Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead-based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast.Plasmodiumfalciparum , the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour-coded microsphere (Luminex™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings  were analysed.Recombinant proteins or peptides derived from liver or blood stage antigens of 41 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post-treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50 % of the parasite antigens tested.Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97 % (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite-derived recombinant proteins or peptides. Prevalence of responders to LSA1The multiplex MBA-Magpix technology assay provides an accurate high throughput monitoring of parasite-specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies. Plasmodium. In Ivory Coast, malarial endemicity has been shown to be heterogeneous, depending upon various bio-ecological areas  as already described [The custom-made Magnetic Bead-based MAGPIXescribed . Plates IgG responses were quantified by ELISA in duplicate plasma samples diluted 1:100 as previously described using whole parasite extract Ag from schizonts 07/03 Dielmo strain adapted to culture , 29, 30.® 9.3 Procedures Guide. Cary, NC: SAS Institute Inc.) and R .Antibody levels and prevalence of responders in different groups were compared using the Mann–Whitney signed rank test, the Spearman rank correlation test for non-normally distributed paired data and the Fisher exact test. In case of multiple associated comparisons, Bonferroni correction was applied. Statistical analyses were performed using Statview 5.0  to each clinical sign and setting  and antibody responses.Recruitment was done after the rainy season for 30 patients per village, a limited proportion of children under 5 years old were included, except in Man . Mean age of the three groups was similar without any significant difference in the age distribution between the three settings Table .−3). These three settings are high risk for malaria, the prevalence of clinical malaria in Abobo is approximately 50 % lower than the mean national level and 150 % lower compared to the rural villages. Of note, urban patients recruited in Abobo suffered highly parasitic clinical episodes: 10 and 30 % of the patients harboured <10,000 or >100,000 trophozoites per μL, respectively, compared to 26 and 16 % in Man, and 56 and 0 % in Korhogo for these cut-off parasitaemia levels.As shown in Table Importantly, treatment was highly efficient: 55, 42 and 3 % of patients overall cleared their parasitaemia in 24, 48 and 72 h, respectively. As detailed in Table Prevalence of antibody responses in the three settings against the 12 biomarkers is summarized in Table 41. In Korhogo, prevalence of antibody responses was significantly higher compared to the two other villages for CSP, LSA141, SALSA, AMA1, MSP4p20, and P. malariae CSP. Prevalence of responders to PF13 was significantly lower in Abobo (45 %) compared to the two other settings .As detailed in Table −3). When comparing responses to biomarkers in Abobo vs Man, no significant differences appeared except for PF13 (P = 0.04). Of note, the IgG response against whole parasite extract  did not significantly discriminate any significant high level of antibody response in Korhogo as did the set of biomarkers.Levels of antibody responses are summarized in Table −3). As summarized in Table Results of analysis for inter-relation between antibody responses against the different targets and the age of individuals are detailed in Table −3) rho = −0.35 and PF13 (P < 10−4) rho = −0.41.There was no correlation between parasitaemia observed at recruitment (or log-transformed parasite density) with antibody responses against all antigens except a significant negative correlation with IgG responses to LSA3  in individuals with high levels of parasites . A significant relationship was found between parasitaemia and thrombocytaemia (P < 0.001), thrombocytaemia decreased when parasitaemia increased.PmCSP.The relationship between immune responses and the efficiency of treatment was analysed by stratifying clearance of parasitaemia between early <24 h) and later (48–72 h) delay of blood smear negativation. As shown in Fig. 4 h and lWhen investigating correlates with the records of clinical signs, a significant relationship was found only between parasitaemia and nausea (P < 0.01), parasitaemia was higher in patients with nausea. In the multivariate analysis, studying the relationship of parasitaemia with PF13, LSA3, thrombocytaemia, nausea, village, the last model was based only on PF13 and thrombocytaemia. Parasitaemia decreased with PF13 level and increased when thrombocytaemia decreased.P. falciparum malaria and its recent history in Ivory Coast. However, observed P. falciparum prevalence in previous surveys from central and south-central Ivory Coast [A. gambiae to polluted water [The present study was undertaken to draw a first profile of antibody responses to a large array of antigens in three sentinel settings for malaria national surveillance and treatment in Ivory Coast, a country with a high level of endemicity . There ary Coast , 32, 33 ry Coast . Transmiry Coast , as welled water  or the sed water .A. gambiae sub-population can add a possible source of increasing transmission [In this context, affordable tools for evaluation and monitoring of malaria control interventions, such as immune biomarkers, can play a valuable role –11, 14. smission .P. falciparum strongly stimulates immune responses against all components of the parasite. Thus, all patients had a comparable, individual, clinical susceptibility to infection that necessitated consultation and hospitalization, independently of the setting and the individual natural history of infection. There was a clear and highly significant, unexpected, difference of antibody responses between one of the setting (Korhogo) and the other two.Recruitment of patients with non-severe clinical malaria was a first pilot approach, regarding which clinical infection by Most sero-epidemiology studies rely on cross-sectional or longitudinal follow-up of individuals before or after clinical episodes, including prospective analysis to establish relationship between antibody responses against one antigen –23, 38 oInterestingly, the whole parasite extract from 07/03 in vitro-adapted strain from Dielmo has been already used as a relevant marker for follow-up of immunity levels , 42, 43,41 and the levels of antibody responses to PF13. PfEMP1–PF13 antigen was shown as a relevant biomarker of cumulative acquisition of immunity in endemic setting [One main question raised by the measure of multiplex reactogenicity profile on day of consultation is the search for potential immune marker(s) able to predict a differential susceptibility to the observed level of infection. Only two antigens induced antibody responses significantly different between the sites of Abobo and Man: the incidence of responders to LSA1 setting , 44 and Susceptibility to infection can be reflected by the level of parasitaemia and by the degree of individual severity of clinical signs. The level of parasitaemia was significantly lower in Korhogo compared to the two other sites and there was a clear decreasing gradient of high level parasitaemia from Abobo (30 %) to Man (16 %) and Korhogo (0 %). The multivariate analysis investigating correlation between level of parasitaemia, according to each clinical signs and setting, underlined only nausea as a significant clinical sign. This is in line with observation that severity of clinical outcome in a mild malaria episode is not related to the level of parasitaemia or acquired immunity, once susceptible . HoweverAnopheles salivary peptide gSG6-P1 antigen, have been shown to be a new relevant marker of risk for malaria transmission [Importantly, this study shows clearly the potential impact of host immunity on malaria treatment outcome. A fast clearance of circulating parasite 24 h) was associated with higher level of immunity. This general trend, shown in Fig. 4 h was asmission –7, 51–53Accurate analysis of immune responses from malaria symptomatic individuals can underline heterogeneity in individual degree of exposure and bring important information on the effective application of control measures at a community level.Plasmodium species. However, further investigations are required, including antigen targets from other species, enlarged to multiple sites and comprising enrolment of young individuals. Immunity profiling using biomarkers has the potential for immunological mapping of intervention and help with prevention measures towards malaria elimination.This pilot study suggests that measures of antigen reactivity profiles using the MBA can conveniently contribute to follow-up and monitoring of integrated malaria control measures. A panel of antigens, covering targets from different parasite stages is preferentially required, including antigens from other


Although malaria control intervention has greatly decreased malaria morbidity and mortality in many African countries, further decline in parasite prevalence has stagnated in western Kenya. In order to assess if malaria transmission reservoir is associated with this stagnation, submicroscopic infection and gametocyte carriage was estimated. Risk factors and associations between malaria control interventions and gametocyte carriage were further investigated in this study.Plasmodium falciparum parasite presence and density were determined by stained blood smear and by 18S mRNA transcripts using nucleic acid sequence-based amplification assay (NASBA), gametocyte presence and density were determined by blood smear and by Pfs25 mRNA-NASBA, and gametocyte diversity by Pfg377 mRNA RT-PCR and RT-qPCR.A total of 996 dried blood spot samples were used from two strata, all smear-positives (516 samples) and randomly selected smear-negatives (480 samples), from a community cross-sectional survey conducted at peak transmission season in 2012 in Siaya County, western Kenya. Of the randomly selected smear-negative samples, 69.6 % (334/480) were positive by 18S-NASBA while 18S-NASBA detected 99.6 % (514/516) smear positive samples. Overall, 80.2 % of the weighted population was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the weighted population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Children 5–15 years old were more likely to be parasitaemic and gametocytaemic by NASBA than individuals >15 years old or children <5 years old while gametocyte density decreased with age. Anaemia and self-reported fever within the past 24 h were associated with increased odds of gametocytaemia. Fever was also positively associated with parasite density, but not with gametocyte density. Anti-malarial use within the past 2 weeks decreased the odds of gametocytaemia, but not the odds of parasitaemia. In contrast, recent anti-malarial use was associated with lowered parasite density, but not the gametocyte density. Use of ITNs was associated with lower odds for parasitaemia in part of the study area with a longer history of ITN interventions. In the same part of study area, the odds of having multiple