Insecticide Treated Nets (ITNs) have been shown to reduce morbidity and mortality, but coverage and proper utilization continues to be moderate in many parts of sub-Saharan Africa. The gains made through a nationwide free distribution were explored as well as the effect on malaria prevalence in semi-urban and rural communities in south western Cameroon. A cross sectional survey was conducted between August and December 2013. Information on net possession, status and use were collected using a structured questionnaire while malaria parasitaemia was determined on Giemsa-stained blood smears by light microscopy. ITN ownership increased from 41.9% to 68.1% following the free distribution campaign, with 58.3% (466/799) reportedly sleeping under the net. ITN ownership was lower in rural settings  and at lower altitude  compared to semi-urban settings and intermediate altitude respectively. Conversely, ITN usage was higher in semi-urban settings (p = 0.002) and at intermediate altitude (p = 0.002) compared with rural localities and low altitude. Malaria parasitaemia prevalence was higher in rural  compared to semi-urban settings and in those below 15 years compared to those 15 years and above. Overall, participants who did not sleep under ITN were more susceptible to malaria parasitaemia . Despite the free distribution campaign, ITN ownership and usage, though improved, is still low. As children who reside in rural settings have greater disease burden (parasitemia) than children in semi-urban settings, the potential gains on both reducing inequities in ITN possession as well as disease burden might be substantial if equitable distribution strategies are adopted. Plasmodium falciparum remains one of the most important causes of morbidity and early mortality in endemic regions of sub-Saharan Africa [Malaria due to n Africa . Currentn Africa . Malarian Africa . Neverthn Africa . Furthern Africa , with run Africa , 6.Insecticide treated nets (ITNs) and indoor residual spraying (IRS) have both been demonstrated to reduce malaria –10 and, However, bed nets as a tool for malaria control can present challenges, such as coverage, proper use and replacement of old and torn nets . Recent While challenges to increasing ITN ownership may diminish as a result of the expansion of large-scale distribution efforts, ITN impact on transmission will be minimized if they are not properly and consistently used, especially among populations vulnerable to increased malaria morbidity and mortality, such as children and pregnant women . In addiP. falciparum infection and reported individual use of an ITN the previous night in Benin [Although ITNs have been shown to reduce morbidity and mortality in numerous controlled trials , the proin Benin  and Tanzin Benin . Furtherin Benin .This study uses observational data from large cross sectional surveys conducted in 2013 to assess net possession and investigate the protective effect of ITNs on malaria infection in semi-urban and rural communities in the South West Region of Cameroon.0: 2013-03-0153) while administrative authorization was obtained from the South West Regional Delegation of Public Health. Written informed consent or assent was obtained from all participants or the parents/guardians for those below 21 years of age.The study was approved by the Institutional Review Board of the Faculty of Health Sciences, University of Buea, Cameroon  [A. gambiae are as high as 287 infective bites/person/year, with overall Entomological Inoculation Rates (EIR) of 3.93 infective bites/person/night [Malaria transmission is intense and perennial in the area, with parasitaemia higher in the rainy seasons  and at lA. nili) ,35. Infeon/night .Although the indigenes of this area are of the Bakweri tribe and part of the Bantu ethnic group , its ferThe government of the Republic of Cameroon undertook a nationwide free LLITN distribution campaign from health facilities to all households in the country at the end of 2011. The objective of the campaign was to provide an ITN, with a lifespan of five years, to all household beds or an LLITN for every two individuals per household, to a maximum of three ITNs per household.This is a cross-sectional community—based study that was conducted between August and December 2013. Communities were first identified as rural and semi-urban and then randomly selected based on differences in altitude. Authorization to conduct the survey was then sought from Chiefs or Quarter heads and a community was only investigated with their approval. The population was then adequately sensitized on the project objectives, methods and possible benefits/risks through elites and/or community leaders. Participants were then invited to a central enrollment point and only enrolled if they or their caregivers/guardians gave written informed consent/assent. All individuals that had been living in the community during the free ITN distribution campaign were eligible for enrollment. Nevertheless, only resident adults were interviewed in either English/French  or pidgin  to document ethnodemography and household ITN ownership and usage.P. falciparum parasites on a thick blood film whereas malaria was defined as current fever together with parasitaemia.A structured questionnaire was used to record ownership, utilisation, status (either torn or good), source and approximate date of acquisition of the bed net as well as gender, age and area of residence of all participants. The participant’s axillary temperature was then measured using a digital thermometer, with current fever defined as body temperature ≥ 37.5°C taken during the survey. A sample of blood (2 ml) was then collected from each participant, by venepuncture, into an EDTA tube  for parasitological analysis. An ITN was defined as any long-lasting insecticidal net, any bednet factory-treated with insecticide and obtained less than 36 months ago, or any bednet treated with insecticide less than 36 months ago. ITN use were defined as reportedly sleeping under a bednet or ITN the previous night. Coverage was defined as the proportion of individuals reportedly having an ITN over their sleeping space. Ethnicity was defined as the self-reported ethnic group of the mother (or father if the mother’s could not be obtained). Parasitemia was defined as presence of asexual Plasmodium species was identified on the thin blood smear. A smear was only considered negative if no malaria parasites were seen in 50 high power fields. With each positive smear, the level of parasitaemia was estimated by counting the parasites against at least 200 leucocytes and assuming a leucocyte count of 8000 per microlitre to calculate the number of parasites/μl blood [Thick and thin blood smears were prepared following standard procedures and stained with 10% Giemsa . The malaria parasitaemia status and density were determined under oil immersion with the 100x objective, 10x eyepiece of a binocular Olympus microscope  while the μl blood .2 test whereas the differences in group means were assessed using Student’s t—test or analyses of variance (ANOVA). Association analysis of malaria prevalence and ITN usage was undertaken by logistic regression, using as covariates altitude, location, gender and age group. A difference giving a P value ≤ 0.05 was considered statistically significant.All data were entered into Excel and analysed using SPSS Statistics 20 for windows . Levels of parasitaemia were log-transformed before analysis. The significance of difference in prevalence were explored using the Pearson’s χThe geographical and parasitological characteristics of the study sites are shown in The overall reported ITN ownership and usage was 69.3% (554/799) and 58.3% (466/799) respectively. ITN ownership and usage was independent of age and gender . ITN ownThe overall prevalence of parasitaemia was 20.1% (161/800), with a geometric mean parasite load of 1721 parasites/μl blood . 4.5% (36/798) of the study participants actually had malaria in the community. Additionally, parasitaemia prevalence was higher (p<0.001) in participants below 15 years  compared to those above 15 years . The parasitaemia density was independent of gender, ethnicity and locality but varied with age group and altitude . The preP. falciparum parasitaemia prevalence is shown on P. falciparum prevalence, the analysis was further stratified by season. The protective effect of ITN was still evident in the rainy season , after excluding the survey conducted in the rainy to dry season transition in Tole. There was, however, no association between ITN usage and malaria parasitaemia prevalence across the different age groups, gender, ethnicity and altitudes in both localities  and usage by all persons (58.3%) has increased in the area after the campaign as reported elsewhere ,24. The ITN ownership and usage was lower in rural areas compared to semi-urban settings despite the free distribution through health facilities. This finding is in agreement with other studies –44 whereIn the south western region, the prevalence of malaria parasitaemia is still high (29.6%) in children of all age groups below 15 years, confirming that malaria remains a major cause of illness during childhood. Although higher malaria prevalence have been reported elsewhere , the ageThe greater risk of malaria parasite infection in rural settings reported here may be due to their specific characteristics that may facilitate human–mosquito contacts. This may accrue to the differences in housing types since house construction has previously been linked to malaria risk ,45,51. TITNs have been shown to reduce morbidity and mortality in numerous controlled trials . In our This study has a number of limitations. First, it is a cross-sectional study, and inherently there are limitations. Though the sample size is large, Cameroon is a populous country and there is a definite possibility of sampling error. There are also possibilities of non-sampling errors depending on the interviewer, the questions and the respondents. As with all survey data, the findings are limited by recall and social desirability biases. Some answers to questions such as sleeping under ITNs were reported by the parents and not observed. Likewise, it was not possible for the team to verify the status of nets during the survey. However, the recall period for most questions was relatively short (e.g. the previous night), so that the response will reflect the prevailing circumstances. Secondly, the surveys were undertaken along the slope of Mount Cameroon in the South West Region, one of ten regions in Cameroon. Although, these localities are geographically diverse, the findings are not necessarily representative of the whole region or the country. Thirdly, malaria microscopy is subject to limitations in sensitivity at low parasitaemia typical of asymptomatic infections  which coDespite the free distribution campaigns, ITN ownership and usage, though improved, is still low. As children who reside in rural settings have greater disease burden (parasitemia) than children in semi-urban settings, the potential gains on both reducing inequities in ITN possession as well as disease burden might be substantial if equitable distribution strategies are adopted. Prior equity analyses of ITN distribution campaigns may therefore be necessary to reduce inequities in ITN possession. There may be more negative consequences on health of rural populations beyond the scope of this study due to disparities in access between rural and urban populations.


