the majority of anti-malarial drugs were prescribed to patients in whom we did not detect Plasmodium parasitaemia.Febrile illness misdiagnosis can be significant in malaria endemic settings, resulting in a myriad of downstream issues including improper treatment, chronic suffering of patients as the underlying cause of illness remains unknown, and drug wastage . MoreoveThe discrepant prescribing patterns between the two study sites may be due to the over three times greater numbers of patients with microscopically positive malaria observed at Sena Health Centre than at Tom Mboya Health Centre during the respective sampling periods. Accessibility to other health facilities may also have influenced prescribing practices. Compared to Tom Mboya Health Centre on Rusinga Island, Sena Health Centre on Mfangano Island is more isolated from the mainland where patients access other nearby and specialized health centres that they may be referred to for assessment of differential diagnoses. Due to the relatively limited accessibility to such referral clinics for patients on Mfangano Island, clinicians may have had a stronger tendency to prescribe the more readily available anti-malarials at Sena Health Centre.Plasmodium infection are likely to have endemic stability to malaria infection and may be able to tolerate a low degree of parasitaemia with absence of clinical disease [Plasmodium infections for the underlying differential diagnosis of their febrile symptoms [Plasmodium infection, may contribute to integrated malaria elimination/control efforts by reducing the size of the infectious reservoir within communities [Individuals with extremely low-parasitaemia  disease . Therefosymptoms . Nonethemunities –11.Plasmodium infections and other differential diagnoses. Some non-malaria febrile patients are diagnosed and treated for malaria based on their clinical symptoms, and may be inadvertently integrated into epidemiological statistics [Plasmodium infections go unrecognized [This study demonstrates how improper febrile illness diagnoses confound prevalence estimates of atistics –6, whilecognized . As a recognized . The comPlasmodium infection rates will translate into the development of better rural malaria diagnostics and treatments, ultimately reducing gametocyte incidence rates in asymptomatic populations with low-parasitaemia malaria [Plasmodium transmission rates to mosquito vectors [Improved understanding of  malaria . While i vectors , 42, the vectors , 42, 43, vectors  and indi vectors . In cont vectors . These uPlasmodium detection and species differentiation is a valuable molecular tool that can be used to complement microscopy and RDT and enhance understanding of specific Plasmodium infection rates. While sensitive and specific, nPCR-HRM was not designed as a diagnostic tool for health clinics, as it requires real-time PCR infrastructure, specialized training to avoid the cross-contamination risks associated with nested PCR assays, and more man power in comparison to RDTs. Recent developments in both loop-mediated isothermal amplification (LAMP) [Plasmodium infections. Nonetheless, nPCR-HRM can be employed for monitoring low-parasitaemia infection rates within malaria endemic regions to assess the progress of regional malaria elimination efforts.This study’s approach to low-parasitaemia n (LAMP)  and isotn (LAMP)  assays sPlasmodium diagnostics to quantify both the over-prescription of anti-malarial drugs in non-malaria febrile patients and under-prescription of anti-malarial drugs in low-parasitaemia malaria patients. While patient febrile illness symptoms cannot be attributed to any of the sub-microscopic Plasmodium infections identified in this study, consistent anti-malarial treatment limited to such cases should contribute to regional malaria elimination efforts [Plasmodium infection prevalence, we further demonstrate the inadequacy of rural diagnostics in detecting low-parasitaemia infections.This is the first study that employs low-parasitaemia  efforts –11. Othe efforts , 23, 24, efforts , alongsi efforts , is the  efforts . More rePlasmodium infections may not suffer from acute febrile illness as a result of malaria, they may contribute to a significant proportion of the infectious reservoir during their asymptomatic state. Conversely, the lack of diagnostics and treatment for unrecognized differential febrile illness diagnoses may have severe health implications that cannot be addressed properly. Epidemiological estimates need to take into account the considerable proportions of asymptomatic low-parasitaemia Plasmodium infections that contribute to the infectious reservoir, while also considering the health impacts of unrecognized differential diagnoses of febrile illness in malaria endemic regions.While patients with undiagnosed and untreated low-parasitaemia


The leading cause of death in end stage renal disease is cardiovascular disease (CVD). Kidney transplantation is associated with improved survival over dialysis. We hypothesized that arterial stiffness, a marker of CVD, would improve in patients post kidney transplant, potentially explaining one mechanism of survival benefit from transplant.After obtaining Institutional Review Board approval and informed consent, we performed a longitudinal prospective cohort study of 66 newly transplanted adult kidney transplant recipients, using aortic pulse wave velocity (PWV) to assess arterial stiffness over a 12 month period. All patients were assessed within one month of transplant (baseline) and 12 months post transplant. The primary outcome was change in PWV score at 12 months which we assessed using Wilcoxon Signed Rank test. Secondary analyses included correlation of predictors with PWV score at both time points.The median age of the cohort was 49.7 years at transplant, with 27 % Black and 27 % female. At baseline, 43 % had tobacco use, 30 % had a history of CVD, and 42 % had diabetes. Median baseline calcium was 9.1 mg/dL and median phosphorus was 5.1 mg/dL. Median PWV score was 9.25 and 8.97 m/s at baseline versus month 12, respectively, showing no significant change . In multivariable regression, subjects with increased age at transplant (p = 0.008), diabetes (p = 0.002), and a higher baseline PWV score (p < 0.001) were at increased risk of having a high PWV score 12 months post transplant.Aortic arterial stiffness does not progress in the first year post kidney transplant. Increasing age, diabetes, and higher baseline PWV score identify patients at risk for increased arterial stiffness. Further research that assesses patients for greater than one year and includes a control dialysis group would be helpful in further understanding the change in arterial stiffness post transplantation. Cardiovascular disease (CVD) exerts a heavy burden in patients with chronic kidney disease (CKD), and it is the leading cause of death in the end stage renal disease (ESRD) population –3. ContrIncreased aortic arterial stiffness is strongly associated with the development of CVD and is an independent predictor of cardiac and all-cause mortality . Aortic We hypothesized that aortic arterial stiffness would be improved in patients receiving a kidney transplant, potentially explaining one mechanism of survival benefit from transplant. In this study we examined a prospective cohort of 66 kidney transplant recipients with aortic PWV measurements and assessed the changes in aortic arterial stiffness over the first year post kidney transplant.All study procedures were approved by the Institutional Review Board (IRB) of Vanderbilt University Medical Center (VUMC). Informed consent was obtained