gametocyte alleles were also lower in individuals using ITNs than in those not using ITNs and parasite density was positively associated with gametocyte diversity.A large proportion of submicroscopic parasites and gametocytes in western Kenya might contribute to the stagnation in malaria prevalence, suggesting that additional interventions targeting the infectious reservoir are needed. As school aged children and persons with anaemia and fever were major sources for gametocyte reservoir, these groups should be targeted for intervention and prevention to reduce malaria transmission. Anti-malarial use was associated with lower parasite density and odds of gametocytaemia, but not the gametocyte density, indicating a limitation of anti-malarial impact on the transmission reservoir. ITN use had a protective role against parasitaemia and gametocyte diversity in western Kenya.The online version of this article (doi:10.1186/s12936-016-1482-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum in children below 5 years of age as diagnosed by blood smears declined from 60 % in 2003 to 26 % in 2008, but rose to 41 % in 2009 [Scale-up of malaria control interventions in endemic countries has resulted in drastic declines in malaria morbidity and all-cause mortality in many African countries. In western Kenya, after decades of malaria prevention and treatment measures, such as insecticide-treated nets (ITNs), intermittent preventive treatment in pregnancy (IPTp) and artemisinin-based combination therapy (ACT), the prevalence of P. falciparum gametocytes (stage I–IV) are sequestered in organ tissue, mainly in the bone marrow [Malaria transmission relies on sexual stage parasites, the gametocytes that derive from a small fraction of asexual parasites. Immature forms of e marrow . The game marrow , 4, and e marrow –7 using e marrow . The mate marrow . In thise marrow . The Pfse marrow . It has e marrow , 10.Gametocyte production and epidemiology could be associated or influenced by several factors that include transmission intensity, exposure to interventions like ITNs and anti-malarial drugs, host age/immunity, asexual parasite density, anaemia, multiplicity of infection , 11–15. Anti-malarials are also found to affect malaria transmission , 23, 24.P. falciparum gametocytes can persist three times longer than those from single-clone infections [ITNs have been deployed for over 16 years and remain the primary malaria intervention in western Kenya. ITNs significantly reduce malaria morbidity and all-cause mortality in children less than 5 years old , 31. ITNfections . Multiplfections , 34 and fections . HoweverGiven the complex biology and epidemiology of gametocytes that could be influenced by multiple factors including ITN use and anti-malarial treatment, the objectives of this study were: (1) to estimate the level of submicroscopic infection and gametocyte carriage in circulating blood, measured by molecular tools, in a region of western Kenya, where malaria transmission has stagnated since 2009, (2) to assess the risk factors associated with gametocytaemia, and (3) to determine the associations between malaria control interventions and gametocyte carriage. Results from this study could be useful in understanding and improving the interventions on transmission reservoirs and in providing the information for development of new strategies for transmission reduction and elimination of malaria.This study was part of a community-based annual cross sectional survey to evaluate impact of malaria control interventions on malaria parasitaemia and anaemia within the KEMRI/CDC Health and Demographic Surveillance System (HDSS) . The croThe sampling frame for year 2012 survey included all households in the study areas with at least one child under 5 years old. From this sampling frame, households were selected randomly via probability sampling and all individuals above 1 month of age were sampled in each selected household. A total of 1779 samples was collected for this survey. Self-reported information on age, fever, ITN and recent anti-malarial use were collected based on the study questionnaire. Participants were categorized into three age groups . Fever was defined as self-reported fever within the past 24 h. ITN use was defined as having slept under an ITN the night prior to the survey. Recent anti-malarial (AM) use was defined as receiving anti-malarials (96 % had taken AL) in any dosage at any time within the 2 weeks prior to the survey. During the survey, finger prick blood sample was collected to prepare blood smears to determine the presence of parasitaemia and gametocytaemia by microscopy . HaemoglIn addition, 50 µL of whole blood was collected into each spot of a Whatman 903 filter paper and dried overnight at room temperature. The dried blood spot (DBS) was sealed tightly with desiccants and a moisture indicator, and shipped to the CDC laboratory in Atlanta, USA within 2 months after sample collection and stored at −80 °C until use.For the present study, a total of 999 DBS samples from 1779 samples collected during the survey were tested to determine submicroscopic infection and gametocyte carriage. Among them, 446 samples were from Asembo, including all 221 smear-positive and a random selection of 225 from 600 smear-negative samples, and 553 samples were from Karemo, including 298 smear-positive and 255 from 660 randomly selected smear-negative samples. Random sampling of the smear negative samples was performed by Random Sample Excel Professional plus 2010 in Excel, based on the sample size calculated by the program online .Total RNA was extracted from DBS samples using QIAshredder and RNeasy mini kits  according to the QIAGEN protocols. A whole spot from a DBS sample was cut and used for RNA extraction. After RNA extraction, DNase digestion on one of three aliquots of RNA sample was performed with Ambion DNA-free DNA removal kits  for use in Pfg377 reverse transcription polymerase chain reaction (RT-PCR) and RT-qPCR assays. The other two RNA aliquots without Ambion DNA-free kit treatment were used for 18S-NASBA and Pfs25-NASBA assays.P. falciparum were measured for detection of malaria infections and submicroscopic parasites. The primers and molecular beacon probes of 18S-NASBA used were based on previously published methods [4–10−2 parasites/µL of blood) from cultured 3D7 ring stage parasites. The limit of quantification (LOQ) for 18S-NASBA was 0.01 parasites/µL blood based on the standard curve established in this laboratory.The mRNA transcripts of 18S small subunit rRNA gene of  methods . The ass methods . In orde4–1.8 × 10−2 gametocytes/µL) in culture blood . The limit of quantification (LOQ) for Pfs25-NASBA was 0.018 gametocytes/µL based on the standard curve established in this laboratory.Pfs25 mRNA was used to detect stage V gametocytes circulating in the host blood and Pfs25-NASBA was performed as previously described , 40, 41.For detection of gametocyte diversity, Pfg377 reverse transcription polymerase chain reaction (RT-PCR) was performed according to a published protocol . The Pfg4 to 100 gametocytes per µL of blood. All specimens were tested in duplicate; the mean Ct and number of gametocytes were obtained automatically on the Stratagene Mx3005P qPCR system . The limit of quantification (LOQ) for Pfg377 RT-qPCR was one parasites/µL based on the standard curve established in this laboratory.For quantification of Pfg377 mRNA, quantitative reverse transcription (RT-qPCR) of Pfg377 was performed as described previously . The staThe 18S-NASBA was used for measuring total parasite load and Pfs25-NASBA was used for measuring gametocyte carriage in both Asembo and Karemo study areas. In addition, Pfg377 RT-PCR and RT-qPCR were performed only on the samples collected in Asembo area for exploring the gametocyte diversity due to funding constraints. Because NASBA uses different approaches for determination of positivity and quantification of parasite density (see NASBA method section above), the positive values for samples with extremely low parasite density could have been below the cut-off of quantitative standard curve, in such positive samples a density was assigned at the midpoint between the cut-off and zero.All data management was performed in SAS 9.3 . Population weighted prevalence of parasitaemia and gametocytaemia was calculated via PROC SURVEYFREQ, accounting for field and laboratory sampling scheme and within-household clustering via Taylor series linearization . Model sMultivariable analysis was performed to evaluate participant characteristics associated with parasite or gametocyte presence and density, as well as characteristics associated with presence of multiple gametocyte alleles. Model selection was based on the generalized linear model (GLM) with either Gaussian distribution for continuous outcomes or binomial distribution with a logit link as appropriate for binary outcomes. Best fitting models were selected by lowest Bayesian information criterion (BIC) , and ful10 transformed prior to analysis, so estimates are presented as geometric means. Study area was included as a class variable in all models. Where there was no interaction or main effect of study area, overall estimates were presented. Where there was significant impact of study area, area-specific results were presented. Age category of participants was included in all models. The sample collection time was included as a class variable where lowest BIC models included week to account for trends across the sampling timeframe. Anaemia was evaluated as a potential predictor in models for gametocytaemia only. Interactions were not evaluated for gametocyte diversity due to small sample size.Association between gametocyte presence and participant characteristics was restricted to individuals who were 18S positive and, therefore, considered infected. Data on parasite and gametocyte density were logThree smear-positive samples from Karemo were excluded from analysis due to absence of information on fever in previous 24 h, leaving 550 samples from Karemo in this analysis. Thus, characteristics of participants for the study described here were from 996 individuals with complete data. Among 996 participants, 19.8 % of participants reported fever in the past 24 h while 67.5 % of individuals reported sleeping under an ITN the night prior to the survey. Among the 996 participants, 17.8 % had used anti-malarials within the 2 weeks prior to the survey, of which more than 96 % used was AL. Using the haemoglobin cut-off of <11.0 g/dl, 40.3 % of individuals were considered anaemic.Out of the 996 samples from Asembo and Karemo, 848 85.1 %) were 18S-NASBA positive  samples tested were gametocyte positive by Pfs25-NASBA (Table Pfg377 positivity was detected in 124 of 221 (56.1 %) smear-positive and in 6 of 225 (2.7 %) smear-negative individuals tested in Asembo. Six single gametocyte alleles were identified based on band size (range from 273–378 bps)  for parasite or gametocyte presence and association with predictors were calculated based on the data of parasite and gametocyte presence in 996 individuals tested   Fig. a. Parasi2 parasites/µL) was associated with higher probability of having multiple gametocyte alleles   Fig. . High pa26) Fig. , but higP. falciparum measured by sensitive