Malaria symptoms are unspecific in acute undifferentiated fever. Seropositivity against various previous infections might be high in exposed populations. Serological results in a cohort of malaria patients in India are presented.et al., Malaria Journal 2013). At the monitoring site Dengue IgM capture ELISA (MAC-ELISA), Scrub typhus immunofluorescence (IFA) on Scrub ELISA positive samples and Leptospira Microscopic Agglutination Test (MAT) on Leptospira ELISA positive samples were also performed. Convalescence samples for test of rise in titer were not collected due to logistical challenges. Malaria PCR was considered gold standard for the diagnosis of malaria.Serological tests from patients with malaria in an observational fever study were analyzed. During April 2011-November 2012, 1564 patients aged ≥5 years with fever for 2-14 days were consecutively included at seven secondary community hospitals in Tezpur (Assam), Raxaul (Bihar), Mungeli (Chhattisgarh), Ratnagiri (Maharashtra), Anantapur (Andhra Pradesh), Oddanchatram and Ambur (Tamil Nadu). Scrub typhus IgM ELISA , Leptospira IgM ELISA , Chikungunya IgM ELISA , Dengue rapid NS1 antigen and IgM Combo test  were performed at the hospital sites. At the monitoring site , malaria genus mitochondrial PCR was performed on all samples and 18S species-specific PCR or sequencing performed on genus positive samples with methods as described previously , 15% (173/268) had positive Dengue MAC-ELISA, 14% (37/268) positive Leptospira MAT, 8% (29/233) positive Chikungunya IgM ELISA and 35% (25/71) positive Scrub typhus IFA. Both Chikungunya ELISA and Dengue MAC were positive in 6/268, both Leptospira MAT and Dengue MAC in 4/268, both Scrub typhus IFA and Leptospira MAT in 6/268, both Scrub typhus IFA and Chikungunya ELISA in one and both Scrub typhus IFA, Dengue MAC and Chikungunya ELISA also positive in one patient.Serological tests generally considered gold standard for the diagnoses of scrub typhus, leptospirosis, Dengue fever and chikungunya were positive in a high number of malaria patients. Multiple etiologies of fever in addition to malaria to such an extent are not likely, while persistent antibodies after previous infections or cross reactivity more likely explain the findings. Results of a single serological test should be interpreted with caution when treating patients with acute undifferentiated fever in India and malaria should be considered a differential diagnoses also when IgM antibodies against other pathogens are detected.


Iron accumulation within the arterial wall has been hypothesized to promote atherosclerosis progression. Aim of this study was to evaluate whether the hormone hepcidin and iron stores are associated with arterial stiffness in subjects with essential hypertension.Circulating hepcidin, ferritin, and mutations in the hemochromatosis gene were compared between subjects included in the first vs. third tertile (n=284 each) of carotid-femoral pulse wave velocity (PWV) in an unselected cohort of patients with arterial hypertension.At univariate logistic regression analysis, high PWV was associated with higher ferritin levels (p=0.010), but lower hepcidin (p=0.045), and hepcidin ferritin/ratio (p<0.001). Hemochromatosis mutations predisposing to iron overload were associated with high PWV (p=0.025). At multivariate logistic regression analysis, high aortic stiffness was associated with older age, male sex, lower BMI, higher systolic blood pressure and heart rate, hyperferritinemia , and lower circulating hepcidin concentration . In subgroup analyses, high PWV was associated with indices of target organ damage, including micro-albuminuria , lower ejection fraction , cardiac diastolic dysfunction (p=0.004), and lower S wave peak systolic velocity (p<0.001). Ferritin was associated with cardiac diastolic dysfunction, independently of confounders (p=0.006).In conclusion, hyperferritinemia is associated with high aortic stiffness and cardiac diastolic dysfunction, while low circulating hepcidin with high aortic stiffness. Atherosclerosis, the leading cause of cardiovascular disease and mortality worldwide, is a chronic inflammatory disease characterized by the progressive formation of neo-intimal lesions and lumen narrowing of affected arteries. Development of atherosclerotic lesions is caused by retention of low-density lipoprotein cholesterol within arterial intima, favored by activation of immune cells with induction of oxidative stress . HyperchArterial stiffness, a physical phenotype of the vascular wall, can be estimated by measurement of aortic pulse wave velocity (PWV) . PWV refHowever, even in patients with hypertension there is a great variability in the progression of arterial damage, and in a considerable proportion of cases vascular disease is not fully explained by classic risk factors. Iron accumulation in macrophages within the arterial wall has been hypothesized to represent a novel determinant of atherosclerosis progression by inducing oxidative stress and the release of proinflammatory mediators , 9. IndeAim of this study was therefore to evaluate whether body iron stores and circulating hepcidin are associated with arterial stiffness in individuals with arterial hypertension.The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by Institutional Ethics Review Committees of the Institution involved . All participants provided informed written consent.Aim of the study was to evaluate whether serum hepcidin and iron stores were associated with high vs. low PWV (third vs. first tertile) in a previously described cohort of 827 consecutive unselected patients with arterial hypertension for whom serum and DNA samples are available .From September 2006 to October 2008 827 consecutive outpatients aged 18–80 years old, followed at the Hypertension Center of San Gerardo Hospital, Monza, Italy, for essential hypertension with adequate blood pressure control were enrolled.For the purpose of this study, subjects in the upper and lower PWV tertiles were selected, obtaining two groups of 284 subjects each. Exclusion criteria were age younger than 18 years old, pregnancy, secondary hypertension, stage 4–5 chronic kidney and advanced pulmonary disease, substance abuse, a history of cancer or of cardiovascular events in the month before the study.For all subjects, a comprehensive medical history was collected and complete physical examination was performed. Height and weight were obtained to calculate the patient body mass index (BMI) and waist circumference was assessed halfway between the lower ribs and the iliac crest. Fasting serum glucose, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) cholesterol, triglycerides, creatinine, interleukin-18 (IL-18), Serum Amyloid A (SAA) e High Sensitivity C-reactive Protein (CRP) were measured.Aortic stiffness was evaluated by estimating PWV between the ipsilateral carotid and the femoral arteries with the patient in the supine position. The pressure pulse waveforms were simultaneously obtained at the two arterial sites on the right side using an automatic device  and their distance (between the hip and neck) calculated by a rigid ruler. Measure was corrected by a 0.8 factor accordingly to the PWV measurement methods consensus document .The mean of two acquisitions was used for the analysis. The intra-session within- and between- operator variability of PWV were 2% and to 4%, respectively. The corresponding value for the inter-session between- operator variability was 4% .Hepcidin-25 serum levels were measured by Liquid Chromatography-Mass Spectrometry (LC-MS) on the LTQ analyzer (Thermo Scientific). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.5 ng/mL and 1.0 ng/mL, respectively. Linearity of quantification was confirmed in the following range: 1.– 500 ng/mL.Serum Ferritin was measured by the COBAS 8000 automated analyzer (Roche Diagnostics) by ECLIA (Electro-Chemiluminescence ImmunoAssay) . Hyperferritinemia was defined as ferritin levels higher than 240 ng/ml in females and 320 ng/ml in males .Serum IL-6 and IL-18 were measured by BEP 2000 the automatic analyzer (Siemens Diagnostics) by ELISA, SAA by immunonephelometry  by the BN II Analyzer (Siemens Diagnostics), hs-PCR was measured by the COBAS 8000 automated analyzer (Roche Diagnostics) by immunoturbidimetry.Proteinuria was quantified in a subset of 126 subjects (65 in the upper and 61 in the lower PWV tertiles) on urines collected in the preceding 24 hours.CCA-IMT was measured in a subgroup of 175 individuals (85 in the upper and 90 in the lower PWV tertiles). With the subject supine and the neck in partial extension, right CCA was scanned by the Philips Sonos 5500 ultrasonography device . Two-dimHFE genotype (C282Y and H63D variants) was assessed by sequence allele specific PCR as previously described [HFE genotypes at risk of iron overload was defined according to previous literature data [DNA was extracted from peripheral blood by phenol—chloroform method. Success rate in extracting DNA was 100% for each study group. escribed . Random ure data .For descriptive statistics, continuous traits were summarized as means±SD. Highly skewed variables—such as circulating hepcidin-25 and ferritin concentration—were summarized as medians and interquartile range. Categorical variables are shown as percentages. Analyses were performed by fitting data to generalized linear regression models. Logistic regression models were fit to examine binary traits (high vs. low PWV). Hepcidin and ferritin levels were log-transformed before entry into the models.Statistical analyses were carried out with JMP 11.0 . A two-sided P value <0.05 was considered statistically significant.The clinical features of patients stratified according to PWV tertiles (third vs. first tertile) are shown in HFE genotypes . Independent predictors of hepcidin levels at multivariate generalized linear model are shown in As expected, the hepcidin/ferritin ratio was lower in patients carrying HFE genotypes predisposing to iron overload was associated with high PWV (p = 0.025).At univariate logistic regression analysis (HFE genotypes at risk of iron overload (p = 0.041) remained associated with high PWV.At multivariate logistic regression analysis corrected for age and sex . At generalized linear regression, there was no significant association between hepcidin or ferritin levels with albuminuria (p>0.1).Cardiac function parameters stratified by PWV tertiles are shown in Hepcidin levels were inversely correlated with the E/A ratio , and low circulating hepcidin is associated with high aortic stiffness.S1 Fig(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S1 TablePrevalence  are shown. Wt: wild-type.(DOCX)Click here for additional data file.S2 TableComparisons were made by fitting data to a logistic regression model, adjusted for age, sex, BMI, SBP, heart rate, hepcidin levels, and presence of hyperferritinemia. OR: odds ratio; c.i.: confidence interval; BMI: body mass index; SBP: systolic blood pressure; bpm: beats per minute.(DOCX)Click here for additional data file.S3 TableComparisons were made by fitting data to a generalized linear model, adjusted for age, PWV, hepcidin, and ferritin levels. SE: standard error; PWV: pulse wave velocity.(DOCX)Click here for additional data file.