from all study participants. We conducted a single-center, prospective cohort study of 66 newly transplanted adult kidney transplant recipients, using pulse wave velocity (PWV) to assess arterial stiffness over a 12 month period. Kidney transplant recipients were consecutively recruited from the VUMC Renal Transplant Clinic from August 2009 through September 2012. Inclusion criteria included patients aged ≥ 18 who were undergoing or had recently undergone kidney transplantation. Participants were excluded if they were unwilling to participate or had atrial fibrillation, since this condition makes pulse wave velocity testing uninterpretable. Induction therapy was alemtuzumab with methylprednisolone in 85 % of patients. All patients were maintained on tacrolimus and mycophenolate. Maintenance steroids were used in 9 %. Tacrolimus was dosed to a 12 h trough blood concentration of 8–10 ng/ml the first 6 months, then of 6–8 mg/ml for months 6 through 12.The study was composed of two visits, with patients assessed within 1 month of transplant (baseline) and 12 months post transplant. Demographic, medical, and social data was collected from each participant and confirmed in the electronic medical record. Participants underwent pulse wave velocity testing by a trained professional at the Vanderbilt Clinical Research Center using the SphygmoCor CPV Pulse Wave Velocity system . Briefly stated, pulse wave velocity is the time it takes the arterial pressure wave to travel between two arterial sites, where PWV = distance (meters)/transit time (seconds). A higher PWV positively correlates with increased arterial stiffness. A more detailed description of pulse wave velocity procedure has been previously published . In thiswww.r-project.org) and used a two-sided 5 % significance level for all statistical analyses.We expressed descriptive statistics as proportions for categorical variables and as means and standard deviations, or medians and interquartile ranges for continuous variables, depending on the distribution of the variables. The primary outcome was change in PWV score at 12 months from baseline, which we assessed using Wilcoxon Signed Rank test. We further assessed the change in PWV score stratified by subjects’ diabetes status at baseline. Secondary analyses included predictors of PWV score at baseline and at 12 months post transplant. Spearman’s rank correlation was used to test the association of age at baseline with PWV score at baseline and 12 months as well as the association of PWV score at baseline and at 12 months. We further conducted multivariable regression to determine predictors for both baseline and 12 month PWV measures. Age at transplantation, race, gender, diabetes status, and time in months on dialysis at baseline were included as potential predictors. Baseline PWV score was also included in the multivariable model to predict PWV score at 12 month post transplant. Normality of residuals of all linear models was diagnosed figuratively. We conducted all analyses using R-software version 2 · 11 · 1 . Neither time on dialysis nor the calcium-phosphorus product prior to transplant correlated with PWV at baseline. For PWV at 12 months, both PWV at baseline  and age  were significantly correlated. No significant correlation was seen between serum creatinine at 12 months and PWV at 12 months. Results for multivariable regression are shown in Table Patients who undergo kidney transplantation have improved survival and lower cardiovascular risk relative to wait-listed dialysis patients. This suggests that transplant may positively impact cardiovascular risk. Though this effect likely occurs through several processes, here we examined the effect of kidney transplantation on arterial stiffness by assessing PWV. In this prospective study of 66 new kidney transplant recipients, we showed that there was no change in aortic PWV over the first year post kidney transplant, suggesting arterial stiffness does not progress during this time period. Increasing age, having diabetes and higher baseline PWV score were associated with increased PWV score at 12 months and may help identify patients with increased arterial stiffness.Previous studies have suggested that PWV progresses with time on dialysis. For example, Utescu and colleagues showed an annual increase of 0.84 m/s per year (95 % CI 0.50−1.12 m/s per year) in a cohort of 109 hemodialysis patients . In our For kidney transplant recipients, prior cross-sectional studies have shown that transplant recipients have higher aortic PWV measurements than historical healthy controls. The reported median normal values for a large European population of individuals aged 40–49 was 6.9 m/s and aged 50–59 was 8.1 m/s compared to 9.25 m/s in our patient cohort with a median age of 49.7 years . SimilarThese prior studies had single time points of PWV assessments of prevalent transplant recipients, with patients enrolled at varying times post transplantation. Our study was different in that it followed incident patients from time of transplant, with measurements at baseline and one year, adding further information about what happens to PWV over time. Our results indicate that changes in aortic stiffness may be slowed with successful transplantation, consistent with two previous smaller studies over a shorter time period of 3 months , 21. In 2, where the initial measured carotid PWV was 9.1 m/s. In this group, however, PWV increased to 9.8 m/s when measured 24 months later [In contrast to our study, a study in Poland found that carotid PWV increased with time post transplantation, but these patients were already a median time of 36 months post transplantation at time of enrollment, differing from our population of newly transplanted patients. Similar PWV values to those in our study were observed in the 61 kidney transplant recipients with an eGFR of 55 mL/min/1.73 mhs later . One poshs later . These sStrengths of our study included the prospective longitudinal design and duration of follow up, including the fact that all patients started follow up at time of their transplant, so they all had been exposed to transplant immunosuppression the same length of time. In addition, a large percent of the cohort was black, allowing study of a population not frequently available in other cohorts of kidney transplant recipients. A weakness was our inability to adjust for multiple covariates without overfitting the model due to our relatively small sample size, though we were able to adjust for all demographics and important cardiovascular risk factors like diabetes and pre-existing cardiovascular disease. Also due to the small number of cardiovascular events in this study current cohort, we were not able to test associations of PWV with these. In addition, overall our population was a low risk population for acute rejection and low risk for immediate cardiovascular events due to our selection process pre transplant.In a prospective study of new kidney transplant recipients treated with tacrolimus, we showed that there was no change in aortic PWV over the first year post kidney transplantation, suggesting arterial stiffness does not progress during this time period in a cohort of low risk individuals for cardio-renal outcomes. Further prospective research regarding arterial stiffness after transplant would be helpful to better understand the impact of transplant on cardiovascular risk. Specifically studies with longer follow-up time and with adequate number of cardiovascular events are necessary. Ultimately this may help identify kidney transplant patients at higher risk for CVD events and to target for intervention.