RNA-based detection methods and to further assess the risk factors and interventions influencing gametocyte carriage in a community-based cross-sectional survey carried out in western Kenya in 2012 during peak malaria transmission season. Using a subset of samples from the survey, this study revealed an overall high level of submicroscopic parasitaemia (69.6 % of randomly selected smear negative samples) and gametocytaemia . When population level estimates were further calculated taking into account of multiple-layer sampling scheme, again a large proportion of infections were submicroscopic. Overall, 80.2 % of the weighted population sample was parasite positive by 18S-NASBA vs 30.6 % by smear diagnosis and 44.0 % of the population was gametocyte positive by Pfs25-NASBA vs 2.6 % by smear diagnosis. Risk factors for gametocyte carriage included being 5–15 years old, anaemia and self-reported fever. The study further showed that the use of anti-malarials within the past 2 weeks was associated with lower odds of gametocytaemia, but not gametocyte density, relative to untreated individuals. Use of ITNs the night before the survey was associated with lower odds of parasitaemia and lower odds of multiple allele gametocyte infection in Asembo.This study was conducted to estimate submicroscopic infection and gametocyte carriage of The overall high level of submicroscopic infection and gametocyte carriage observed in this study was mainly attributed to using the sensitive 18S- and Pfs25-NASBA methods. NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background . The risPrevious studies have suggested that a high prevalence of submicroscopic gametocytaemia may contribute to malaria transmission , 26, 56.Unlike a clear age-dependent decrease in densities of gametocytaemia Fig. , older cP. falciparum infection due to the destruction of infected erythrocytes, shortened survival of uninfected erythrocytes and dyserythropoiesis [P. falciparum infection [Similar to previous studies , 64, 65,opoiesis –68. It ropoiesis , 70 or dopoiesis  and erytopoiesis  triggeriopoiesis , while snfection , 65. Thenfection . But theAL, a highly effective anti-malarial drug with gametocytocidal properties , was impThis study showed that sleeping under an ITN the night prior to the survey was associated with lower odds of parasitaemia by 18S-NASBA in Asembo, but not in Karemo and also did not influence gametocyte presence among parasitaemic individuals. The different effect of ITN use on the odds of parasitaemia between the two areas could possibly reflect relatively lower level of transmission in Asembo than in Karemo. Entomologic inoculation rates were relatively higher in Karemo in both 2011 and 2012 although the rates were below 10 infectious bites per person per year for both areas . However, these EIR measures were subject to high variation as they were a combined estimate of mosquito densities and sporozoite rates and easily affected by site selection and sampling methods. Parasite positivity as measured by microscopy and by 18S-NASBA in this study was similar between both areas suggesting the differences in EIRs were not substantial. Individual analysis and combined analysis were conducted by study area to explore if there were varying risk factors in these two adjacent areas since they had different ITN implementation time periods (7 years apart). Results from this study confirmed that there were no significant differences in risk factors between Asembo and Karemo except for ITN use on parasitaemia mentioned above.This study showed that the higher parasite density by 18S NSABA was associated with increased odds of gametocyte diversity. Previous studies have reported that high multiplicity of infection (MOI) is correlated with high malaria transmission level in endemic areas –79, and,This study had a few limitations. First, the sampling strategy of cross-sectional survey in 2012 involved randomly selecting a probability sample of compounds with at least one child under 5 years of age. Therefore, no weighing for geographical density of households was done. Second, self-reported ITN use, fever, recent AL treatment might introduce recall bias among study participants. Third, due to funding constraints, the total number of samples tested for 18S- and Pfs25-NASBA was limited, resulting in full coverage of smear positive samples, but a random selection of smear negative samples from the survey. Because of the multi-level stratified sampling scheme, complex analysis was employed for determination of population prevalence estimates. Both household and laboratory selections were addressed by weighted analysis and all weighted estimates were based on the subset of data selected for laboratory analysis. Although this was a subset of the entire epidemiological dataset with a complex study design, the statistical correction used in this study most likely resulted in representative population estimates of the study area. As well, for resource limited reason, Pfg377 diversity was tested in the samples from the one area with a longer history of ITN interventions for exploration. Although the gametocyte diversity was only examined in this area, the results clearly showed that use of ITNs had a protective role against the gametocyte diversity. The exploratory results could lead to further investigation of gametocyte diversity in relation to sex ratio in context of vector control in future.This study showed a large proportion of submicroscopic parasites and gametocytes in western Kenya, which might partially explain stagnation in malaria prevalence and suggests that additional interventions are needed to target the infectious reservoir. As school aged children (5–15 years old) had the highest likelihood for both parasitaemia and gametocytaemia, this age group should be paid more attention in improving the ITN coverage in this area. Gametocyte presence was positively associated with fever and anaemia, indicating patients with clinical malaria or malaria-associated anaemia might be one of major sources for gametocyte reservoir and for potential malaria transmission in western Kenya. Recent AL use reduced parasite density and prevalence of gametocytes, but not the gametocyte density, indicating a limitation of AL in having an impact on transmission reservoir. ITN use played a protective role against parasitaemia and gametocyte diversity in an area with long-standing Asembo area of western Kenya.


This study aimed to investigate the total serum IgE profile A cross sectional observational study composed of microscopically confirmed malaria positive (N = 197) and malaria negative (N = 216) apparently healthy controls with and without helminth infection was conducted at Wondo Genet Health Center, Southern Ethiopia. A pre-designed structured format was utilized to collect socio-demographic and clinical data of the subjects. Detection and quantification of helminths, malaria parasites and determination of serum IgE levels were carried out following standard procedures.Irrespective of helminth infection, individuals infected by malaria showed significantly high levels of serum IgE compared with malaria free apparently healthy controls (with and without helminth infections). Moreover, malaria patients co-infected with intestinal helminths showed high level of serum IgE compared with those malaria patients without intestinal helminths (2198 IU/ml versus 1668 IU/ml). A strong statistically significant association was observed between malaria parasite density and elevated serum IgE levels  with high and low parasitaemia , respectively. Likewise, helminth egg loads were significantly associated with elevated serum IgE levels (P = 0.003).The elevated serum IgE response in malaria patients irrespective of helminth infection and its correlation with malaria parasite density and helminth egg intensity support that malaria infection is also a strong driver of IgE production as compared to helminths. Malaria remains one of the major causes of morbidity and mortality in the developing world and about 70% of the cases and 90% deaths are from Africa ,2. SimilM. tuberculosis and HIV . To detect malaria infections, 200 fields  were examined as described before . BrieflyThe presence of intestinal parasites was detected from stool samples using direct microscopy and formol-ether sedimentation techniques. Moreover, coarse quantification of eggs was obtained by counting the number of eggs on a smear of 41.7 mg of feces, and a quantitative variable scoring  was created for each helminth following the standard procedure used by WHO ,30.Entamoeba histolytica and Giardia lamblia) were not included for IgE determination. This is because of different IgE responses observed in protozoal and helminths infections.The total serum IgE levels were quantified by total IgE ELISA kit  following the manufacturer’s instructions as described earlier . BrieflyThe data was analyzed using to SPSS version 15 statistical package software. Intestinal parasite densities were transformed to log for analysis and geometric mean was used. Non-parametric tests were performed to compare mean values of the different groups. The Mann Whitney test and the Kruskall Wallis tests were used for comparisons between two groups and three or more groups, respectively. Spearman’s correlation was used to check for correlations between parameters. P values were considered significant when found <0.05.The study protocol and design including the consent procedures were approved by The Ethical Review Committee of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University and South Nations and Nationalities Peoples Region Health Department. Written (from those who can read and write) or verbal (from those who can’t read and write) informed consent from all study subjects was also obtained. The written informed consent and the ethical clearance letters are documented in both offices. However, verbal consent was not recorded and documented. The consent procedures were also approved by both The Ethical Review Committee of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University and South Nations and Nationalities Peoples Region Health Department. Patients with malaria and helminthiasis were managed as part of the routine clinical management of patients in the health care facilities following the national guideline .P. falciparum. Plasmodium vivax accounted for 35.2% of malaria cases. Mixed infections with P. falciparum and P. vivax were detected in 6% of the subjects. Irrespective of malaria infection, high rates of helminth infection were observed in the settings. There was no significant difference in magnitude of helminths among malaria patients and malaria free apparently healthy controls (78.2% (154/197) versus 84.3% (182/216)), respectively. The pattern of helminth infection in malaria positive and malaria free apparently healthy controls was similar  and Entamoeba histolytica/dispar (5 patients). The least encountered helminths were Taenia species (7), and Strongyloides stercoralis (2). Twenty-three percent of the subjects from both groups had a single infection. Multiple infection i.e. infection with 2, 3 and 4 different species of intestinal parasites were observed in 37.0% (132/356), 21.6% (77/356) and 3.4% (12/356) patients, respectively. The