Malaria and undernutrition frequently coexist, especially in pregnant women and young children. Nutrient supplementation of these vulnerable groups might reduce their susceptibility to malaria by improving immunity.NCT01239693).Antibody immunity to antigens expressed by a placental-binding parasite isolate, a non-placental binding parasite isolate, merozoites and schizonts at enrolment (before 20 gestation weeks) and at 36 gestation weeks were measured in 1,009 Malawian pregnant women receiving a daily lipid-based nutrient supplement, multiple micronutrients or iron and folic acid, who were participants in a randomized clinical trial assessing the effects of nutrient supplementation on pregnancy outcomes and child development -1.04 , was observed. Similarly, women with higher socioeconomic status had significantly lower IgG and opsonizing antibodies to placental-binding antigens. Neither of these associations was significantly influenced by the supplementation type.In the current cohort nutrient supplementation did not affect anti-malarial antibody responses, but poor and undernourished mothers should be a priority group in future trials.The online version of this article (doi:10.1186/s12936-015-0707-2) contains supplementary material, which is available to authorized users. It is estimated that about 125 million pregnancies worldwide are at risk of malaria annually, significantly increasing maternal and child morbidity and mortality ,4. The cPlasmodium falciparum erythrocyte membrane protein-1 family of variant surface antigens (VSA). These antibodies help protect women against adverse clinical outcomes in subsequent pregnancies [In endemic regions, pregnant women naturally acquire antibodies to malaria with repeated exposure to infectious mosquito bites prior to and during pregnancy. During first pregnancies, women frequently acquire antibodies to the pregnancy-specific antigens, primarily to VAR2CSA of gnancies . HoweverIn non-malaria-related studies both macro- and micronutrient supplementation have been shown to significantly improve pregnancy outcomes and maternal health ,15. LipiThe only study to date that has assessed the effects of maternal nutrient supplementation on malaria antibody levels reported that vitamin A prenatal supplements were associated with a reduction in antibody responses to a placental-binding isolate EJ-24, but no significant changes in antibody responses to non-pregnancy related parasite isolates were observed .In areas with food insecurity and high malaria transmission, nutritional supplements could improve pregnancy outcomes and may also lead to stronger acquired immune responses to malaria. To investigate this, antibody immunity was measured to antigens expressed by placental-binding and non-placental-binding parasite isolates, merozoite antigens and schizont extract in pregnant women from Mangochi, Malawi enrolled in a randomized controlled trial receiving daily LNS, multiple micronutrients  or iron and folic acid supplements (IFA – 60 mg of iron and 400 μg of folic acid). The primary aim was to determine whether LNS supplementation improved antibody responses to malaria in pregnancy compared to IFA or MMN.From February 2011, 1391 pregnant women attending four antenatal clinics in Mangochi District, Malawi were recruited to a single-blinded randomized controlled trial of nutrient supplementation to improve pregnancy outcomes and child development . Parasitized red blood cells (pRBCs) were counted microscopically against 200 leucocytes under 100 X magnification. PCR was performed as previously described [P. falciparum lactate dehydrogenase (pfldh) gene. The samples were assayed in duplicate using a Bio-Rad CFX384 Touch Real-Time PCR detection system. Concentration of PfLDH was estimated using standard curves computed from amplification curves of a series of 10 g DNA extracts of parasite line 3D7 that were included in each assay. RDT was performed using Clearview® Malaria Combo  which detects the proteins PfLDH and histidine-rich protein 2. The prevalence of malaria in the current cohort was 23.2% by RDT at baseline [Blood plasma samples were collected from participants at study entry and at 36 gw. Malaria parasitaemia was diagnosed by light microscopy slide positivity, PCR or rapid diagnostic test (RDT) for asexual escribed . BrieflyP. falciparum reticulocyte binding homologue 2 (PfRh2) (construct PfRh2-2030) [Recombinant merozoite surface protein-1, MSP-1 19 kD (3D7 clone) and full-length MSP-2 FC27 clone) antigens  were kin clone anh2-2030)  and the h2-2030) .In brief, 0.5-2 μg/ml of each recombinant antigen was coated on to 384 well high protein binding black Optiplates . The plates were washed with PBS/Tween 20 and non-specific binding blocked with 0.1% casein on the following day. The plates were then incubated with participant sera diluted at 1:1,000 in 0.1% casein and assayed in triplicates. Alexa Fluor 488-conjugated goat anti-human IgG  was used at 1:2,000 dilution to detect antigen-bound IgG. The fluorescence intensity (FI) of each well, which is proportional to the amount of IgG bound, was measured using BMG POLARstar Omega fluorimeter  in the 485–1 excitation and 520 emission spectra range.Plasmas collected at enrolment and at 36 gw from 150 participants (50 from each supplementation group) were chosen by selecting alternate pairs of samples from 4 boxes containing equal numbers of samples from the three supplementation arms, blind to participant clinical data. These samples were used to measure IgG subclass antibodies to the above antigens. Antigens were coated at the same concentration on 384 well NUNC maxisorp plates  and were incubated in triplicates with plasma on the following day at 1:250 dilution for IgG1and IgG3, and 1:50 for IgG2 and IgG4. Following incubation the plates were washed and incubated with respective mouse anti-human secondary antibodies  at 1:1,000 dilution. Tertiary antibody horseradish peroxidase-conjugated goat anti-mouse IgG was added at 1:2,500 dilution followed by development of the assay using ABTS ; SIGMA-Aldrich). The reaction was stopped using 1% sodium dodecyl sulphate, and absorbance was read on the BMG POLARstar Omega fluorimeter at 410 nm wavelength.The total plasma IgG in the samples of the above 150 pregnant women were measured using the fluorescence assay method described above. Gamma-chain specific anti-human IgG capture antibody (SIGMA-Aldrich) was coated on to the plates at 0.5 μg/ml concentration and incubated with participants’ plasma diluted at 1:50,000. The concentration of IgG in each sample was determined using a purified human IgG isotype control (Life technologies) standard curve (1 μg/ml – 0.016 μg/ml).Serial samples were run on the same assay. The mean FI (MFI) or optical density (OD) were reported as a percentage of the positive control standard curve following adjustment for inter and intra plate variability. The positive control was a pool of plasma collected from malaria-immune individuals. The positivity for IgG was determined as greater than mean plus three standard deviations of the negative control (60 plasma samples collected from Melbourne adults).P. falciparum isolate, CS2 which binds to chondroitin sulphate A (CSA) expressed in placenta tissue with upregulated expression of var2csa [P. falciparum isolate, E8B which binds to ICAM-1 and CD36 [Placental-binding  var2csa  and non-and CD36  were culand CD36 . Pro-monand CD36 .In order to measure antibodies that opsonize pRBCs for phagocytic clearance a high throughput assay previously established in the current laboratory was used , with miUsing published methods ,35 IgG tAll statistical analyses were performed in Stata version 13.0 . Figures were created using GraphPad Prism 5. Statistical analyses were performed according to the pre-planned analyses for the current study approved and published in . AntibodLevels of antibody of each IgG subclass to merozoite and schizont antigens were compared between supplementation groups LNS versus IFA and LNS versus MMN using Mann Whitney U test. Comparisons were also made between antibody levels at enrolment and at 36 weeks using Wilcoxon matched-pair test.2); normal (18.5-25 kg/m2) and increased (>25 kg/m2). All the variables were treated as categorical except for antibody levels at enrolment which was treated as a continuous variable. Likelihood ratio test was used to test whether the association between supplementation type and antibody levels at 36 weeks varied with covariates chosen for the study.The covariates of the association between supplementation type and malaria antibody immunity were determined based on their biological relevance. Covariates chosen were gravidity, maternal age, HIV infection, malaria infection, residence, socioeconomic status (SES), body mass index (BMI) and antibody levels at enrolment. Gravidity, maternal age, HIV and malaria infection were previously shown to associate with malaria antibody immunity ,37. PartNevertheless, all of the above variables  and multivariate logistic regression analyses for categorical outcomes . Differences in antibody levels among women with different BMI and SES groups were determined following multivariate linear regression analysis, adjusting for covariates including supplementation group, as detailed in the table footnotes. Associations were considered statistically significant if the p value is <0.05.2 was 5.7% and MUAC < 23cm was 6.8%, who were identified as undernourished women in the current cohort. Thirteen percent of women were HIV infected. The proportion of participants lost to follow up between the supplementation groups was similar with an overall 6.04% lost to follow up [Overall 1,391 women were enrolled in the iLiNS-DYAD-M study. Malaria immunity was only measured in the 1,009 enrolment and 36 weeks paired plasma samples that were available. The baseline characteristics of the participants across the supplementation arms were comparable  and 42.3% compared to 15.0% at enrolment (p < 0.0001), respectively  increase in BMI is associated with a decrease in relative antibody levels by −1.04% , −0.66%  and −1.22%  respectively , and opsonizing antibodies to non-placental-binding VSA (p = 0.005) Figure D-F. BothThe association between nutrient supplementation and malaria immunity was evaluated in 1,009 pregnant women enrolled in a randomized controlled trial comparing LNS, MMN and IFA. Ingestion of LNS was not associated with significant increases in malaria antibody levels, a faster rate of change of antibody levels from enrolment to 36 weeks, or likelihood of seropositivity to malaria antigens. This study is the first to investigate the relationship between nutrient supplementation and malaria immunity in detail.Antibodies were measured against VSA expressed by a placental-binding and non-placental-binding parasite isolate, merozoite antigens and schizont extract. Antibodies to placental-binding VSA reduced the risk of placental malaria by inhibiting cytoadhesion to receptors , and by P. falciparum dihydrofolate reductase (DHFR) and dihydropteroate (DHPS). In Malawian pregnant women quintuple mutations of DHFR and DHPS are highly prevalent, however the prevalence of more severe sextuple mutant DHPS 581 which is associated with increased placental inflammation is uncommon [A strong increase in both total and opsonizing antibodies to placental-binding VSA from study entry to 36 weeks was observed while antibodies to merozoite anti6gens and non-placental-binding isolates declined. Several explanations are possible. First, most parasite isolates infecting pregnant women express antigens that are unique and are exclusively expressed during pregnancy . Antiboduncommon .et al., unpublished work) during the current pregnancy, thereby eliciting pregnancy-specific immunity. Poorer women could have more exposure to malaria due to poor housing that allows entry of mosquitoes, and they may be less likely to own insecticide-treated bed nets. In addition poverty could lead to poor nutrient intake due to inability to afford food especially during the hunger season.The type of supplementation had no strong or consistent influence on malaria antibody levels, however a regression analysis showed a decline in antibody responses to MSP-2 and PfRh2 from enrolment to 36 weeks, and an increased rate of reduction in antibody levels to MSP-2, in women who received LNS compared to MMN. Given the large number of comparisons reported, these differences in the adjusted models could be due to chance. Similar differences in other anti-merozoite antibodies were not observed, and the significance of the changes is unknown. The current study also reported the effects of nutrient supplementation on antibody responses in subsets of women at high risk of undernutrition and infection; those with poor nutritional status and low SES. A negative relationship was observed between malaria antibody levels and maternal BMI and SES. Overall, women with a low BMI at study entry had higher antibody levels to VSA than did women with normal or increased BMI. Supplementation type did not influence the relationship between antibody responses and BMI. Of all the antibody types being tested the opsonizing antibodies to VSA were the most strongly related to BMI and SES in the current cohort, suggesting that these women might be exposed to more malaria . In addition unavailability of participant data for some of the covariates may have reduced the power of the multiple regression analyses. For analysis of BMI and malaria immunity, stratification of participants reduced the study power: for instance only 57 pregnant women were in the low BMI group.Overall the current study concluded that nutrient supplementation of pregnant women was not associated with broad changes in antibody responses to malaria. This study is the first to investigate interactions between nutrition and malaria immunity in pregnancy and further studies must be conducted to determine the role of nutrient supplementation on other components of host immune system such as cell-mediated immunity, to determine the overall influence of nutrient supplementation on malaria susceptibility and malaria immunity.