Iron deficiency (ID) and malaria co-exist in tropical regions and both contribute to high rates of anaemia in young children. It is unclear whether iron fortification combined with intermittent preventive treatment (IPT) of malaria would be an efficacious strategy for reducing anaemia in young children.A 9-month cluster-randomised, single-blinded, placebo-controlled intervention trial was carried out in children aged 12–36 months in south-central Côte d’Ivoire, an area of intense and perennial malaria transmission. The study groups were: group 1: normal diet and IPT-placebo (n = 125); group 2: consumption of porridge, an iron-fortified complementary food (CF) with optimised composition providing 2 mg iron as NaFeEDTA and 3.8 mg iron as ferrous fumarate 6 days per week (CF-FeFum) and IPT-placebo (n = 126); group 3: IPT of malaria at 3-month intervals, using sulfadoxine-pyrimethamine and amodiaquine and no dietary intervention (n = 127); group 4: both CF-FeFum and IPT (n = 124); and group 5: consumption of porridge, an iron-fortified CF with the composition currently on the Ivorian market providing 2 mg iron as NaFeEDTA and 3.8 mg iron as ferric pyrophosphate 6 days per week (CF-FePP) and IPT-placebo (n = 127). The primary outcome was haemoglobin (Hb) concentration. Linear and logistic regression mixed-effect models were used for the comparison of the five study groups, and a 2 × 2 factorial analysis was used to assess treatment interactions of CF-FeFum and IPT (study groups 1–4).P = 0.89). The adjusted differences in Hb were 0.24 g/dl  in children receiving IPT and −0.08 g/dl  in children receiving CF-FeFum. At baseline, anaemia (Hb <11.0 g/dl) was 82.1 %. After 9 months, IPT decreased the odds of anaemia , whereas iron-fortified CF did not , although ID (plasma ferritin <30 μg/l) was decreased markedly in children receiving iron fortified CF .After 9 months, the Hb concentration increased in all groups to a similar extent with no statistically significant difference between groups. In the 2 × 2 factorial analysis after 9 months, no treatment interaction was found on Hb  contains supplementary material, which is available to authorised users. Plasmodium falciparum malaria for 24 % of all anaemia cases ) and 9 months  , the decrease of malaria prevalence in groups 3 and 4  was significantly higher than in groups 1, 2 and 5   in all five groups showed no major differences at baseline, except CRP in group 5 was significantly higher than in groups 1, 2 and 4.P = 0.78), on AGP concentration (P = 0.18) or on CRP (P = 0.52). In the 2 × 2 factorial analysis, neither intervention significantly affected the P. falciparum prevalence at 6 or 9 months follow-up. CRP concentration was reduced by 47 % after 9 months (95 % CI 2–92 %) in children receiving IPT (Table P = 0.030]), but this effect was not observed after 9 months  no interaction between IPT of malaria and CF on Hb concentration, anaemia or ID; (2) iron fortified CF had no effect on Hb concentration or anaemia prevalence but substantially decreased the prevalence of ID; (3) IPT of malaria modestly reduced anaemia; and (4) iron fortification with FeFum or FePP had the same effect on Hb concentration, although anaemia decreased more in children receiving FePP.Despite covering >90 % of the iron RNI with compounds expected to have high bioavailability, after 9 months, iron fortification had no significant effect on Hb concentration or anaemia prevalence. The results differ from a subgroup analysis from a recently published Cochrane review that reported a positive effect of iron fortification at point of consumption with micronutrient powders on anaemia prevalence in malaria-endemic regions . HoweverID was defined as PF <30 µg/l and was based on recommendations from WHO  for popuP. falciparum parasitaemia [Treatment with SP/AQ clears sitaemia , and is sitaemia , 25. Malsitaemia . Hence aP. falciparum infection [P. falciparum infection [P. falciparum is the predominant risk factor for anaemia in young children.No evidence was found that IPT of malaria improves the efficacy of iron fortified CF by facilitating iron absorption or erythropoiesis, which are reported to be diminished during nfection , 27. Hownfection . The lacnfection . This stP. falciparum infection in the control group did not change during the first 6 months, but it decreased by almost one-third over the last 3 months. Although Hb concentration did not change in the control group during the first 6 months, it increased during the last 3 months of intervention. The effect of IPT on Hb appeared to be stronger at the end of the rainy season (at 6 months). This suggests the abatement of malaria transmission during the dry season likely influenced the impact of the treatments, and may have been responsible for the differences in outcomes at 6 and 9 months.The present study was implemented at the transition zone of tropical rainforest in the South and the savannah in the North  and, altThe present study has several strengths. It was conducted in infants and young children in a rural setting of West Africa where anaemia is common and malaria transmission is intense. Beside the comparison of the five groups, a 2 × 2 factorial design was employed to assess potential interactions between CF and IPT and compliance was high for both interventions. Iron compounds with high bioavailability were used