frequent combinations of helminths diagnosed in single patients were A. lumbricoides - T. trichiura in 28.8%; A. lumbricoides - T. trichiura - S. mansoni in 18.5%, and A. lumbricoides -T. trichiura- Hookworm in 8% of the patients. In 4% of the patients 4 different helminths: A. lumbricoides, T. trichiura, S. mansoni and hookworm were observed.In this cross sectional observational study 197 malaria patients and 216 malaria free apparently healthy controls were included. The mean age of the study subjects in both groups was not different (34 versus 35 years). The male female ration was also 1: 1.2. The demographic characteristics of the pateints, magnitude of malaria and type and destitution of helminthes were described previously . Fifty sAlthough, it is very difficult to have a normal range of IgE even at a population level, a range of values of IgE levels was defined enabling the analysis of the frequency of normal, moderate and high IgE levels in each group of patients. Normal values were adjusted because the groups had a wide range of different total serum IgE level. Thus, the normal value (N) was defined by the median IgE levels from malaria free apparently healthy helminth negative controls from malaria non endemic area. Accordingly, serum IgE values between N and 1.5 N were considered as low, between 1.5 N and 2 N as moderate IgE levels and above 2 N as the highest levels.As indicated in Figure The finding of elevated serum IgE level among malaria patients irrespective of helminth co-infection and the high serum IgE concentration in malaria free helminth infected apparently healthly controls is in agreement with previous studies ,31,32 anAscaris is highly allergenic; if the function of IgE was only to get rid of worms; it would seem detrimental for Ascaris to induce it. However, no significant association was found between presence of individual helminths and serum IgE levels in the current study. The prevalence of intestinal parasites among malaria patients and malaria free apparently health individuals were as high, which corresponds well with previous studies from Ethiopia [in vivo evidence for Th2 activation [The correlation of malaria parasite density and helminth egg intensity with elevated serum IgE levels could also contribute to severity of disease. As it was reported ,16, totaEthiopia -25 and stivation ,34-36. TDespite the low sample size and the cross-sectional nature of the study, a remarkably elevated total serum IgE level in malaria patients irrespective of helminth co-infection was observed. The elevated serum IgE response in malaria patients irrespective of helminth infection and its correlation with malaria parasite density and helminth egg intensity support that malaria infection is a strong driver of IgE production as helminth. Thus, mass deworming may be a cost effective approach in reducing the morbidity of helminths and co-infection where malaria, tuberculosis and HIV are endemic.The authors declare that they have no competing interests.AM: conception of the research idea, study design, data collection, analysis and interpretation and the drafting of the manuscript; YB and BM: reviewing the manuscript; ML and BE: designing the study and reviewing the manuscript; DN: collecting data, reviewing the manuscript; TN, FO, AK and DE: designing the study, reviewing the manuscript. All authors read and approved the final version of the manuscript.


Plasmodium falciparum and Plasmodium vivax. The comparative burden of anaemia in the community caused by these two species is incompletely characterized.Most malarious countries outside of Africa are co-endemic for P. vivax, P. falciparum and mixed parasitaemia in Papua, Indonesia. Adjusted population-attributable fractions were calculated from multivariable logistic regression models. Eight hundred and twenty-five households were surveyed with a total of 5255 occupants, 3890 (74 %) of whom were present and provided a blood sample. Plasmodium falciparum parasitaemia was present in 8.1 % (n = 315) of participants, P. vivax in 6.4 % (n = 250) and mixed infections in 1.9 % (n = 72). Overall, P. falciparum was associated with a mean reduction in haemoglobin of 1.16 g/dL compared to those without patent parasitaemia . The corresponding values for P. vivax and mixed infections were 0.66 g/dL  and 1.25 g/dL , respectively. Overall, 16.7 %  of haemoglobin concentrations <7 g/dL in the community were estimated to be attributable to patent parasitaemia. The fractions for infants and 1–5 years old were 34.4 %  and 23.2 % , respectively. Plasmodium vivax was associated with a greater than threefold higher attributable fraction of anaemia in infants compared with P. falciparum [27.6 %  versus 7.94 % ].A three-stage, cross-sectional, community survey was used to determine the proportion of moderate or severe anaemia (haemoglobin <7 g/dL) attributable to patent P. vivax is an important and preventable risk factor for anaemia during infancy—a probable consequence of relapsing disease prior to the development of immunity.Despite comparatively low-level endemicity, malaria is associated with a significant proportion of all cases of community anaemia in southern Papua. Contrary to its benign reputation,  Plasmodium infection  but a disproportionately high direct malaria-attributable mortality rate  whereas the opposite was observed for all other age groups. After infancy, there was a statistically significant trend to decreasing prevalence of P. vivax parasitaemia with increasing age (p = 0.003 for trend) but no such trend for P. falciparum (p = 0.13). Unlike in highly endemic regions, there was no age-associated decrease in the likelihood of having concomitant fever with parasitaemia for either species . Overall, a higher proportion of Highland and Lowland Papuans were parasitized  than non-Papuans .In total, 5255 individuals resided in the 825 households surveyed of whom 3890 (74 %) were present and consented to providing a finger-prick sample of blood Fig. . Those we parasite density for P. falciparum  whereas for P. vivax there was a weak negative correlation . Those with parasitaemia and fever within the last 24 h (n = 205) had similar mean haemoglobin concentrations as parasitaemic individuals without fever (n = 456) . Forty-two per cent (1629/3890) of the survey participants had a history of fever in the last month and these individuals had both a significantly lower mean haemoglobin concentration and an increased unadjusted odds ratio for moderate or severe anaemia compared to those without a history of fever .The mean haemoglobin in the sample as a whole was 11.0 g/dL (95 % reference range 6.1–15.9 g/dL) Table  with 5.7P. falciparum parasitaemia was associated with an absolute reduction in haemoglobin of 1.16 g/dL   and 1.25 g/dL , respectively (p < 0.001 for both). Although numbers were small, P. vivax was associated with a large mean reduction in haemoglobin of 1.93 g/dL  in infants. There was a smaller and non-statistically significant reduction for infants with P. falciparum .After adjusting for age, gender, ethnicity, pregnancy, and weight for age/gender/ethnicity, presence of 1) Table . The corP. falciparum  and P. vivax, respectively . For infants with P. vivax the AOR was 47.7  and for infants with P. falciparum it was 6.02 .Table Plasmodium was responsible for 16.7 %  of cases of moderate or severe anaemia in this study , 4.94 %  and 3.41 % , respectively. The attributable fraction was greatest in infants  and decreased with increasing age thenceforth. Plasmodium vivax was responsible for greater than three times the proportion of moderate or severe anaemia in infants compared with P. falciparum [27.6 %  versus 7.94 % ] although the precision of these estimates was poor. Figure Patent parasitaemia due to any species of P. vivax is less prevalent than P. falciparum overall, this study has shown that in Mimika District it is the commoner species in children under 5 years of age and that it is associated with a higher population-attributable fraction of anaemia in infants. This study also suggests that mixed species infections are associated with a greater reduction in haemoglobin than P. falciparum or P. vivax infections alone.Despite comparatively low-level endemicity, patent parasitaemia in southern Papua is associated with 17 % of all haemoglobin concentrations under 7 g/dL. In infants and young children, the corresponding proportions rise to 34 and 23 %, respectively. Although In this survey, 17 % of individuals were parasitaemic and approximately one-third of these were febrile. Presence of fever did not confer a greater risk of anaemia. Although parasitaemia was strongly associated with anaemia, the population attributable fractions presented are still likely to represent under-estimates of the true total effect of malaria on haemoglobin concentrations in the community. Full haematological recovery takes several weeks following acute malaria  suggestiP. vivax accounts for a greater fraction of anaemia than P. falciparum is in agreement with results from the local hospital [P. vivax is the most common cause of malaria-related admission in the first year of life and produces an equal or greater reduction in haemoglobin than P. falciparum [P. vivax parasitaemia with age, whereas there was no such reduction for P. falciparum. Second, infants with P. vivax parasitaemia in this study had had significantly more episodes of fever in the last month than infants with P. falciparum . The subsequent decline in the fraction of anaemia attributable to either species of Plasmodium with increasing age is likely to relate to three main factors: the acquisition of some degree of immunity, especially in the case of vivax malaria, the increasing importance of alternative causes of anaemia, such as intestinal helminthiasis and chronic infections, and lastly the increasing likelihood that lack of parasitaemia represents a state of remission or a period between primary infections rather than a state of complete malaria naivety.The estimated adjusted population-attributable fractions of moderate or severe anaemia in infants were based on small numbers. Nevertheless, the finding that hospital , 19, 33 hospital . At the lciparum –38, a phlciparum . In keepP. falciparum or P. vivax alone. This finding is consistent with other work from Papua [P. vivax in northern Thailand has been postulated to attenuate the risk of severe anaemia secondary to P. falciparum infection, possibly due to some degree of cross-species immunity [P. vivax), driving deeper levels of anaemia.This study showed that mixed species infections were associated with a greater drop in haemoglobin and a higher risk of moderate or severe anaemia than infections with om Papua , 19 and om Papua  but in dom Papua . Concomiimmunity , 39. In This study has several limitations. Due to the cross-sectional design it was not possible to draw solid conclusions about the direction of the observed associations. Although anaemia is an established sequel of both falciparum and vivax malaria, there is evidence that iron deficiency anaemia reduces the risk of falciparum malaria  and convThe population-attributable fractions were estimated using odds ratios as approximations of relative risk. Since moderate or severe anaemia was not a particularly