Plasmodium falciparum malaria is highly endemic and remains stable despite the implementation of various malaria control measures. Understanding P. falciparum population structure variations across the country could provide new insights to guide malaria control programmes. In this study, P. falciparum genetic diversity and population structure in regions of varying patterns of malaria transmission in Mali were analysed.In Mali, P. falciparum via quantitative PCR (qPCR). Multiple loci variable number of tandem repeats analysis (MLVA) using eight microsatellite markers was then performed on positive qPCR samples. Complete genotypes were then analysed for genetic diversity, genetic differentiation and linkage disequilibrium.A total of 648 blood isolates adsorbed onto filter papers during population surveillance surveys  in four distinct sites of Mali were screened for the presence of ST < 0.02), even between sites located approximately 900 km apart, thereby illustrating marked gene flux amongst parasite populations. The lack of linkage disequilibrium further revealed an absence of local clonal expansion, which was corroborated by the genotype relationship results. In contrast to the stable genetic diversity level observed throughout the country, mean multiplicity of infection increased from north to south (from 1.4 to 2.06) and paralleled malaria transmission levels observed locally.Of 156 qPCR-positive samples, complete genotyping of 112 samples was achieved. The parasite populations displayed high genetic diversity (mean He = 0.77), which was consistent with a high level of malaria transmission in Mali. Genetic differentiation was low  contains supplementary material, which is available to authorized users. Plasmodium falciparum, which is transmitted by mosquitoes of the genus Anopheles, is the most common and the deadliest human malaria parasite. Despite extensive malaria elimination efforts, 214 million cases of malaria and 438,000 deaths were estimated globally in 2015, which primarily affected the sub-Saharan African population and children under 5 years of age [P. falciparum malaria remains highly endemic; stable incidence of the disease has been reported in several malaria vaccine-testing sites, such as Bandiagara [P. falciparum genetic diversity may be useful to elucidate the mechanisms of transmission persistence and rebound. Such studies would provide insight into human reservoirs of the parasites, including symptomatic cases and those of asymptomatic carriage [P. falciparum genetic diversity would shed light on the characteristics of malaria burden and the expected difficulties hindering malaria control. Indeed, it has been shown that P. falciparum genetic diversity is indicative of the ability of malaria parasites to adapt to their hosts by selection of advantageous traits, such as drug resistance and antigenic variability [In Mali, from the Sahara Desert to the Sudano-Guinean savannah, throughout the Sahel region, the variety of malaria transmission pattern is characterized by a north to south increasing gradient . Malariacarriage , 8, 10 acarriage –15. Addiiability .Plasmodium falciparum genetic diversity can be assessed by analysing genetic polymorphism of the merozoite surface proteins (msp-1 and msp-2), as previously published in Mali [msp-1 and msp-2 genes are under selective pressure and neutral markers such as microsatellites or single nucleotide polymorphism (SNPs) are better suited for population genetics assessment [P. falciparum population genetics via multiple loci variable number of tandem repeats analysis (MLVA) [P. falciparum population genetic features, including parasite migration and linkage disequilibrium. To date, microsatellite markers have never been assessed to study P. falciparum population genetics in Mali. in Mali , 17, 18.sessment . Highly s (MLVA) , 20–23. P. falciparum DNA extracted from blood samples collected from four Malian study sites displaying various malaria transmission patterns. The four study sites were located along a 900-km long north to south axis and included the city of Bamako.In this study, MLVA was performed on Blood samples were collected in Rharous (Timbuktu District), Bamako (Bamako District), Doneguebougou (Kati District), and Bougoula Hameau (Sikasso District) Fig. . Each ofA total of 648 blood samples were collected by finger prick (~50 µL) and adsorbed onto filter paper (Whatman™ 3MM CHR). Dried blood spots on filter papers were transported and stored at ambient temperature at the Malaria Research and Training Centre (MRTC) Central Laboratory in Bamako, Mali. Along with blood sampling, epidemiological and clinical data were reported on a standardized form. Sample collection was performed at the end of the rainy season (October) and at the beginning of the dry season (January to March), which correspond to the highest malaria transmission periods  Table 11. SampleEthical clearance was obtained from the Ethics Committee of the Faculty of Medicine of Bamako (No 2012/81/CE/FMPOS) and blood samples were obtained after individuals provided informed consent.2 using scissors and were incubated for 48 h at room temperature in 800 µL EasyMAG lysis buffer . Nucleic acid extraction was then performed using a NucliSENS EasyMAG system  with an elution volume of 100 µL [P. falciparum was performed using specific primers and probe targeting the 18S rRNA gene via qPCR with a LightCycler 480 PCR system  as previously described [P. falciparum18S rRNA) control. Standard curves were generated from serial ten-fold dilutions of the plasmid for species-specific quantification of parasite density (number of parasites/μL of blood). It was assumed that each genome of P. falciparum has five copies of the 18S rRNA gene (as observed in the 3D7 genome) [Filter papers with dried blood spots were cut into two pieces of approximately 1 cmf 100 µL . Screeniescribed . Each ex genome) .P. falciparum, as previously published [Genotyping relied on the analysis of eight polymorphic microsatellite markers  distributed among five chromosomes of ublished , 29. Micublished . Primer ublished , 29. PeaOnly complete genotypes  were included in the population genetics analysis (complete dataset).P. falciparum  present in an individual. As P. falciparum is haploid in the human stage, multiple peaks or alleles correspond to an infection with multiple genotypes or strains (a polygenomic infection). In a given sample, MOI was scored as the maximum number of alleles observed when taking into account all analysed loci (n = 8). An average MOI was then calculated.Multiplicity of infection (MOI) is defined as the number of genetically distinct clones of P. falciparum is haploid in the human host, the terms ‘genetic diversity’ and ‘Nei’s unbiased genetic diversity index’ were employed instead of ‘expected heterozygosity (He)’. Nei’s unbiased genetic diversity index was calculated using ARLEQUIN v.3.5 [n = number of samples analysed and p = frequency of each allele at a given locus [Because of the frequent occurrence of polygenomic infections, only major peaks were considered to assemble genotypes for genetic diversity analysis, as previously described , 20, 21.IN v.3.5  with theen locus . For a hen locus .ST), a measure of genetic differentiation between populations, using ARLEQUIN v 3.5 [The complete dataset, including genotypes from the four study sites, was analysed to calculate Wright’s F-statistics (FIN v 3.5 .ISA), a quantitative measure of LD. Values range from 0 (no loci in LD) to 1 .The overall multi-locus linkage disequilibrium (LD) was assessed using the LIAN v.3.7 web interface . This geA minimum spanning tree was constructed based on the complete dataset to investigate genetic connections between genotypes. The chosen plug-in and the cluster analysis tool were implemented in BioNumerics v7.1 Software .2). Plasmodium falciparum MOI association with sex, age group, study site, and parasite density was tested via univariate unconditional logistic regression analysis (Logistic procedure). Multivariate unconditional logistic regression analysis with stepwise selection was performed to select the most parsimonious, best-fitting model of P. falciparum MOI. The analyses were performed using two-sided tests with a P < 0.05 significance level using SAS v9.2 statistical software .Parasite densities of the four sites were compared using a negative binomial regression model (Genmod procedure). The influence of categorical variables including gender, age category  and fever on the occurrence of polygenomic infections was assessed via Chi squared test . Mean MOI values ranged from 1.4 (Rharous) to 2.06 (Bougoula) and were intermediate in Bamako (1.5). Similarly, polygenomic infection prevalence significantly differed based on the study sites (P < 0.0001). The lowest polygenomic infection rate was observed in Rharous (37 %), while the highest rate was noted in Bougoula (83 %). Univariate analysis revealed no significant effect of gender, age, fever, and parasite density on the occurrence of P. falciparum polygenomic infections (Table P < 0.001). Accordingly, the risk was significantly lower in Rharous (P < 0.01) and Bamako (P < 0.05) when compared with Bougoula.The proportion of polygenomic infections and mean MOI values per study site are shown in Table  Rharous 7 %, whilGenetic diversity values (He) for each study site are shown in Table ST between the four study sites, which reveals an almost complete panmixia with very low FST values (FST = 0.0011–0.018). Pairwise FST was statistically significant only between Bamako and Rharous (P = 0.03).Table P. falciparum genotypes in the different study sites. The ISA values and associated P values are shown in Table Global multi-locus LD analysis of genotypes showed an absence of significant LD among alleles of Of the 112 complete genotypes, 100 % were unique genotypes as they were found only in a single sample. Most genotypes differed by two, three or more than three loci variants of the microsatellite markers. No major clustering of genotypes was detected between and within study sites as shown in Fig. P. falciparum populations in Mali to date and the first study to employ MLVA genotyping of P. falciparum in this country. Plasmodium falciparum isolates from Mali displayed high genetic diversity (mean He = 0.77) and low genetic differentiation (FST < 0.02). The lack of LD further revealed an absence of local clonal expansion, which was corroborated by the genotype relationship results. These findings highlight the marked gene flux between parasite populations, including within sites of lower transmission rates (Rharous and Bamako) and between sites located 900 km from each other (Rharous and Bougoula). In contrast with the lack of variability between genetic diversity levels observed in the country, mean MOI and proportions of polygenomic infection showed a north to south increasing gradient paralleling local malaria transmission levels [This work represents the most extensive investigation of genetic diversity of n levels .P. falciparum in African regions highly endemic for malaria [Comparable levels of genetic diversity (He = 0.72–0.8) have already been observed in multi-locus microsatellite studies of  malaria , 20, 21. malaria , 35 and  malaria , 20. Ind malaria .msp-1 and msp-2) [P. falciparum genetic diversity remained high despite bolstered malaria control efforts [P. falciparum microsatellite markers in Djibouti between 1998 and 2009 [P. falciparum clonality have also been observed in Senegal (Thies city) during a longitudinal study using a set of 24 SNPs molecular barcode [P. falciparum population genetics in African countries suggest that in Mali, malaria control interventions still exert a limited impact on P. falciparum populations and malaria transmission.Genetic diversity metrics have been useful to assess malaria control