that had previously demonstrated efficacy . An effeLimitations of the present study include possible confounding by seasonal variations in malaria transmission, the relatively high drop-out rate (attrition bias), and the use of a single iron biomarker (i.e. PF) to define iron status. Inherited haemoglobinopathies are unlikely to play a major role in anaemia in this population, as previous studies in this region reported 83 % of subjects to have normal Hb genotype, and only 8, 7 and 1 % carried an S allele, C allele, and had sickle cell anaemia, respectively . LikewisThe results of the present study confirm that anaemia is a major public health problem in young children in Côte d’Ivoire and requires urgent action. About half of the anaemic children were iron deficient and the iron fortified CF almost eliminated ID, but against the authors’ expectation, it did not decrease anaemia prevalence. This observation is in line with a recently published Cochrane review including anemic children living in malaria-endemic regions receiving iron supplementation and IPT for a 12-week period . The aut


Unlike other herpes viruses, Kaposi's sarcoma-associated herpes virus (KSHV) is not ubiquitous worldwide and is most prevalent in sub-Saharan Africa. The reasons for this are unclear. As part of a wider investigation of factors that facilitate transmission in Uganda, a high prevalence country, we examined the association between antimalaria antibodies and seropositivity against KSHV.P. falciparum merozoite surface protein (PfMSP)-1, P. falciparum apical membrane antigen (PfAMA)-1 and KSHV antigens (ORF73 and K8.1) were measured in samples from 1164 mothers and 1227 children.Antibodies against PfMSP-1 and PfAMA-1, respectively (P < 0.0001). Among children, only antibodies to PfAMA-1 were significantly associated with KSHV seropositivity, (P < 0.0001). In both mothers and children, anti-ORF73 antibodies were more strongly associated with malaria antibodies than anti-K8.1 antibodies.Kaposi's sarcoma-associated herpes virus seroprevalence was 69% among mothers and 15% children. Among mothers, KSHV seroprevalence increased with malaria antibody titres: from 60% to 82% and from 54% to 77%, comparing those with the lowest and highest titres for The association between malaria exposure and KSHV seropositivity suggests that malaria is a cofactor for KSHV infection or reactivation. Kaposi's sarcoma-associated herpes virus (KSHV) is the causative agent of Kaposi's sarcoma (KS) ,2. In geMansonella perstans and malaria parasitaemia were independently associated with KSHV seropositivity; in children, only malaria parasitaemia showed an association with KSHV seropositivity, but prevalence of the other parasites was very low [As all other known human herpes viruses are ubiquitous worldwide, the geographic variation in prevalence of KSHV is anomalous and the factors that sustain higher rates of transmission in sub-Saharan Africa than elsewhere are unclear. In a cross-sectional study of women, hookworm infection, very low ,8. Helmivery low ,10; thisvery low ,12. BothRepeated malaria exposure has detrimental effects on immune function –16. ThesThis was a cross-sectional study carried out within the context of a clinical trial, the Entebbe Mother and Baby study (EMaBS) (ISRCTN32849447). EMaBS is an ongoing birth cohort that originated as a double-blind, randomised placebo-controlled trial designed to determine the impact of helminth infections and their treatment on vaccine responses and infectious diseases outcomes; the details have been reported elsewhere ,20. A toThis study was approved by the Science and Ethics Committee (SEC) of the Uganda Virus Research Institute, Uganda National Council for Science and Technology and the London School of Hygiene & Tropical Medicine Research Ethics Committee.Stored plasma samples taken from 1164 mothers in the early post-partum period, and from 1227 of their 5-year old children, were screened for the presence of KSHV antibodies using an enzyme-linked immunosorbent assay (ELISA) for recombinant proteins to a lytic structural glycoprotein, K8.1 and a latent nuclear protein latency-associated nuclear antigen (LANA) encoded by ORF73. Each plate contained three positive and three negative controls. Each assay cut-off was calculated based on the performance of the negative controls. This procedure has been reported elsewhere ,22.P. falciparum antigens: merozoite surface protein (MSP)-1 and apical membrane antigen (AMA)-1 [The same plasma samples were tested for malaria antibodies using two  (AMA)-1 . A pool  (AMA)-1 .P-values; all P-values are two-sided. To determine the effect of malaria antibodies on KSHV seropositivity, we grouped malaria antibody titres into three groups, for mothers and children separately: low, middle and high. The selection of the groups was not based on any biological or clinical criteria but on the frequency distribution of the titres. Antibody titres from below the 25th quartile were grouped as low, from the 25th to 75th quartile as middle and above the 75th quartile as high. As grouping continuous risk factors leads to loss of information [Statistical analysis was performed using Stata-12 software . Separately for mothers and children, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the Mantel–Haenszel test and logistic regression to obtain crude and adjusted odds