rare outcome, this may have resulted in slight overestimation of the attributable fractions, particularly for 1–5 years olds who had a prevalence of moderate or severe anaemia of 9.4 %.Selection bias may have affected the population-attributable fractions due to their heavy reliance on the prevalence of parasitaemia in the sample. Those who did not provide a blood sample (mostly due to absence at the time of the survey) tended to be older males. Overall, there was relatively little effect of increasing age or gender on the odds of parasitaemia, however those who did not provide a blood sample could conceivably have been at greater risk of malaria acquisition due to behavioural or lifestyle factors. If this were true, the fraction of anaemia attributable to malaria may have been underestimated.Several potentially important confounders could not be controlled for in analyses. Infestation with intestinal helminths has been shown to cause an additive reduction in haemoglobin concentrations in malaria co-infected children . AlthougPlasmodium infection is less clear and may differ between species [Haemoglobin and red cell abnormalities as a whole are protective against severe malarial anaemia –53 but t species , 54, 55.Plasmodium infection, any residual confounding is likely to have biased the results towards the null. Finally, the effects of chronic disease and bacteraemia could not be controlled for in this analysis. The former is unlikely to have been important in the younger age groups and bacteraemia would be expected to be rare in the community setting.Incorporating weight for age/gender/ethnicity into the multivariable regression models should have accounted for at least some of the potential confounding caused by iron deficiency anaemia. Since iron deficiency is thought to be protective against Plasmodium endemicity, patent parasitaemia (whether symptomatic or not) is an important and preventable cause of anaemia in southern Papua. Young children bear the brunt of this burden but the haematological effects also extend into adulthood. Plasmodium vivax is an especially important cause of anaemia in infants, probably because it causes recurrent disease prior to the onset of immunity. Since infancy is a time of increased susceptibility to infectious diseases as well as rapid physical and neurological development, anaemia associated with vivax malaria may be an important and under-estimated contributor to indirect malaria mortality and developmental morbidity in regions where this species is prevalent.Despite comparatively low-level


Children represent a high-risk group for malaria worldwide. Among people in Thailand who have malaria during childhood, some may have multiple malaria attacks during their lifetime. Malaria may affect neurological cognition in children, resulting in short-term impairment of memory and language functions. However, little is known regarding the long-term effects of malaria infection on cognitive function. This study examines the long-term impact of malaria infection on school performance among school children living in a malaria-endemic area along the Thai–Myanmar border.A retrospective cohort study was conducted among school children aged 6–17 years in a primary-secondary school of a sub-district of Ratchaburi Province, Thailand. History of childhood malaria infection was obtained from the medical records of the sole malaria clinic in the area. School performance was assessed by using scores for the subjects Thai Language and Mathematics in 2014. Other variables, such as demographic characteristics, perinatal history, nutritional status, and emotional intelligence, were also documented.A total of 457 students were included, 135 (30 %) of whom had a history of uncomplicated malaria infection. About half of the malaria-infected children had suffered infection before the age of four years. The mean scores for both Mathematics and Thai Language decreased in relation to the increasing number of malaria attacks. Most students had their last malaria episode more than two years previously. The mean scores were not associated with duration since the last malaria attack. The association between malaria infection and school performance was not significant after adjusting for potential confounders, including gender, school absenteeism over a semester term, and emotional intelligence.This study characterizes the long-term consequences of uncomplicated malaria disease during childhood. School performance was not associated with a history of malaria infection, considering that most students had their last malaria infection more than two years previously. These findings indicate that the impact of uncomplicated malaria infection on school performance may not be prolonged. Malaria infection remains a major public health problem worldwide. The World Health Organization has estimated that malaria affects 198 million individuals, and globally 584,000 persons died from the disease in 2013 –13. MalaThailand is considered as a malaria-endemic country. Although the overall endemicity is low nationwide, the transmission is most intense in rural areas along the international borders. In these areas malaria remains the major cause of illness among children, many of whom may experience multiple malaria attacks during their childhood . HoweverThe study was conducted in Tanousri, Suanphuang, Ratchaburi Province, located on the western border of Thailand. This is a mountainous area bordering Myanmar, where malaria transmission persists throughout the year. The Rajanagarindra Tropical Diseases Center (RTIC) operated by Mahidol University, the sole malaria clinic in this area, has provided free diagnosis and treatment of malaria to people in this district for more than 15 years. The vast majority of malaria cases in the area are detected at this center. There is only one primary-secondary school in Tanousri, which provides free tuition and education to approximately 1000 students.Children aged 6–17 years old who were studying in 2014 at the Rujirapat primary-secondary school of Tanousri, Suanphuang, Ratchaburi Province, Thailand, and were permanent residents in the area, were enrolled in the study. Students who had a history of meningitis, encephalitis, and other central nervous system infections were excluded.A retrospective cohort study was conducted among students in the school. History of malaria infection since birth was obtained from the medical records of the RITC. All malaria cases were based on microscopic diagnosis by trained and experienced staff. The participating students were classified into malaria and non-malaria groups according to their history of malaria infection. History of intestinal parasitic infection was obtained from the most recent mass stool examination survey in 2013 performed by trained personnel. Information on school performance (outcome) and other variables such as demographic characteristics, perinatal and birth history, nutritional status, and emotional intelligence were primarily collected in 2014.School performance was assessed using pupils’ scores in the subjects Mathematics and Thai Language, graded by class teachers and ranging from 0 to 100. The education tests were routinely developed following a standard protocol of the school, which regularly received quality assurance by the Ministry of Education, Thailand. Students in the same grade were tested with the same set of paper examination. The level of school performance was classified according to a standardization protocol: poor school performance was identified when a z-score test was below zero . This stWeight and height of students were collected from school records, documented routinely by the school. The measurement was performed by trained teachers following standard procedures provided by the Ministry of Public Health. Nutritional status was assessed following the standard guidelines of WHO, including weight for age and height for age. Poor nutritional status was considered when weight for age and height for age was below −2.0 of the standardized z-score , 22.Emotional intelligence was assessed by a standard emotional intelligence test (EQ test) developed by the Ministry of Public Health of Thailand. This standard questionnaire for EQ screening was tested among children in different parts of Thailand, and shown to have high validity and reliability . FollowiThe outcome of the EQ test was classified according to the guidelines of the Ministry of Public Health of Thailand. Students were classified as having low emotional intelligence if the test score was below 40 out of 100 for students aged 6–11 years and below 140 out of 208 for those aged 12–17 years , 25.Students and their parents were interviewed using a structured questionnaire. Questions concerned basic demographic characteristics and socioeconomic status. Perinatal and birth histories of students were obtained from the review of the perinatal log book (if available).All analyses were performed using SPSS version 18.0 . Significance was considered at an alpha level of less than 0.05 (p < 0.05). The Student t-test and analysis of variance were used to analyse mean differences between groups. Logistic regression was used to determine crude odds ratio (OR) and 95 % confidence interval (CI) for predictive factors of school performance. Factors that showed significant associations were then included in multivariate analysis to estimate adjusted ORs and 95 % CIs.The study was approved by the Ethical Review Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand. Informed consent and assent forms were obtained from parents and students who participated in the study.A total of 457 school children were enrolled. About half of the students were male (52 %) and in the age group 6–11 years (53 %). The majority of students (82 %) were Karen, an ethnic minority that live along the Thai–Myanmar border. Most fathers and mothers of students were uneducated , and 67 % of students were from low-income families . More than 70 % of students were term delivered with a birth weight of 2500 g or more  had low emotional intelligence. Most students attended the school regularly; only 19 % accumulated absenteeism of seven days or more. A total of 202 students were tested for intestinal parasitic infection in 2013. Among them, 98 (49 %) had been infected with intestinal parasites and subsequently received treatment  had experienced 1–2 malaria attacks and 13 (17 %) had had five or more malaria attacks since birth. Almost half of the malaria-infected students (45 %) had their first malaria infection during the first four years of life. Recent infection (within <1 year) was observed in only 4 students. For the majority of malaria-infected students (80 %) it had been three years or more since their last malaria infection , all of whom had uncomplicated malaria. The majority of malaria cases were infected with either Mean scores for both Mathematics and Thai Language significantly decreased in relation to the increasing number of malaria attacks Table . StudentSchool performance was identified according to the standardized score. Of 457 students, 211, 220, and 168 students had poor scores in Mathematics, Thai Language, and both, respectively. Factors associated with poor school performance were firstly identified