interventions and to highlight the disparities in malaria control programme results in endemic African countries , 21, 37.d msp-2) , 17, 18 and 2009 . This se barcode . Clonal P. falciparum populations located approximately 200 km apart was depicted as the marker of substantial parasite flux between the two locations. In the present study, a similar pattern was observed between study sites located up to 900 km apart (Rharous and Bougoula). A possible explanation could be the serious political crisis that occurred in 2012 and 2013 in Mali, which led to significant deterioration of security in the northern regions as well as displacement of large populations to neighbouring countries and the southern areas of Mali. Importantly, these findings imply that the effectiveness of malaria control efforts may be impaired if all malaria foci are not tackled simultaneously. High parasite flux from one site to another may also favour the diffusion of anti-malarial drug-resistant strains.Findings highlight pronounced gene flux between parasite populations. As illustrated in Rharous , the association between a high proportion of monogenetic infections (63 %) and high genetic diversity (He = 0.76) suggest that genetically diverse parasite strains have been imported from highly endemic regions via extensive human migration events. A similar trend was observed in the capital city of Bamako, thereby suggesting that many malaria cases are being imported from rural areas. These hypotheses are also supported by the lack of genetic differentiation between the different study sites. In Nigeria , a lack P. falciparum genotype was unique, with an absence of population clustering, even in the remote low-transmission site of Rharous. Human migration events might play a role in this phenomenon by boosting genetic diversity amongst P. falciparum populations. The absence of local clonal expansion was further corroborated by the lack of LD, which revealed a panmixia parasite population. Nevertheless, other markers, such as SNPs, might be more prone to show clonal expansion of parasites [P. falciparum populations has been observed in Thies city using SNP molecular barcode [The analysis of the relationship between genotypes further revealed that each arasites . For ins barcode , 5. TherThe lowest levels of MOI and proportion of polygenomic infections were found in the areas exhibiting low malaria transmission, namely the urban site of Bamako and the Sahelo-Saharan site of Rharous. In contrast, the highest levels were found in the Sudano-Guinean site of Bougoula, where malaria transmission is almost perennial. A similar trend has been observed in two Malawian districts characterized by different transmission settings . AlthougP < 0.0001). Nevertheless, the significantly lower proportion of polygenomic infections observed in Rharous is probably not due to differences in case detection, as the univariate analysis revealed no association between proportion of polygenomic infections and fever, patient sex, age group, or estimated parasitaemia. Variations in proportion of polygenomic infections correlated with the study site only (P < 0.001). Additionally, disparities between malaria cohorts, such as fever prevalence, seem not to have impacted genetic diversity level, as it was similar in the different study sites. Likewise, this observation was further supported by a comparable high level of allelic richness.A limitation of this study may be that the human cohorts of each study site were not totally comparable. Indeed, a passive sampling procedure was used in Rharous due to security issues that precluded active case detection Table . This exP. falciparum genetic diversity level is particularly high in Mali. Such diversity may constitute an obstacle to control malaria as genetic polymorphism may facilitate parasite populations to adapt to their host and counteract certain interventions, such as anti-malarial vaccination. The pronounced parasite gene flux mediated by human migration events likely represents another hindrance for malaria control. Indeed, high parasite flux from one site to another could favour the diffusion of parasite strains, including those resistant to anti-malarial drugs. Therefore, these findings argue that malaria control measures should be intensified in Mali and that all malaria foci must be tackled simultaneously to reduce P. falciparum genetic diversity. As the measures currently in place (ACT and LLINs) seem to have a limited impact on P. falciparum populations, malaria control programmes aiming at reducing both malaria burden and parasite populations are greatly warranted.Overall, these results reveal that


Plasmodium falciparum infection in Port Harcourt, Nigeria reporting to malaria clinics. A total of one hundred and thirty-six (136) male patients were recruited. QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count was used. The mean values of the haematological parameters in each quartile of parasite densities were determined using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination (R2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters Neutrophil count had a negative exponential relationship with the parasite density and is related to the parasite density by a polynomial equation model: ynm = -7E-07x2 - 0.0003x + 56.685. The coefficient of determination (R2) was 0.6140. This means that the rate of change of the parasitemia will depend on the initial value of the neutrophil. As the neutrophil increases, the parasitemia will tend to decrease in a double, triple and quadruple manner. The relationship between lymphocyte count, monocyte count and eosinophil count and parasite density was logarithmic and expressed by the following linear equation models: ylm = -2.371ln(x) + 37.296, ymm = 0.6965ln(x) + 5.7692 and yem = 0.9334ln(x) + 4.1718 in the same order. Their respective high coefficients of determination (R2) were 0.8027, 0.8867 and 0.9553. This logarithmic relationship means that each doubling of monocyte count and eosinophil count will cause the same amount of increase in parasitemia whereas each doubling of lymphocyte count will cause the same amount of decrease in parasitemia. The best fitting regression model for total white cell count (WBC), haemoglobin concentration, packed cell volume (PCV)(haematocrit) and mean cell haemoglobin concentration (MCHC) and parasite density was a linear model and expressed by the following linear equation models: yWBCm = 1.2314x + 8533.8, yHbm = -0.0014x + 13.004, yPCVm = -0.0046x + 41.443 and yMCHCm = -0.0008x + 32.336. Their respective coefficients of determination are 0.7397, 0.6248, 0.9758 and 0.8584. This linear relationship means that as the parasite density is increasing that there is a corresponding decrease in haemoglobin concentration, PCV and MCHC and a corresponding increase in total white cell count. The best fitting regression model between platelet count and parasite density is a power model with a very high coefficient of determination (R2=0.9938) and expressed by: yPltm = 278047x-0.122. These equation models could be very useful in areas where there may not be functional microscopes or competent microscopists and in medical emergencies.This study examined the experiential relationship between the parasite density and haematological parameters in male patients with  Plasmodium. The female anopheles mosquito is the carrier of the parasite and transmits it to man by next blood meal. Malaria is widespread in tropical and subtropical regions because of the significant amounts of rainfall and consistent high temperatures and high humidity, along with stagnant waters which provide mosquitoes the environment needed for continuous breeding , quantitative buffy coat (QBC) technique and microscopic method using peripheral blood thick and thin film. These techniques are all with some limitations particularly in a resource limited environment like Nigeria.P. vivax, P. ovale, or P. malariae. These tests are not quantitative and will not provide any information concerning levels of parasitemia. Antigen persistence is also a problem. PCR testing and QBC method offer a rapid, sensitive, and less subjective methods to determine the presence and species of Plasmodium. Unfortunately, these malaria tests are expensive and not routinely used in Nigeria.Several rapid diagnostic tests have been developed which detect malaria parasite antigens in lysed blood using monoclonal antibodies. However, there are currently no known rapid diagnostic tests which can differentiate The gold standard used for malaria parasite diagnosis in Nigeria is the peripheral blood thick and thin film method. This is because it is most economical. It is also reliable, sensitive and specific but the reliability, sensitivity and specificity of this method depend on the efficiency of the microscopist. Unfortunately in many laboratories in Nigeria, particularly in the rural areas, this manpower is lacking.On the contrary, a good number of laboratory scientist and technicians in Nigeria can easily assess haematological parameters. Many malaria cases particularly children under 5 years come as medical emergency and as such little time is needed to diagnose, treat and save their lives. To reduce malaria-related deaths in future particularly in rura006C Africa, strategies on quick diagnosis and treatment should be adopted. Based on these, this study examined the experiential relationships between some haematological parameters and the parasite density of the malaria infected male subjects in Port Harcourt, Nigeria. These relationships can be used to, given any of the haematological parameter, predict the malaria parasitemia within the shortest time even in the absence of a functional microscope or a competent microscopist. There are sex differences in some haematological parameters hence the need to develop these regression models according to sex.The study was carried out in Port Harcourt located between latitudes 4° 2′ North and 4° 47′ North and longitudes 6° 55′ East and 7° 08′ East has continued to record a high incidence of malaria infection despite the federal government efforts to roll back malaria in Nigeria. The reason is that Port Harcourt is a typical coastal zone located in the Niger Delta of Nigeria. High temperatures and humidity as well as marked wet and dry seasons characterize the climate. The mean annual rainfall is estimated at about 2,405mm while the mean monthly temperature varies between 24°C and 32°C throughout the year . The meaThe laboratory study was carried out for a period of six months during which a total of one hundred and thirty- six (136) males were recruited. The inclusion criteria were male out-patients to the participating clinic site within the age of 1- 60 years queried for malaria infection with the presentation of at least one of the following: an oral temperature of 38°C, headache, or a history of fever within the past 72 hours and who must not have commenced any treatments for malaria. Exclusion criteria were male out-patients with pathological conditions outside malaria such as protozoan or helminthes infection, typhoid fever and HIV/AIDS, congenital manifestations such as sickle cell disease and history of allergy. All enrolled patients were interviewed for information on current symptoms and previous malaria episodes and treatments and informed consent obtained from them before blood sample collection.A volume of 2.6 ml of the venous blood sample was drawn into monovette tubes containing the anticoagulant potassium ethylenediamine-tetra acetic acid (EDTA) for QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count. QBC Autoread Plus manufactured by QBC Diagnostic Incorporated, USA provided a diagnostic haematology profile of the following quantitative values from a single tube of blood; packed cell volume (haematocrit), haemoglobin