ratios and Plasmodium falciparum malaria using two antigens, PfMSP-1 and PfAMA-1. As expected, antibody titres to both PfMSP-1 and PfAMA-1 were much higher in mothers compared to children. The median antibody titres to PfMSP-1 and PfAMA-1 among mothers were 737.5 and 600.2, respectively, while the median antibody titres to PfMSP-1 and PfAMA-1 among children were 103.4 and 17.2, respectively. To establish the seroprevalence of KSHV among mothers and their children, we tested for KSHV-specific antibodies using two KSHV antigens, K8.1 and ORF73. Individuals with antibodies to either ORF73 or K8.1 were considered to be KSHV seropositive, and individuals with antibodies to neither ORF73 nor K8.1 were considered to be KSHV seronegative. The seroprevalence of KSHV was 69% (806/1164) among mothers and 15% (185/1227) among children. Among mothers, 64% (744) were positive for anti-ORF73 antibodies and 46% (539) were positive for anti-K8.1 antibodies. Among children, 8% (96) were positive for anti-ORF73 antibodies and 12% (148) were positive for anti-K8.1 antibodies.To determine the level of malaria exposure, we quantified antibody titres to P = 0.01) among mothers, but the association was lost when we adjusted for household socio-economic status and location. Because all children were 5 years old, age was not included in the analysis of children's data.Tables PfMSP-1and PfAMA-1 were strongly associated with KSHV seropositivity. The odds of being KSHV seropositive among mothers with high malaria antibody titres, compared to those with low malaria antibody titres, were 2.67 (P < 0.0001) and 2.43 (P < 0.0001) for PfMSP-1 and PfAMA-1 antibodies, respectively   for PfMSP-1 antibody titres and 60% (P < 0.0001) for PfAMA-1 antibody titres. In the children, for every fourfold increase in PfAMA-1 antibody titres, the odds of being KSHV seropositive increased by 53% (P < 0.0001). PfMSP-1 antibody titres were not associated with KSHV seropositivity among children.Treating antimalaria antibody titres as a continuous variable, among mothers, for every fourfold increase in malaria antibody titre, the odds of being KSHV seropositive increased by 67% (PfMSP-1 and PfAMA-1 in the mothers. (2) In the children, KSHV seropositivity was highly associated with malaria antibody titres to PfAMA-1 but not PfMSP-1. (3) In both mothers and their children, the association between malaria antibodies and KSHV antibodies was much stronger with ORF73 antibodies than K8.1 antibodies.To our knowledge, this is the first study to investigate malaria antibodies in relation to KSHV seropositivity. Its major findings are as follows: (1) KSHV seropositivity was strongly associated with malaria antibodies to both Kaposi's sarcoma-associated herpes virus seropositivity was strongly associated with both malaria antibodies in the mothers. In this study, we have used malaria antibodies as a proxy measure of malaria exposure. Continuous exposure to malaria infection leads to the development of an antibody response, some of which is associated with protective immunity to clinical disease –28. The The association between KSHV seropositivity and malaria antibodies could also be directly due to the effects of malaria parasite infection. We have previously shown that asymptomatic malaria parasitaemia is associated with KSHV seropositivity ,8. PossiWe found that, in both mothers and their children, the association between KSHV antibodies and malaria antibodies is much stronger with ORF73 antibodies than with K8.1 antibodies. ORF73 encodes LANA (latently associated nuclear antigen), which is expressed during latency to facilitate KSHV episomal replication, segregation of the KSHV genomes to daughter cells and viral oncogenicity . K8.1, iPfAMA-1 but not PfMSP-1 in the children. Possible explanations  could be that (i) the KSHV seroprevalence was very low in children (15%); this might have hindered detection of significant associations. (ii) The PfAMA-1 used is a larger molecule, will have more epitopes and therefore, is potentially a more sensitive biomarker for exposure [PfMSP-1 take longer to develop, and hence their levels during early life are less well correlated with malaria exposure [Unlike in the mothers, we observed that KSHV seropositivity is associated with antibodies to exposure . Also, aexposure .Our sample sizes were large enough to allow detection of statistically significant associations, at least in the mothers. Also, data on possible confounders were available; therefore, we were able to adjust for them. We were also able to detect specific antibody responses to more than one antigen per infection. Potential weaknesses were the lack of any information on the temporality, which could have effects on malaria infection distribution, and the cross-sectional study design, which did not allow us to ascertain when malaria antibody titres increased and when KSHV primary infection occurred.Repeated exposure to malaria infection has detrimental effects on immune function, for example by leading to B- and T-cell exhaustion. We have shown that exposure to malaria infection, detected using malaria antibodies, is highly associated with KSHV seropositivity. Therefore, it is possible that malaria exposure is facilitating reactivation of KSHV, and perhaps also increasing the susceptibility to infection, which in turn may lead to increased transmission.