separately for each Mathematics or Thai Language test. The results of each individual analysis showed similar patterns regarding the predictive factors. Therefore, only the results of school performance defined by both Mathematics and Thai Language tests are presented here. Overall, approximately 37 % of the students (n = 168) had poor school performance in test scores of both Mathematics and Thai Language. A history of malaria infection, in terms of malaria infection, number of malaria attacks, age at first malaria infection, and duration since the last attack, was not significantly associated with school performance, even after adjusting for potential confounders . Poor school performance was also observed more among younger students (aged 6–11 years) than in those 12–17 years old. In addition, students who had more school absenteeism (≥1 week) were about 1.7 times more likely to have poor school performance. Low emotional intelligence was also associated with poor school performance . However, ethnicity of students, parents’ education, family income, perinatal history, history of parasitic infection, and nutritional status were not significantly associated with poor school performance  for poor school performance were also explored in this study, including socioeconomic status, parents’ education, perinatal history, history of intestinal parasitic infection, and current nutritional status. These findings suggested that these factors were not associated with the school performance of students. In this geographic area the population is fairly homogeneous in terms of socioeconomic status, culture, and lifestyle; these factors may therefore not make a difference in the performance of children in school. Although the school participating in this study is the only one in this rural area, it has very good school health programs. Intestinal parasitic infection has been periodically detected and treated. To improve their nutritional status, it is routinely assessed yearly for all students; therefore, chronic malnutrition is rare. Almost 80 % of students in this school had normal nutritional status, which is higher than that reported for other schools in rural areas of Thailand . This faHowever, the cause of poor school performance is multifactorial. Although several potential confounders have been explored and adjusted in this study, some variables may be left unadjusted, such as underlying diseases of students and the learning environment outside school. However, most of the students in the study were healthy; chronic diseases were not observed in this study population. In addition, variation in the learning environment outside school in this rural area is less likely than in urban areas where competition and learning opportunities are greater. This study was conducted in a setting of low malaria transmission where the majority of the population is Karen, a minority group. Therefore, results from this study may not be generalizable to children living in hyperendemic areas or in other urban settings.The test scores and nutritional status data collected in this study were based on school reports. Although the paper examinations were developed following a standard protocol and a same set of paper examination was given to students in the same grade, the test scores might still vary depending on teachers’ assessment. This would affect the reliability of test scores when comparing among different classroom. However, the standardized scores, rather than the raw scores, were used to classified school performance status of students to avoid bias due to variation of grading by different teachers. Errors may also occur when measuring weight and height, especially when performed by different persons or different equipment. However, weight and height data in this school were collected by trained teacher using the same measurement tools; this would ensure the reliability of nutritional status data collected in the study.In this study, data on history of malaria infection in students were obtained from the only malaria clinic in the area. The village census record that has been regularly updated was used to identify students who permanently live in the area but did not have malaria infection. Using these data sources helped to ensure the validity of the past malaria status of the subjects. In addition, school performance status was classified using a standardized score to avoid bias that might occur because of variation in standard grading by different teachers in different classrooms. Moreover, this study tracked the history of malaria infection since birth, which provides information on the long-term consequences of uncomplicated malaria in children living in an area of low malaria transmission.Childhood malaria infection may affect cognitive function and lead to impairment of memory and language functions. However, the association between childhood malaria infection and school performance was not significance in this study, when the majority of malaria infected children previously had uncomplicated malaria infection in the past three years. These findings suggested that, in low malaria endemic area, the educational consequence after uncomplicated malaria may be less and may not be prolonged.


Multiple studies in various parts of the world have analysed the association of nutritional status on malaria using anthropometric measures, but results differ due to the heterogeneity of the study population, species of the parasite, and other factors involved in the host and parasite relationship. The aim of this study was to perform a systematic review on the inter-relationship of nutritional status based on anthropometry and malarial infection.Two independent reviewers accessed the MEDLINE and LILACS databases using the same search terms related to malaria and anthropometry. Prospective studies associating anthropometry and malaria (incidence or severity) were selected. References from the included studies and reviews were used to increase the review sensitivity. Data were extracted using a standardized form and the quality of the prospective studies was assessed. Selected articles were grouped based on exposures and outcomes.The search identified a total of 1688 studies: 1629 from MEDLINE and 59 from LILACS. A total of 23 met the inclusion criteria. Five additional studies were detected by reading the references of the 23 included studies and reviews, totaling 28 studies included. The mean sample size was 662.1 people, ranging from 57 to 5620. The mean follow-up was 365.8 days, ranging from 14 days to 1 year and 9 months, and nine studies did not report the follow-up period. Prospective studies assessing the relationship between malaria and malnutrition were mostly carried out in Africa. Of the 20 studies with malarial outcomes, fifteen had high and five had average quality, with an average score of 80.5 %. Most anthropometric parameters had no association with malaria incidence  or parasite density . However, the impact of malnutrition was noted in malaria mortality and severity . Regarding the effects of malaria on malnutrition, malaria was associated with very few anthropometric parameters .P. falciparum endemic areas, with a significant heterogeneity in studies’ design. Apparently malnutrition has not a great impact on malaria morbidity, but could have a negative impact on malaria mortality and severity. Most studies show no association between malaria and subsequent malnutrition in P. falciparum areas. In Plasmodium vivax endemic areas, malaria was associated with malnutrition in children. A discussion among experts in the field is needed to standardize future studies to increase external validity and accuracy.This systematic review found that most of the evidence associating malaria and malnutrition comes from  Nutritional status is a closely tied to immune responses to infection, being on the one hand, an important determinant of the risk and prognosis of infectious diseases, and on the other hand, being directly influenced by infection . This biMalaria is a preventable, diagnosable and treatable disease. With recent innovations and roll out of multinational interventions, there are fewer people dying from malaria today than 10 years ago. Nonetheless, malaria is still a deadly disease with approximately 219 million cases and 660,000 deaths in 2013 . MalnutrNutritional status impacts on mortality among children under 5 years due to diarrhoea, respiratory diseases, malaria and measles . In relaAlthough several studies in various parts of the world analysed the impact of nutritional status on malaria using anthropometric measures, their results differ due to the heterogeneity of the study population, species of the parasite, and other factors involved in host and parasite relationship. The aim of this study was to perform a systematic review on the evidence for relationship between malaria and subsequent chronic or acute malnutrition based on anthropometry, for the protective effect of malnutrition on malaria outcomes, and for malnutrition increasing the risk of adverse malaria outcomes.A systematic review was performed in order to identify the available published data on malaria and malnutrition causal interactions. A broad free text search was made using the terms  AND [(anthropometry) OR (anthropometric) OR (nutrition)) for PUBMED and  AND (anthropometry) OR (anthropometric) OR (nutri$)] for LILACS. Potentially relevant papers in all languages were accessed from MEDLINE (September 2014) and LILACS (September 2014) in order to review full texts. Additional articles were obtained through citation tracking of reviews/opinion articles and original papers. The titles, abstracts, and studies identified in the literature search were assessed by two reviewers. All studies matching the inclusion criteria were reviewed by the authors and disagreement in conclusion was settled through discussion. Articles written in English, Portuguese, Spanish, German and French were included.Plasmodium species were included. Only original prospective cohort studies were included, presenting one of the following characteristics of exposure and outcome:Malnutrition assessed by anthropometric measurements as the independent variable and morbidity or mortality from malaria as the outcome.Malaria as an exposure variable and malnutrition assessed by anthropometric measurements as the outcome.For this study, observational studies in non-pregnant populations and without co-morbidities  were included. Studies on any of the The anthropometric variables gathered in the articles were anthropometric indices , measures of skinfold thicknesses, body circumferences and increments in weight, height or anthropometric measures throughout follow-up. As malarial variables, we looked for frequencies of malaria such as incidence or prevalence, parasitaemia, complications and mortality as outcomes. Severity criteria recommended by the World Health Organization  were used .Plasmodium spp., confounders adjusted in the analysis and type of association were extracted. Data on the type of association  between the exposure and the outcome were retrieved. The association was classified as neutral when the exposure had no impact on the outcome, as risk when the exposure increased the outcome and as protection when