concentration, mean corpuscular haemoglobin concentration, platelet count, white blood cell count, granulocyte count (% and number) and lymphocyte-monocyte count (% and number). Daily quality assurance checks were performed and recorded.For QBC malaria parasite detection analysis, the centrifuged tube was examined under a fluorescence microscope in the region between the light red blood cells and granulocytes and lymphocytes/monocytes, where the parasites are most abundant. Examination of the centrifuged blood under a fluorescence microscope readily permits the detection of malaria parasite in the infected cells and plasma. Since the parasites contain DNA which takes up the acridine orange stain, they appear as bright specks of light in the dark background of non-fluorescing red cells.For QBC malaria parasite species identification, at magnification of 600X, all parasites in the red blood cells were easily visualized and their morphologies identified. Species identifications were made based upon the size and shape of the various stages of the parasite and the presence of stippling (i.e. bright red dots) and fimbriation (i.e. ragged ends). Plasmodium parasites are always intracellular, and they demonstrate, if stained correctly, blue cytoplasm with a red chromatin dot.The parasite densities were obtained by multiplying the average number of parasites in 10 QBC fields by a factor of 10.5 .For a reliable differential leucocytes count, a thin blood film was prepared by a standard manual technique as described by 2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters.The data obtained were grouped into four parasite densities and analyzed using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination  to 61.41±4.17 % in very heavy infection (4+). There was increase in neutrophil count with increase in parasitemia. Other haematological parameters in the P.falciparium malaria infected subjects include monocyte count which ranged from 6.42±1.87% in scantly infected male subjects to 10.59±2.13% in very heavily infected subjects.nm) is related to the parasite density x, by the polynomial equationAs shown in nm = -7E-07x2 - 0.0003x + 56.685y (1)2) was R² = 0.6140.The coefficient of determination  + 37.296y (2)lm is the lymphocyte count in males. The coefficient of determination was very high (R² = 0.8027).Where ymm) and eosinophil counts in males (yem) and. their respective coefficients of determination were 0.8867 and 0.9553.As also shown in mm = 0.6965ln(x) + 5.7692y (3)em = 0.9334ln(x) + 4.1718y (4)As shown in WBCm)This relationship is defined by the equation 5 for WBC Count in males (yWBCm = 1.2314x + 8533.8 (5)yAs shown in Hbm is the Hb Concentraion in malesThe relationship is expressed in equations 6 where yHbm = -0.0014x + 13.004 (6)yAs shown in PCVm is the PCV in malesThe relationship is expressed in equations 7 where yPCVm = -0.0046x + 41.443 (7)yAs shown in MCHCm = -0.0008x + 32.336 (8)yMCHCm is the MCHC in malesThe coefficient of determination was 0.8584 where y2=0.9938). The relationship is expressed in equations 9 where yPltm is the platelet count in males.As shown Pltm = 278047x-0.122 (9)yThe mean neutrophil counts in most of the infected cases studied were within the normal range of 40-75%. This finding is similar to the works of The inverse relationship between lymphocyte count and parasite density from this study suggests an increased apoptosis at higher parasitemia. The best predictive regression models for lymphocyte count and parasite density was logarithmic. This logarithmic relationship means that each doubling of lymphocyte count will cause the same amount of decrease in parasitemia.P. falciparum (The trend of decreasing platelet count with increasing levels of parsitemia observed in this study has been previously noted for The empirical relationship between the parasite density in the male subjects and their mean monocyte counts were expressed by logarithmic regression model. This means that each doubling of monocyte count will cause the same amount of increase in parasitemia.Mean eosinophil counts in the studied group showed a consistent significant increase with increasing parasite density. This agrees with earlier works by Only 5.88% of the infected males showed leucocytosis. No specific diagnostic indications are given by the white blood cell count during malaria attack and the few cases of leukocytosis may reflect the presence of concomitant bacterial infections. This is in agreement with previous work done by Mean Hb concentration and PCV in the studied group showed a consistent decrease across the quartiles of parasite density. This observation agrees with the report of There was no significant variation between the mean values of MCHC in the studied group from the normal range of 32-36. This suggests probably that the Hb content of the circulating non-infected erythrocytes remained intact. The empirical relationship between the parasite density and the mean MCHC was expressed by a linear regression model in the studied group.falciparum (The trend of decreasing platelet count with increasing levels of parsitemia observed in this study has been previously noted for P. Malaria parasitemia has been shown to have effects on some haematological parameters from this study while some haematological parameters are more predictive of malaria infection than others. Eosinophilia, thrombocytopenia and lymphopenia were identified as the key haematological indicators of malaria infection in the studied population. Eosinophilia can be used to predict the intensity of malaria infection. Thrombocytopenia is strongly associated with malaria infection and malaria parasite density. Lymphopenia is however more indicative of hyperparasitemia. Decrease in Hb and PCV in children is strongly suggestive of malaria infection. Neutrophil, monocyte and basophil counts do not have any significant relation with malaria parasitemia and are therefore not reliable indices for predicting malaria infection.Experiential equation models have been developed from this study for estimating the values of parasite densities from haematological parameters. This could be very useful in areas where there may not be functional microscopes or competent microscopists and in medical emergencies.nm neutrophil count in malaria infected malesylm lymphocyte count in malaria infected malesymm monocyte count in malaria infected malesyem eosinophil count in malaria infected malesyWBCm total white cell count in malaria infected malesyHbm haemoglobin concentration in malaria infected malesyPCVm packed cell volume in malaria infected malesyMCHCm mean cell haemoglobin concentration in malaria infected malesyPltm platelet count in malaria infected malesy


HLA-G, a non-classical HLA class I antigen, is of crucial interest during pregnancy by inhibiting maternal immune response. Its role during infections is discussed, and it has been described that high levels of soluble HLA-G during childhood increase the risk of malaria. To explore more precisely interactions between soluble HLA-G and malaria, latent class analysis was used to test whether distinct sub-populations of children, each with distinctive soluble HLA-G evolutions may suggest the existence of groups presenting variable malaria susceptibility.A study was conducted in Benin from 2010 to 2013 and 165 children were followed from birth to 12 months. Evolution of soluble HLA-G was studied by the latent class method.Three groups of children were identified: one with consistently low levels of soluble HLA-G during follow-up, a second with very high levels and a last intermediate group. In all groups, low birth weight, high number of malaria infections and high exposure to malaria transmission were associated with high level of soluble HLA-G. Placental malaria was not. Presence of soluble HLA-G in cord blood increased the probability of belonging to the highest trajectory.These results, together with previous ones, confirm the important role of HLA-G in the individual susceptibility to malaria. Assaying soluble HLA-G at birth could be a good indicator of newborns more fragile and at risk of infections during childhood. Human Leukocyte Antigen-G (HLA-G) is a non-classical HLA class Ib antigen, with important immune-regulatory functions , that diHLA-G 3′UTR were associated with susceptibility to human African trypanosomiasis and could influence the clinical and immunological responses directed to Plasmodium falciparum .A general model’s formulation is:πj(zi) that a child i with the covariates vector zi belongs to the group j is: zi and its corresponding coefficients vector θj.The posterior probability The analysis was performed using three steps.Firstly, each sHLA-G trajectory has been modelled, by means of a polynomial function of time of degree 3, without introducing any covariate. The most appropriate number of trajectories was selected using the Bayesian information criterion (BIC). The group prevalence, that should not be less than 5 % of the total number of the sample, was used as additional criteria, for practical interpretation .p < 0.20 during univariate analysis were entered in the multivariate step. Statistical significance in the final model was set at p < 0.05. The effect of covariates on HLA-G trajectories can be identical for each trajectory or different according to them. During this step a particular attention was paid to the stability of the trajectories determined during the first step. Indeed, as mentioned by Nagin [Secondly, the effect of the covariates on each trajectory was studied using univariate and multivariate analysis. Only covariates associated with by Nagin  the addiIn the final multivariate model each individual was assigned to the trajectory group for which he had the highest posterior probability. Within each group, posterior probability values greater than or equal to 0.7 indicate adequate internal reliability . All theFinally, in the third step, the predictor effect of some covariates to belong to one or more trajectory has been tested. This concerned placental malaria, parity, maternal anaemia, LBW and sHLA-G in cord blood.