Malaria is a major disease in Africa and leads to various public health problems. A study was carried out at the Aviation Medical Clinic Laboratory, Murtala Mohammed Airport, Ikeja, Lagos State, Nigeria, in 2014. The work aimed to determine the prevalence of malaria among patients attending the laboratory. Blood samples were therefore collected from 51 patients and subjected to both blood smear microscopy and a rapid immunochromatographic diagnostic test  for detection of, respectively, malaria parasites and antigens. At the end of the study, 22% of the patients were detected positive by the microscopic examination while 9.8% were tested positive when using SD BIOLINE Malaria Ag. The outcomes of the study show a high prevalence of malaria at the airport. This represents a serious risk factor leading to a high likelihood of spread and occurrence of malaria in other countries including Western countries whereby the disease is nonendemic. It also pointed out that the blood smear microscopy seems to be better than Rapid Diagnosis Test (RDT) for malaria diagnosis. Malaria is one of the most common infectious diseases and a great public health problem worldwide. It is one of the world's deadliest diseases affecting people particularly in tropical and subtropical regions of the world, especially in sub-Saharan Africa and Southeast Asia.Pf-pLDH and/or pan-pLDH), a commercial immunochromatographic test based on plasmodium species-specific antigens that allow differential detection of P. falciparum and other species . It is the commonly used and the one available at this health care centre.Malaria's mortality rates have reached 42% globally since 2000 and 49% in the African region . Of the This study was carried out in the Aviation Medical Clinics, which is situated within the Murtala Mohammed International Airport, Ikeja, Lagos, Nigeria, in 2014. In order to achieve the objective of this study which was to investigate the possibilities of malaria transportation from Nigeria to other countries via travellers, this aviation clinic was chosen. It is under the Medical Department of the Federal Airports Authority of Nigeria (FAAN). It provides medical facilities to staff of FAAN, Nigeria Airspace Management Authority (NAMA), and Accident Investigation and Protection Bureau (AIB). It also responds to emergencies involving passengers and other airport users.Patients who visited the clinic during the study period with malaria symptoms were recruited for this study regardless of their age and sex. Participants were children (4-5 years old), adolescents (6–17 years), and adults (≥18 years) with severe and uncomplicated malaria. These samples were stratified by age so as to be able to assess the relationship of age with these parameters. Regarding ethical clearance, approval from the management of Aviation Medical Clinic was obtained before the implementation of the study. Moreover, an informed consent form was obtained from subjects participating in the study. For children <15 years, the consent was obtained from their parents.Venous blood was collected using 5 mL plastic syringe attached to a 20 SWG needle. In case the patient's veins are small a 23 SWG needle was used. Blood was collected in tubes containing ethylene diamine tetra acetic acid (EDTA). For finger prick, blood was collected on slides using the needle of a stylet from the fingertip of participants. Thick and thin blood films of the blood samples were made and air dried. Thin films were fixed with methanol and both thick and thin films were then stained with Giemsa. The slides were microscopically analysed for the presence of species of malaria parasites.The blood samples collected by finger prick were assayed using SD BIOLINE Malaria Ag test kits. It is an immunochromatographic test that detects the presence of pan malaria specific antigen (pLDH: plasmodiumlactate dehydrogenase) for the detection of all malarial parasites.μL of blood and is readable after 15 minutes following the manufacturer's instructions . The results were recorded in comparison with the control line as positive if a unique Pf-pLDH line appears indicating P. falciparum infection and/or pan-pLDH line indicating either an infection with P. falciparum or a mixed infection with P. falciparum and one or more of the nonfalciparum species.The test uses approximately 5 Data were entered in Microsoft Excel 2013. Distribution of participants' characteristics and malaria parasite prevalence were assessed using contingency tables. Taking blood slide microscopy as the gold standard, the performance of the Rapid Diagnostic Test was compared to it by computing the sensitivity, specificity, the negative predictive value, and the positive predictive value of the test as per Wayne .Fifty-one persons of both sexes from 4 to 73 years old were recruited for the study . The groOut of the 51 persons recruited for this study, 11 patients were tested positive for malaria in microscopic examination, while 5 samples were tested positive with the RDT. The actual prevalence of malaria among patients attending this clinic is therefore about 22% using microscopy and 9.8% in the case of RDT . Further P. falciparum using RDT.The RDT used in this study is for malaria's antigen detection in order to assess its effectiveness by comparing it to the gold standard. However, only 5 out of the 11 positives (microscopy) were tested positive forFrom Plasmodium spp. antibodies. Such situation can therefore compromise the health safety of Western countries' travellers in case they are not diagnosed and do not receive adequate malaria treatment before going back to their home countries, whereby they may not have the chance to receive specific malaria treatment. Nevertheless, RDT detected only 9.8% of malaria positive cases at the same facility on the same sample types. According to previous comparative studies between RDT and microscopic malaria diagnostic tests, RDT usually provides greater prevalence than microscopy [Malaria prevalence detected by slide microscopy in this study was about 22%. This prevalence is relatively high but normal because of the endemicity of malaria in the study area. However, it shows a threat of malaria spread from Nigerian to other foreign airport users who might probably be coming from nonendemic malaria countries. As a matter of fact, Medina Costa et al.  reportedcroscopy , 7. With Plasmodium falciparum specific histidine rich protein-2 (HRP-2) [ Pf-pLDH. The difference between these two types of antigens targeted during the RDT that could lead to differences in the detection proportion was previously explained by Murray et al. [ Pf-pLDH rapidly fall to undetectable levels after initiation of effective therapy. Likewise, Kocharekar et al. [ Pf-pLDH based RDT was giving higher sensitivity (95%) than HRP-2 based RDT (70.3%). Fifteen days later, while patients were undertaking treatment, the sensitivity of Pf-pLDH based RDT falls to 7.92%, while the one of HRP-2 based RDT still gave as high sensitivity as 58.41%. In other words, positive cases detected by the PfHRP2 based test can be regarded as false positive cases since people do not normally have the parasite in their blood stream anymore. Furthermore, considering that some of the patients used in this study might be undergoing malaria treatment before the sampling has been done, the RDTbased on Pf-pLDH might fall to signal them as positive. This justifies the low prevalence of malaria given by the RDT in this study compared to the slide microscopy. Moreover, there was a sexual distribution of the prevalence, showing that male patients were more infected by malaria parasite than female patients. This suggests that males may probably be at higher risk of mosquito bites than females, which could be explained by the fact that African women sleep under mosquito nets freely received at health care centres during pregnancy and for their children protection.For instance, most of studies that came out with higher prevalence from RDT than microscopy are based on (HRP-2) , while i (HRP-2)  the RDTsy et al. . They dey et al. . Accordiy et al. , the HRPr et al.  testifieMoreover, the sensitivity of RDT in this study was low (45.45%). The probability of RDT to detect positive cases among microscopically positive cases is therefore much less. This low sensitivity can be due to either the very low parasitaemia in people not seeking treatment or the fact that the RDTs were possibly defective or handled inappropriately causing them to lose sensitivity.Factors like products, storage temperature, and humidity can affect the diagnostic accuracy . HoweverOut of this study, it has been noticed that the prevalence of malaria among people around airports is not negligible and must be further investigated since these places are like interface between foreign people and natives. They are also considered as a risk factor for spread of malaria in developed countries. Highly sensitive and specific diagnostic tools are therefore needed in these areas. Besides, based on the results of this study, well-conducted blood slide microscopy for malaria diagnosis remains the preferred option. The low level of the agreement between RDT and slide microscopy for malaria diagnosis warrants further investigations in clinical facilities and aviation clinic laboratories especially.