the exposure decreased the outcome.One independent reviewer (EDF) supervised by a senior reviewer (MAA) extracted the relevant data using a predesigned data extraction form. Disagreements between the two reviewers were resolved by referring to a third reviewer (WMM). Information for article identification, exposure variables and outcome measurement, sample size, age group, level of malaria transmission in the study site, follow-up time, species of Check List for Measuring Quality proposed by Downs and Black was applied [To assess the quality of the included studies, an adapted questionnaire originated from the  applied . The valThe search identified a total of 1688 studies: 1629 from MEDLINE and 59 from LILACS. After screening titles and abstracts, 950 publications were excluded due to a lack of information regarding an association between malaria and malnutrition, not being original articles or being duplicates, reviews, reports, case–control, ecological or cross-sectional studies, unavailable articles and presence of co-morbidities in participants  studies were conducted in Africa, three (10.7 %) in Oceania (two of Papua New Guinea and Vanuatu), one (3.5 %) in Asia (Vietnam) and one (3.5 %) in South-America (Brazil). African countries with the largest number of studies were Gambia (4), Congo (3) and Ghana (3). Of the 28 included studies, one was done in the 70 s, one in the 80 s, ten in the 90 s, nine in the 2000s and seven in the decade of 2010. Among the 28 included studies, 20 presented anthropometric variables as exposure and had malaria as the outcome, while 13 had malaria as the exposure and malnutrition as the outcome. Five studies conducted evaluations in both directions and were included in both analyses.P. falciparum prevails. Fourteen studies had participants with falciparum malaria, one with falciparum and vivax, one with falciparum, malariae and vivax and one with vivax/falciparum malaria. The setting was characterized as hyperendemic in nine studies, as holoendemic in two, endemic in four, hypoendemic in two, mesoendemic in one and endemicity was not reported in 10 studies. The mean sample size was 662.1 people, ranging from 57 to 5620. The mean follow-up was 365.8 days, ranging from 14 days to 1 year and 9 months, and nine studies did not report the follow-up period. The characteristics and results of these studies are shown in Tables Eleven studies did not report the agent responsible for malaria, all from Africa where Twenty studies with malaria-related outcomes were included, twelve evaluating incidence, seven mortality and six parasitaemia. Five studies evaluated more than one type of outcome.The most assessed anthropometric measures were W/A, with 25 evaluations from 14 studies, and H/A, with 27 evaluations from 11 studies. The W/H measure was obtained in 24 evaluations from 10 studies. The measure AC/A was obtained in four evaluations from two studies. All the other measures  were obtained in two evaluations from one study each. Thus, a total of 92 different anthropometric measures were obtained from the 20 studies, being 14 using National Center Health Statistics 1977  and one Plasmodium falciparum malaria, two evaluated Plasmodium vivax malaria, two evaluated Plasmodium malariae malaria and one evaluated specifically non-severe malaria. Most of the evaluations failed to show any association between malnutrition and malaria incidence; 45/50 (90 %). However, three evaluations showed a risk association between malnutrition and malaria incidence in children: (1) underweight on P. vivax incidence in children above 10 years with undisclosed follow-up time [P. falciparum incidence in children under 9 months years with 3 months of follow-up [P. falciparum incidence in children between 0 and 5 years with 20 weeks of follow-up [P. vivax incidence in children up to 14 years with 1 year of follow-up [The impact of malnutrition on malaria incidence was assessed in 50 statistical comparisons from 12 studies, on parasite density in 25 comparisons from six studies and on mortality in 17 comparisons from seven studies  AC/ollow-up ; (3) Stuollow-up . Two evaollow-up .P. falciparum malaria, three evaluated high parasite density as ≥10,000 parasites/mL specifically in P. falciparum malaria, one evaluated time to peak parasitaemia and one evaluated time to peak gametocytaemia.The 25 assessments of the relationship between malnutrition and malaria parasite density were highly heterogeneous: ten evaluated high parasite density (≥5000 parasites/mL), three evaluated high parasite density as ≥10,000 parasites/mL, three evaluated high parasite density as ≥5000 parasites/mL in asymptomatic malaria, three evaluated high parasite density as ≥300 parasites/mL, three evaluated high parasite density as ≥5000 parasites/mL specifically in P.falciparum parasite density in children of undisclosed age and 28 days of follow-up [P.falciparum parasite density above 300/µL in children above 5 years with 25 weeks of follow-up [P.falciparum gametocytaemia in children of undisclosed age and 28 days of follow-up [P.falciparum parasite density of above 5000/µL in children above 5 years with undisclosed follow-up time [P.falciparum parasite density of above 5000/µL in children between 0 and 5 years with 1 year of follow-up [Although the majority of studies showed no association between malnutrition and parasite density 20/25 (80 %); two evaluations showed a risk association: (1) Weight increments on time to peak ollow-up ; while t-up time  and (3) ollow-up .Of the 17 comparisons between malnutrition and mortality, three evaluated malaria, two focused on severe malaria, one in cerebral malaria, and one evaluated three forms: malaria, cerebral and malarial anaemia. There were 10/17 52.8 %) evaluations with no association between malnutrition and mortality. Seven assessments showed a risk association between malnutrition and mortality in children: (1) underweight on falciparum clinical, cerebral and malarial anaemia in children between 0 and 5 years with undisclosed follow-up time .8 % eval; (3) undThirteen studies assessed the impact of malaria in nutrition, of them 10 assessed the role of malaria incidence (yes/no) while the remaining three used parasite density as the exposure of interest. The impact of malnutrition on malaria incidence was measured in 39 assessments from 10 studies and on parasite density in six assessments from three studies . Eight showed a risk association between malaria and subsequent malnutrition in children: (1) incidence of unspecified malaria on increment in weight in children between 0 and 5 years with 1 year of follow-up ; (2) IncTwo out of six (20 %) assessments showed a positive association between parasite density and malnutrition in children: (1) mean falciparum/mixed malaria parasite density on H/A in children 6–9 months and 14–20 months with 1 year and 9 months of follow-up ; (2) meaOf the 20 studies with malarial outcomes, fifteen had high and five had average quality according to the questionnaire of 12 items, with an average score of 80.5 %. The main methodological problem in these studies was not describing the main outcome measures . Of the fifteen high-quality studies, eight presented a risk association. Of the five medium quality, one found a risk association, totaling nine studies that found malnutrition as a risk factor for malaria  Tables , 5.TableOf the 13 studies assessing nutritional status as the outcome, eleven had high and two medium quality according to the questionnaire of 12 items, with a mean score of 84.8 %. The main methodological problem in these studies was not clearly describing the exposure and the outcome . Of the eleven high quality studies, six had positive associations. Of the two medium quality, one found a positive association, totaling seven studies that found malaria as risk factor for malnutrition .Previous studies on the association of malaria and malnutrition delivered inconsistent results. These conflicting results could be explained by different levels of confounding and to considerable methodological dissimilarities. Randomized controlled trials are impractical and previous observational studies have not fully controlled for potential confounding including nutritional deficiencies, breastfeeding habits, other infectious diseases and socioeconomic status . For theThe majority of studies showed no association between malnutrition and subsequent malaria incidence or parasite density. One study suggested a protective effect against malaria for malnutrition diagnosed by W/A . In contMeasuring the association between measures of malnutrition and the risk of malaria is complex and is hampered by the multiplicity of metrics used for malnutrition. Stunting is generally considered an indicator of chronic malnutrition, wasting generally reflects a more recent and severe process, and being underweight is likely the result of both factors . The resThe impact of malnutrition was noted mostly in malaria mortality and severity. Death or recovery with a neurological deficit on cerebral malaria was significantly associated with malnutrition in Nigerian children . UnderweP. falciparum and its associated growth-depressing immune response [Anthropometric interaction of malaria and nutrition is confounded by micronutrient status, which also impacts on malaria severity , but unfresponse . Thus, aresponse . Resultsresponse .Plasmodium vivax infection has been associated with severe malaria and death [P. vivax infection. In particular, concomitant malnutrition is suspected to increase the risk of severe vivax disease, but this is not well understood. The demographic risk of severe vivax malaria in regions of relatively high endemicity is skewed towards early infancy (a stage when severe anaemia is a major cause of morbidity). A clearer picture of severe vivax malaria is emerging, but further studies are required to refine existing knowledge of the spectrum of syndromes and its association with malnutrition [nd death , althougnd death . Co-morbutrition .The relative insensitivity of anthropometry to detect changes in nutritional status over short periods of time limited conclusions from studies of short follow-ups. One speculates that hypercatabolism and inflammatory status induced by malaria may have an effect on nutritional well-being. TNF is a known mediator of anorexia and cachexia seen in many human disease states and is elevated during acute malaria –69. In tP. vivax contributes with a great percentage of the malaria cases, malaria has been associated with malnutrition in children [P. vivax would not be associated with malnutrition due to the misleading information that vivax malaria is benign [P. vivax may lead to a degree of chronic inflammation. Whether relapsing malaria significantly impairs nutritional status in P. vivax affected areas warrants further research.Interestingly, in Vanuatu and in the Brazilian Amazon, where children , 39. In s benign . Howevers benign . One espP. falciparum endemic areas, with a significant study heterogeneity in terms of sample size, follow-up time and exposure and outcome definitions. Considering malnutrition as exposure, the results presented in this review support that malnutrition has not a great impact on malaria incidence and parasitaemia, but a greater negative impact of malnutrition was noted in malaria mortality and severity. A scarcity of prospective studies have been carried out aiming to establish the relationship between nutritional status and severity of vivax infection, this being a prioritary research topic. Most of the assessed studies showed no association between malaria incidence and malnutrition in areas of P. falciparum predominance, although the anthropometric parameters used were aimed mostly at chronic malnutrition, possibly suggesting that the impact of malaria on nutritional status may be of little significance in the long term. Interestingly, in areas where P. vivax contributes with a great percentage of the cases, malaria was associated with risk for malnutrition in children. A discussion among experts in the field is needed to standardize the observational studies considering external validity in order to allow for more accurate conclusions.In conclusion, this systematic review found that the majority of studies assessing malaria and malnutrition were carried out in African