® Software, Version 12 , using the syntax dedicated to generalized linear latent and mixed models (GLLAMM).All analyses were performed with the StataThe study’s protocol was approved by the Ethics Committee of the “Faculté des Sciences de la Santé (FSS)” of Cotonou and the “Institut de Recherche pour le Développement (IRD)” Consultative Ethics Committee. All mothers and fathers were informed during antenatal care visits and meetings conducted in villages before the beginning of the study. Mothers signed informed consent written in French and explained in local language.At delivery sHLA-G was measured for all mothers and all cord blood samples (n = 165). During the follow-up, a total of 445 measurements were performed from 3 to 12 months distributed as follows: 109/165 at 3 months, 113 at 6 months, 114 at 9 months and 109 at 12 months. Overall, a total of 775 measurements  were performed. In most cases, an HLA-G measurement was missing because the mother had not attended a quarterly visit. Only 10 children (6.1 %) were present for a single visit.The mean age of the mothers was 26.75 years . Most women were multi-gravidae , and 40 % were suffering from anaemia at delivery.The mean birth weight of newborns was 2,964 grammes (SD = 448.31) and 10.9 % (n = 18/165) of children had a LBW. Eighty-eight children (53.33 %) were female and eighteen children were premature. During the follow-up, 50.9 % (n = 84/165) of children had at least one malaria infection, and 10 % more than two. The most infected child had seven infections.At delivery, 78.2 % of women had detectable levels of sHLA-G n = 129/165). The mean level in the peripheral blood for all women was 19.12 ng/ml . Sixty percent of children (n = 99/165) had detectable sHLA-G in cord blood and the mean level was 16.22 ng/ml . A strong correlation has been described previously between the mother/newborn (cord blood) sHLA-G levels, with a significantly higher level in mother’s blood at delivery . The sHL29/165. TDuring the first step of analysis, groups of children following the same pattern of sHLA-G have been identified. Using the BIC as criteria, the two-group model showed a higher BIC than both three- and four-group models. The four-group model was selected when compared to the three groups Table . In the −3). Similarly, detectable sHLA-G level in cord blood, or in mother’s peripheral blood at delivery, was associated with higher levels of sHLA-G from 3 to 12 months of life (p < 10−3 in the both case). Having more than one malaria attack during the follow-up was significantly associated with higher levels of sHLA-G (p = 0.008). In the same way, the higher levels of malaria exposure were significantly related to the high levels of sHLA-G (p = 0.01).Using the three-group model, placental malaria (p = 0.59), parity (p = 0.31), gender (p = 0.95), prematurity (p = 0.43), maternal anaemia (p = 0.51) and parasite density (p = 0.52) have no significant effect on the dynamics of sHLA-G. In contrast, LBW appeared to be significantly associated with higher levels of sHLA-G (p < 10−3). High exposure to malaria and having more than one malaria infection during the follow-up were significantly associated with high levels of sHLA-G . In the final model, the proportions of the groups were 50.9, 27.2 and 21.9 %, respectively for the low, intermediate and high trajectories . Accordingly, a child born with sHLA-G in cord blood would have a probability of 76.25, 20.40 and 3.35 % to belong to the high, intermediate or the low trajectory, respectively.When the same analysis was performed using the four-group model, convergence was not achieved, reinforcing the instability of this model. Taken together, the results are consistent with the existence of three-trajectory model. With this model 97 % of children from the low trajectory, 91.7 and 83 % of children from respectively the intermediate and high trajectories were classified as belonging to these groups with a posterior probability greater than 70 %.Table The present work is complementary to study of Sadissou et al., describing the evolution of sHLA-G in a population of African infants . In the The present analysis, confirms these results, and provides new understanding by identifying new determinants of sHLA-G evolution. Indeed, although in the first study, the level of sHLA-G increased the risk of infection in the following quarter, any association has not been highlighted between the level of sHLA-G and the overall number of malaria infections during the follow-up. In this complementary analysis, this association is strongly significant. The difference between these two results could be due to the complexity of the effect of malaria infection that cannot be pointed out under the hypothesis that all children belong to the same homogeneous population. Using latent class approach allows hypothesizing that distinct subpopulations of children are presents in the overall population, each with its own characteristics, with distinctive sHLA-G evolutions. This approach permitted us to explore more precisely the complex association between malaria and HLA-G.HLA-G gene expression is regulated are only partially understood [P. falciparum could upregulate the HLA-G secretion through the stimulation of cytokines such as IL-10 and IFN-γ. HLA-G is known to inhibit the function of T, NK and B cells through direct interaction with ILT2. Recently, Naji et al. showed that HLA-G inhibits B cell proliferation, differentiation, and immunoglobulin (Ig) secretion in both T cell–dependent and –independent models of B cell activation. HLA-G also acts as a negative B cell regulator in modulating B cell antibodies secretion in a xenograft mouse model [P. falciparum infection, leading to a lower B cell response. As IgG antibodies play a pivotal role in anti-malarial protection [P. falciparum mediated by HLA-G, not only allows the parasite to escape the immune system but also is responsible of the higher susceptibility to infection showed previously.The mechanisms by which derstood . Concernderstood –42. Althse model . Taken totection –46, the However, other factors could influence HLA-G secretion during malaria infection. It has been shown that hypoxia could play a role in HLA-G regulation during pregnancy , in someMore than 60 % of the children born with a LBW belong to the high and intermediate trajectories. It is widely accepted that birth weight is a key indicator of fetal and neonatal health –54 and oLatent class analysis approach is more and more frequently used in biomedical research . It is aHLA-G and malaria has already been pointed out. Indeed, it has been shown that some polymorphisms of the HLA-G 3’UTR were associated with variable risk of infection [P. falciparum. These associations have been identified in another population from Senegal. It has also been demonstrated that a strong molecular signature of balancing selection at HLA-G 5’URR exist and that this region may certainly be the direct target of selection [A genetic association between nfection , but alselection . These rSummary, this study allowed a better understanding of the complex interaction between HLA-G and malaria infection and pointed out the potential interest of sHLA-G as marker of frailty.


Arthralgia and myalgia were associated with patients over the age of 14 years (P < 0.001). Having experienced at least eight malaria episodes prior to the study was associated with a decreased risk of chills and fever and an increased risk of sore throat (P < 0.05). None of the symptoms showed an association with gender or with species of Plasmodium. The clinical spectrum of malaria in semi-immune individuals can have a broad range of symptoms, the frequency and intensity of which are associated with age, past exposure to malaria, and parasitemia. Understanding the full spectrum of nonsevere malaria is important in endemic areas to guide both passive and active case detection, for the diagnosis of malaria in travelers returning to non-endemic areas, and for the development of vaccines aimed to decrease symptom severity.Malaria is a disease that generates a broad spectrum of clinical features. The purpose of this study was to evaluate the clinical spectrum of malaria in semi-immune populations. Patients were recruited in Mâncio Lima, a city situated in the Brazilian Amazon region. The study included 171 malaria cases, which were diagnosed via the use of a thick blood smear and confirmed by molecular methods. A questionnaire addressing 19 common symptoms was administered to all patients. Multiple correspondence analysis and hierarchical cluster analysis were performed to identify clusters of symptoms, and logistic regression was used to identify factors associated with the occurrence of symptoms. The cluster analysis revealed five groups of symptoms: the first cluster, which included algic- and fever-related symptoms, occurred in up to 95.3% of the cases. The second cluster, which comprised gastric symptoms , occurred in frequencies that ranged between 35.1% and 42.7%, and at least one of these symptoms was observed in 71.9% of the subjects. All respiratory symptoms were clustered and occurred in 42.7% of the malaria cases, and diarrhea occurred in 9.9% of the cases. Symptoms constituting the fifth cluster were vomiting and pallor, with a 14.6% and 11.7% of prevalence, respectively. A higher parasitemia count (more than 300 parasites/mm Malaria features a broad spectrum of clinical outcomes that vary from asymptomatic infection  to severThe most easily recognizable symptoms in nonsevere malaria are chills, fever, and sweating, which are frequently observed in patients experiencing the first episode of malaria, a stage during which they have no immunity . HoweverIn general, diagnostic methods applied in malaria-endemic areas are heterogeneous in nature. In some countries, malaria is treated after a clinical diagnosis because of limited access to laboratory tests. To this effect, sensitivity and specificity of symptoms and signals are crucial for medical care because certain symptoms can be nonspecific and may be associated with other diseases; as a result, a patient may be misdiagnosed and receive the wrong treatment, which can have detrimental effects on his or her health .Other malaria-endemic areas use rapid tests, but such tests can yield false negative results, especially in the cases of low parasitemia counts, commonly seen in patients with asymptomatic infections . In mostThroughout Brazil, thick smears or rapid tests are administered at no cost to patients, and cases are detected using either passive or active methods. Passive methods are applied when a patient seeks diagnosis and treatment after experiencing a few days of sickness. Active methods are used by healthcare workers when examining potential cases among both sick and asymptomatic individuals. The Brazilian Ministry of Health  recommenPlasmodium infection during a short prodromal asymptomatic period or during a long-lasting asymptomatic case , rural or riparian (37.9%), and indigenous (4.8%) areas  areas . The cit%) areas . Its mon%) areas . In 2010falciparum malaria and 70.3% vivax malaria; only 0.6% of the cases were of mixed species . The slides were then reviewed by another experienced microscopist in an alternate city.Thick blood smears were stained with Giemsa. According to Ministry of Health guidelines , at leas18S rRNA gene of human malaria parasites, as described by Venous blood was collected in sterile vacuum tubes with ethylenediaminetetraacetic acid (EDTA). Samples were centrifuged, and the sera were separated and stored at −20 °C until tested. In some cases, when the patient refused to have a venous blood sampling, digital blood from a finger prick was spotted onto FTA Micro Cards  and was prepared as recommended by the manufacturer using FTA Purification Reagent . Blood samples were also examined using nested polymerase chain reaction (PCR)-based amplification of a species-specific segment of the Symptoms associated with malaria episodes were confirmed and assessed using microscopy and PCR, as described by vivax malaria were treated with chloroquine and primaquine for 7 days, and those with falciparum malaria were treated with a combined regimen of mefloquine, artesunate, and primaquine or with a combination of artemether and lumefantrine (Coartem®) whenever possible, according to Brazil’s current malaria therapy guidelines . Parasite loads were stratified into two levels: (a) low parasitemia, ≤100 parasites in 100 fields (roughly corresponding to 1–300 parasites/µl of blood) and (b) moderate and high parasitemia, with >100 parasites in 100 fields (>300 parasites/µl of blood). Age was classified into <14 years and ≥14 years, in order to compare children and adolescents with adults. The number of previous confirmed malaria episodes was divided per ≤8 and >8 episodes, corresponding to the median number of previous episodes experienced by the subjects included in the study. The duration of symptoms in days was also divided per ≤3 and >3 days, corresponding to the median number of days during which symptoms were experienced. The distribution of the independent variables was identified using the student’s  measure  was useda priori  matrix in which each (n) line corresponds to a patient and each (p) column refers to a studied characteristic. Each patient presents a  profile, defined by his/her characteristics; likewise, a  profile can be drawn for each variable on the basis of the patients’ answers  matrix as a set of n points within a space of the p dimension, the center of gravity within the mass of data corresponds to