Plasmodium falciparum malaria. The present study was intended to determine if the antibody and inflammatory responses of children living in a malaria-endemic area varied depending on the RESA-1, RESA-2 or RESA-3 proteins and the severity of the disease, two groups of severe and uncomplicated malaria cases being considered.The three members of the ring-infected erythrocyte surface antigen (RESA) proteins family share high sequence homologies, which impair the detection and assignment to one or another protein of some pathogenic processes inherent to Two synthetic peptides representing predicted B cell epitopes were designed per RESA protein, all located outside of the 3′ and 5′ repetition blocks, in order to allow an antibody detection specific of each member of the family. Recombinant rRESA-1B and rRESA-3B proteins were also engineered. Two groups of Beninese children admitted to hospital in 2009 for either uncomplicated or severe malaria were compared for their plasma levels of IgG specifically recognizing each recombinant RESA protein or synthetic peptide, and for their plasma inflammatory cytokine levels , taking into account host and parasite genetic factors.P = 0.007). Increased IL-10 plasma levels were found in children harbouring multiclonal P. falciparum infections on the basis of the T1526G resa2 gene polymorphism (P = 0.004).The absence of IgG cross-reactivity between rRESA proteins and their protein carrier as well as between each RESA peptide and a non-epitopic RESA control peptide validated the use of the engineered recombinant proteins and peptides for the measurement of plasma IgG. Taking into account age, fever duration and parasitaemia, a multiple logistic regression performed on children clustered according to their antibody responses’ profiles concluded to an increased risk of severe malaria for P2 (representative of RESA-1) responders  contains supplementary material, which is available to authorized users. Although estimated incidence rates of malaria have declined by 30% globally between 2000 and 2013, and mortality rates by 47%, malaria is still the most widespread of all human infectious diseases, responsible for an estimated 5,84,000 deaths in 2013  − [ODm NC])/([OD PC] − [OD NC]) where ODm is the mean reactivity of PC or NC for all plates for a defined peptide and OD PC or NC is the mean reactivity of the control plasma samples for a defined peptide of a defined plate. The thresholds for positivity were determined from the mean OD reactivity + 2 SD of 18 plasma samples from non-immune individuals and were 0.741 (MBP), 0.137 (rRESA-1B), 0.000 (rRESA-3B), 0.026 (P Ctl), 0.387 (P1), 0.459 (P2), 0.487 (P3), 0.482 (P4), 0.185 (P5), and 0.352 (P6).−1 by reference to standard curves prepared in each plate with recombinant cytokines. Thresholds of sensitivity were 2 pg mL−1 for IFN-γ, 13 pg mL−1 for TNF-α and 0.5 pg mL−1 for IL-10. Zero was assigned to values below the thresholds.Plasma samples were assayed in duplicate for IFN-γ, TNF-α and IL-10 using ELISA assay according to the manufacturer’s instructions . Results were expressed in pg mLHuman and parasite DNA was extracted from blood spots as described previously .MseI RFLP. A short resa2 gene fragment containing the target base was first amplified by PCR using 5′-TGATGCCGTAAAAGATGGTG-3′ (sense) and 5′-TCATATCTGCATTTATATCGACACCT-3′ (anti-sense) primers. Then enzymatic digestion of the PCR products by MseI  left uncut those fragments carrying the T1526G single nucleotide polymorphism  favoured the occurrence of severe malaria. Both axillary temperature at enrolment and fever duration were kept in the model even if associations were not significant . The fever duration (3–4 days compared to 1–2 days) was weakly associated with a higher risk of SM  and a high axillary temperature (≥39.5°C) appeared indicative of an UM . No impact of haemoglobin genotypes or resa2 mutation was recorded at this final stage of the analysis. In the following analyses, association between anti-RESA antibody responses and severe malaria were adjusted on age, parasitaemia and fever duration. As axillary temperature could not be considered itself as a predictor of severity of malaria infection it was not included in models in order to avoid over fitting.When clinical  and genetic  while the same sera did not cross-react with the MBP control protein .ELISA performed with a rRESA protein can detect not only antibodies raised against this protein but also against the other RESA proteins. The ELISA assay for the measurement of anti-rRESA IgG was validated by the fact that sera recognized and cross-reacted with both rRESA-1B and rRESA-3B . In the same way, IgG against P2 and P4, P2 and P5 as well as P2 and P6 were highly correlated for the UM group only . No correlation was observed between anti-rRESA IgG and IgG directed to the associated peptides.Because the hypothetic B cell epitopes were chosen in non-homologous parts of each RESA protein, the measurement of anti-peptide antibodies was RESA protein-specific. Similarly, a validation of the ELISA assay for the measurement of anti-peptide IgG was brought by the absence of recognition by sera of the non-epitopic peptide P Ctl, as well as by high variability in the sera recognition of distinct peptides. Plasma IgG directed to P1 and P3, P1 and P4, P3 and P4 as well as P5 and P6 were also highly correlated, either for the whole group or for SM and UM groups considered separately  inversely to what was observed for P4, with 3.7% of responders among SM patients vs 18.8% among children with UM (P = 0.01). Responders from both clinical groups had similar IgG levels, as presented in Table P = 0.02].Prevalence rates of IgG responses to RESA antigens according to the clinical presentation of malaria are illustrated in Figure P = 0.013) with a greater risk for Group C  and a trend for Group A .The cluster analysis concluded to an optimal partition of children into three groups presenting particular patterns of their anti-RESA antibody responses Figure . Thirty-P = 0.02]  = 4.10 , % CI = 4.