Plasmodium malariae is not very common in Senegal, as more than 98% of malaria cases are suspected to be due to Plasmodium falciparum. However, it is possible that other malarial species are being under-reported or misdiagnosed. This is a report of a case of P. malariae in a 30-year-old man previously hospitalized with acute kidney injury after treatment with quinine and re-hospitalized three months later. He was diagnosed with renal cortical necrosis post malaria treatment. Plasmodium malariae was identified with light microscope and confirmed using species-specific small-subunit rRNA (ssrRNA) amplification.According to current estimates, P. malariae infection, it should be systematically looked for, even if the predominant species is P. falciparum in Senegal.The patient was treated for malaria with intravenous quinine for seven days, followed by three days of oral treatment; the bacterial infection was treated using ceftriaxone during the first hospitalization and ciprofloxacin associated with ceftriaxone the second time. He also had four rounds of dialysis after which he partially recovered the renal function. Given the complications that can be caused by  Plasmodium falciparum. Over the few last years, the malaria burden in Senegal has declined but the disease remains endemic. Other species such as Plasmodium malariae, and Plasmodium ovale are present in Senegal, but recent data on their frequencies is lacking. Although, infections with non-falciparum species generally result in mild disease [P. malariae is known not to cause severe symptoms, but is sometimes associated with renal pathology [P. malariae does not cause relapse, but low level parasitaemia can persist for years and recrudescent infections are well documented [P. malariae diagnosed in a patient living in Dakar and previously hospitalized with acute kidney injury.Senegal is a malaria endemic region with a predominance of  disease ,2, theirathology -6. Evidecumented  and can cumented . As falcA 30-year-old man first presented at a health centre in Dakar, Senegal  with fever, headaches, myalgia, and vomiting. He was hospitalized and treated for malaria with quinine, based on clinical diagnosis although malaria diagnosis was not confirmed by biological test. The patient failed to recover and he was referred to the Infectious Disease Clinic  where he was treated with furosemide, metronidazole, metopimazine, dycinone and omeprazole, he presented with anuria for three days. He was negative for malaria by microscopy at Fann Hospital. He was transferred to the Nephrology Clinic  with creatinine of 152.52 mg/L and urea of 2.52 g/L. At admission, he received a first round of dialysis at Aristide LeDantec Hospital, where the molecular diagnostic unit is located.The patient presented fever, anaemia, ascitis of low abundance, insomnia and asthenia. He received ceftriaxone 1 g/day, and the furosemide continued to be administered. During the course of hospitalization, he received four rounds of dialysis and three blood transfusions. Creatinine, urea and potassium were measured to monitor the renal function during hospitalization (Table +), 132 mmol/L; potassium (K+), 5.1 mmol/L; chloride (Cl-), 108 mmol/L; total protein, 71.3 g/L.The patient was hospitalized again three months later in another health centre ; suffering from diarrhoea, fever, chills, asthenia and anorexia for a week. Given his renal condition, he was referred to Aristide LeDantec Hospital again, with creatinine of 67 mg/L and urea of 2.40 g/L. The potassium was 6.4 mmol/L; therefore, a dialysis was performed in emergency. After a month of treatment the laboratory test results were creatinine, 34.5 mg/L; creatinine clearance, 27.7 ml/min; sodium  during 7 days, then by oral route for three days and paracetamol intramuscular was administered for the pain. Renal biopsy was prescribed and showed parenchyme fragments with 80% nephrotic reduction. Secondary focal segmental glomerulosclerosis (FSGS) lesions were seen for 30% of residual glomeruli, and an extended mutilating interstitial fibrosis and atherosclerosis lesions of variable distribution between severe to moderate was also noted.He was treated with ceftriaxone 1 g per day and ciprofloxacin 1 g per day because typhoid was suspected and the doses were adapted to his renal condition. But a non-fermentative Gram negative bacillus was isolated from blood culture and typhoid investigation by Widal test and stool culture was negative. Malaria diagnosis by microscopy was performed one week after his admission and was positive for The appearance evoked a subtotal cortical necrosis probably on severe nephroangiosclerosis lesions and pyelonephritis was probably associated. The diagnosis of cortical necrosis post malaria was, therefore, established.At the end of hospitalization, creatinine was 29.4 mg/L, urea 0.48 g/L and the glomerular filtration rate (GFR) 30 ml/min, which means the renal function was not fully recovered and is in agreement with the renal necrosis. The patient is still being monitored in the nephrology clinic.Plasmodium malariae was diagnosed with a parasite density of 18.402 trophozoites/μl , which is specific to P. malariae, DNA was extracted from whole blood with the QIAamp DNA blood mini kit (Qiagen). The ssrRNA gene was amplified as previously described [Plasmodium genus was performed using the following primers rPLUf 5’- TTA AAA TTG TTG CAG TTA AAA CG and rPLUr 5’- CCT GTT GTT GCC TTA AAC TTC. The second round PCR with primers specific to 4 Plasmodium species:To confirm the diagnosis of escribed ,10 with P. malariae rMALf5’- ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC and rMALr AAA ATT CCC ATG CAT AAA AAA TTA TAC AAAP. falciparum rFALf 5’- TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT and rFALr 5’- ACA CAA TGA ACT CAA TCA TGA CTA CCC GTCP. ovale rOVAf 5’- ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA and rOVAr 5’- GGA AAA GGA CAC ATT AAT TGT ATC CTA GTGP. vivax rVIVf 5’- CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC and rVIVr 5’- ACT TCC AAG CCG AAG CAA AGA AAG TCC TTAPCR reactions were performed in a final volume of 20 μl: GoTaq (Promega Green Master Mix 2X) 6 μl, forward primer 10 pmol/μl (1 μl), reverse primer 10 pmol/μl (1 μl), DNA 2 μl, and 10 μl of distilled water.The PCR programme was as follows: initial denaturation at 94°C for 4 min, then 35 cycles of 94 for 30 sec, 55°C for 1 min  or 58°C for 1 min , 72°C for 1 min, final extension at 72°C for 4 min, and 4°C at the end.Plasmodium malariae was diagnosed at the size band of 144 base pairs (bp)  Figure . PlasmodP. malariae does not cause life-threatening infection, it has been associated with kidney injury [P. falciparum, P. malariae infection is chronic and parasitaemia is often low which makes microscopic diagnosis difficult. In Senegal, most rapid diagnosis tests (RDTs) used in public settings only detect the Histidine Rich Protein 2 (HRP2) of P. falciparum and PCR is primarily performed for research purposes. Therefore, patients presenting with P. malariae can easily misdiagnosed and are often treated for bacterial infections.Although y injury -15, whicPlasmodium species but requires extensive training and specialized expertise. Different types of RDTs exist and the choice of RDT implemented depends on the species that circulate in the area [Plasmodium species causing clinical malaria in Senegal is P. falciparum, which represents more than 98% cases. While other species are present, they are predicted to be at much lower frequencies. Additionally, co-infections occurring in P. falciparum endemic areas are often difficult to detect by microscopy, in a setting of high falciparum parasitaemia. The low parasite density of P. malariae compared to P. falciparum could partially explain why it is under-diagnosed even by light microscopy [The recommendation of the National Malaria Control Programme is to combine RDTs with the gold-standard microscopy in endemic areas; however, sometimes only RDTs are used, often in rural areas where the expertise and the equipment for performing microscopy are lacking. RDTs were developed particularly for use in low resource settings to facilitate the diagnosis of malaria in the absence of microscopy. Light microscopy can detect all the area . The moscroscopy ,18.P. malariae is not known to cause relapse, infected red blood cells can persist for long periods of time at low densities (<500 parasites/μl). These low densities are due to the fewer merozoites released after schizont rupture, increased erythrocytic cycle duration , erythrocyte preference for invasion (mature red blood cells) and the host factors such as immunity. One study reports P. malariae parasites isolated from a patient infected 50years earlier while in China [Although in China .P. malariae can cause public health problems as it is associated with kidney injury which can be complicated and costly to treat in low income countries. Given the complications, it is time to re-evaluate the current diagnostic of choice, in order to prevent such pathologies associated with P. malariae infection.Although not a common disease in Senegal, Plasmodium infection was responsible of the injury remains unclear because there is not enough evidence to establish the aetiology.For this case the first diagnosis of malaria was only based on the clinic because biological tests were not available when the patient came to the hospital. During his first hospitalization he has been treated for malaria with quinine, after which he developed kidney injury but whether quinine or the Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.ssrRNA: Small-subunit rRNA; FSGS: Focal segmental glomerulosclerosis; GFR: Glomerular filtration rate; RDT: Rapid diagnosis test; HRP2: Histidine rich protein-2; Bp: Base pairs.Authors declare that they have no any competing interests.ASB confirmed and interpreted the biological diagnosis and wrote the manuscript. KD and SD made the microscopic diagnosis. AAN gave the clinical examination. CKD and ABD made the molecular diagnosis. MN, MCS, TD, ON helped for the literature and biological diagnosis. SM and DN gave constructive advice and reviewed the manuscript. All authors have read and approved the final version of manuscript.