−1 (IQ25–75) = 23.5 (0–142.5) vs 0 (0–8.2), P = 0.004], as shown in Table resa2 T1526G mutation was associated with IL-10 plasma levels, the highest cytokine levels being recorded for children with a mixed infection compared to children infected by parasites harbouring only wild type resa2 T1526G alleles .Plasma levels of inflammatory cytokines  were measured at enrolment in the study and revealed higher TNF-α levels among SM cases than UM ones [median content in pg mlThe present study was intended to correlate the presence of immune responses against the three members of the RESA protein family with the severity of malaria attacks. For this purpose, recombinant proteins were engineered for the two repetitive RESA-1 and RESA-3 proteins and peptides representing putative B epitopes of each RESA protein were designed and synthesized. In most previous studies performed on the RESA antigen, peptides were representative of central or C-terminal repetitive RESA-1 sequences , 21, 24.Plasmodium life cycle and therefore to relate its presence on the red blood cell surface to the different clinical presentations of malaria. Globally, the antibody responses reported in this study reinforced the interest in the already known immunogenicity of the RESA-1 antigen, as no relation to malaria severity was put forward for antibody responses directed to RESA-2 peptides and RESA-3 peptides or RESA-3 recombinant protein. Indeed, only the anti-P2 IgG response was related to malaria severity. This observation was further supported by a cluster analysis, which allowed distributing children into three groups with distinct profiles of their anti-RESA antibody responses: the group of children responders to P2 and inconsistently responders to rRESA-1B presented an association with malaria severity. This association was independent of age, fever duration and parasitaemia levels.No correlation was observed between the IgG recognition of rRESAs and synthetic peptides. Since RESA proteins have highly homologous sequences, they should induce cross-reacting antibodies. Thus, the observed IgG reactivity to a defined RESA protein results from a contribution of the IgG responses directed to identical sequences in RESA-1, RESA-2 and RESA-3, but does not reflect the immunogenicity of a defined protein. Moreover, because no RESA three-dimensional structure has been solved, all designed peptides may not have been optimized for their antigenicity and for being fully accessible to the humoral immune system. A single peptide can by no means recapitulate the complete antigenic properties of a full-length protein. Nevertheless, the serum recognition of a pertinent peptide allows estimating the ability of a protein to induce antibodies during the resa2 gene mutation was associated with parasitaemia >4%, leading to hypothesize that the restoration of a full-length RESA-2 protein could contribute with the other members of the RESA protein family to remodelling the erythrocyte membrane [It was previously shown among the same population group that the T1526G membrane  and thermembrane . In thismembrane –31; howemembrane . A subtlmembrane .resa2 alleles. This observation was based on only six samples but still suggests the following hypotheses. Even if the multiplicity of infection (MOI) was not evaluated in this study by genotyping polymorphic parasite genes such as msp1 or msp2, the presence of concomitant wild and mutant resa2 T1526G alleles was indicative of polyclonality. There seems to be a consensus that high MOI are found during symptomatic malaria episodes of young children from endemic areas, who have a low level of acquired immunity [In link with an explanation towards IL-10 as a marker of severity was the observation in the present study of the highest IL-10 plasma levels among children infected by mixed isolates presenting wild and mutant immunity . A studyimmunity . As no aP. falciparum proteins exported to the erythrocyte membrane in order to help the infected erythrocyte to escape elimination by the spleen, must be deciphered. It is the case of the three components of the RESA protein family, whose respective roles in increasing red blood cell resistance to febrile conditions are difficult to distinguish due to great sequence homologies. This study offered the first opportunity to dissect the antibody recognition of each RESA protein using synthetic peptides representing specific sequences of each RESA protein. The measurement of naturally acquired IgG in the context of clinical malaria indicated a possible relationship to severe malaria of anti-RESA-1, but not anti-RESA-2 nor anti-RESA-3 IgG. As the design of the study was transversal, the protective role of these IgG cannot be ascertained. Increased IL-10 plasma levels in children harbouring multiclonal P. falciparum infections on the basis of the T1526G resa2 gene polymorphism may be suggestive of a pathogenic role of the full-length restored RESA-2 protein. Nevertheless, the small sample size in this study implies the same observations to be made in an independent study or a larger sample in order to be confirmed. At term, the deepening of the understanding of the malaria pathogenesis will provide new possibilities for interventions aimed at reducing parasite survival in the human host.To better understand malaria pathogenesis, the role of