the mean value of all profiles and therefore can be identified as the profile expected value. The distances between each point and the center of gravity represent those between observed and expected values, which are called χ2 distances .In the present study, it was expected that the graphical representation of the two dimensions would illustrate the grouping areas of the categories of variables, which were included in the analysis on the frequency of malaria-related symptoms. This would allow the identification of symptoms that are grouped according to specific characteristics , using the FactoMineR package . Written informed consent was obtained from each participant or by his/her parent prior to the study.P. vivax or P. falciparum. Samples from all patients were submitted for a molecular diagnosis, of which 171 cases revealed single-species malaria . Two cases, diagnosed as P. vivax based on a thick smear, reported a molecular diagnosis of mixed infection  and were therefore excluded from the analysis.A total of 173 malaria cases were selected on the basis of a positive thick smear, indicating either 3); 39.40% had parasitemia between 200 and 500 parasites/mm3; and only two cases (1.20%) had parasitemia higher than 10.000 parasites/mm3 . The majority of the people were born in the Amazon, and the average duration of residence in the area was 28.4 years. Only 3.03% of the participants did not have malaria prior to the study, and, for 7.88%, the malaria episode experienced during the present study was their second occurrence. Almost 50% of the patients reported having at least eight malaria episodes prior to the study. Forty percent of the cases had patent, but low, parasitemia  have more symptoms (represented in the left side of dimension 1), and those with a lower parasitemia count (<300 parasites/mm3) tend to have fewer symptoms (represented on the right-hand side of dimension 1). Finally, patients with more than eight previous malaria episodes generally experienced fewer symptoms, except for sore throat, which was more pronounced in these patients.3) was associated with the presence of fever , vomiting , dizziness , and weakness . A higher parasitemia count, however, was not associated with other symptoms. The occurrence of arthralgia  and myalgia  was associated with age, being more likely to occur in patients older than 14 years. Having experienced at least eight malaria episodes prior to the study was associated with a decreased risk of fever  and chills  and with an increased risk of sore throat . None of the symptoms showed an association with gender, Plasmodium species, or duration of symptoms.Using the Wald test for logistic regression , a higheSevere headache, myalgia, chills, and fever were observed in more than one third of the cases, but all the other symptoms were mild or moderate . There wP < 0.005). A higher parasitemia count was associated with more intense fever (P < 0.001), while myalgia was more intense in patients over the age of 14 years (P < 0.001)  .P = 0.002), headache (P < 0.001), nausea (P < 0.001), abdominal pain (P < 0.001), diarrhea (P < 0.008), and arthralgia (P = 0.032), which tended to be more intense in patients with shorter durations of illness than in those experiencing the disease for more than 3 days.When analyzing the intensity of symptoms with the duration of symptoms, there were associations with chills, fever, sweating . These symptoms have been widely reported in many clinical and experimental studies. The remaining three clusters focus on respiratory symptoms and diarrhea, vomiting, and pallor.In this study, headache was the most frequent symptom, a finding that has been reported in previous studies of African children  and pregThe analysis of symptom intensity showed that a higher parasitemia count is associated with higher fever, and a lower parasitemia count is associated with mild fever or, sometimes, an absence of fever. Studies have reported a relationship between parasitemia and symptom intensity . The lacArthalgia and myalgia were common and were more likely to occur in patients over the age of 14 years. Other studies  have convivax malaria. In a study of 15 adults with uncomplicated vivax malaria, a total of 40% reported limited dry cough   . Thus, dx cases) .Nausea, inappetence, bitter mouth, and abdominal pain were relatively frequent (up to 68.5%); however, few studies have cited them as frequent symptoms. In the present study, both the frequency and intensity of certain symptoms varied by past exposure to malaria. The higher the number of previous malaria episodes, the less intense the presentation of certain symptoms, such as fever, chills, and sweating. The data suggest that previous episodes confer some amount of clinical immunity, and symptoms can therefore become less pronounced.P. vivax (The quantification of symptoms in nonsevere malaria is of increasing importance, given that several vaccines are being tested on human subjects. These vaccines may reduce the intensity of symptoms, and thus the optimization of an algorithm to assess symptoms can provide better estimates of vaccine efficacy, especially in the case of developing a vaccine for P. vivax .vivax and falciparum malaria, although a limited number of P. falciparum cases were assessed. However, falciparum malaria showed higher incidence rate of fever than vivax malaria; however, this was related to the parasitemia levels. In areas where P. vivax and P. falciparum co-exist, the clinical pictures may overlap, making it impossible to differentiate between the two parasites without diagnostic tests.Nevertheless, this study has certain limitations. We were unable to identify symptoms that could differentiate noncomplicated Plasmodium species. This information is important for healthcare workers in endemic areas who perform both passive and active diagnoses. In addition, a complete assessment of malaria-related symptoms is useful for the diagnosis of malaria in travelers returning to nonendemic areas. The evaluation of effects of the parasite-host relationship on clinical presentation can be useful for studies assessing immunological response and the development of vaccines aimed at decreasing symptom severity. New statistical techniques, such as multiple correspondence analysis, as shown in this study, can help to identify groups of patients with similar clinical presentation according to variables of interest, in malaria studies as well as in studies of other diseases.Malaria is a complex disease that presents with multiple symptoms. The nonsevere form features a broad range of symptoms that can be grouped into clusters, with some symptoms appearing more frequently than others. The existence and intensity of symptoms are related to the patient’s age, as well as to parasitemia level and exposure to the 10.7717/peerj.1325/supp-1Supplemental Information 1Click here for additional data file.


Plasmodium species that cause human malaria, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, are transmitted between humans by mosquito vectors belonging to the genus Anopheles. It has recently become evident that Plasmodium knowlesi, a parasite that typically infects forest macaque monkeys, can be transmitted by anophelines to cause malaria in humans in Southeast Asia. Plasmodium knowlesi infections are frequently misdiagnosed microscopically as P. malariae. Direct human to human transmission of P. knowlesi by anophelines has not yet been established to occur in nature. Knowlesi malaria must therefore be presently considered a zoonotic disease. Polymerase chain reaction is now the definitive method for differentiating P. knowlesi from P. malariae and other human malaria parasites. The origin of P. falciparum and P. vivax in African apes are examples of ancient zoonoses that may be continuing at the present time with at least P. vivax, and possibly P. malariae and P. ovale. Other non-human primate malaria species, e.g., Plasmodium cynomolgi in Southeast Asia and Plasmodium brasilianum and Plasmodium simium in South America, can be transmitted to humans by mosquito vectors further emphasizing the potential for continuing zoonoses. The potential for zoonosis is influenced by human habitation and behavior as well as the adaptive capabilities of parasites and vectors. There is insufficient knowledge of the bionomics of Anopheles vector populations relevant to the cross-species transfer of malaria parasites and the real extent of malaria zoonoses. Appropriate strategies, based on more research, need to be developed for the prevention, diagnosis, and treatment of zoonotic malaria.The four main  Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax, transmitted between humans by mosquito vectors of the genus Anopheles are primarily responsible for human malaria. The World Health Organization in its latest malaria report  from chimpanzees  monkeys in Southeast Asia, has been known to be possible for half a century  and circumsporozoite protein (csp) was essential for correct species identification in Kapit , Macaca nemestrina (the pig-tailed macaque), Trachypithecus obscuras (dusky leaf monkey or spectacled langur), and Presbytis melalophus (banded leaf monkey or brown langur) [Ref.  [Ref.  and refeys [Ref.  and refeys [Ref. ]. Within in Ref. , 20, 21]n region . BecauseP. knowlesi inoculations have been used in the past to treat neurosyphilis  and cytochrome b (cytb) and the ssrRNA and csp genes from worldwide isolates of P. vivax and a limited number of P. simium isolates from South America showed that the two parasites were very closely related  It was suspected that platyrrhine howler monkeys (Alouatta fusca) were the original source of the infection, and Anopheles cruzi the vector, in this instance of zoonosis. It was suggested that human P. simium infections contracted in forests may often be misdiagnosed as P. vivax during routine microscopy due to the presence of similar patterns of stippling or Schuffner’s dots in infected red blood cells, and because many microscopists might be unaware of the possibility of simian malaria parasites infecting humans  parasite Plasmodium reichenowi  in West and Central African forests. Analysis of the sequences of the mitochondrial cytb showed that the P. falciparum-related ape parasites could be phylogenetically classified into six host-specific clades  and nuclear lactate dehydrogenase (ldh) genes, and the entire mitochondrial genome, yielded a phylogenetic relationship compatible to that obtained with cytb. Therefore, P. falciparum most probably arose through the cross-species transfer of an ancestral P. praefalciparum-like parasite from gorillas that then successfully adapted itself to humans  and squirrel (Saimiri spp.) monkeys are also infectible with P. falciparum, but a period of adaptation in splenectomized animals is required , from parasite isolates in different parts of the Old World showed that genetic diversity in P. falciparum decreased significantly with the distance from sub-Saharan Africa  and/or ancestral Homo species to whom P. falciparum had become adapted.Analysis of single nucleotide polymorphisms (SNPs) in two n Africa . This fiPlasmodium parasites genetically very similar to human P. vivax are common in wild chimpanzees (P. troglodytes) and western (G. gorilla) and eastern gorillas (Gorilla beringei) but not bonobos (Pan paniscus) or monkeys  also produced results that were consistent with this conclusion  serological phenotype in homozygous individuals. The Fy(a-b-) phenotype has been commonly associated with near complete resistance to P. vivax in most of Africa  parasite Plasmodium rhodaini was very similar, if not identical, to the human quartan malaria parasite P. malariae, with both parasites being readily transferable between humans and chimpanzees [reviewed in Ref.  will facilitate identification of zoonotic infections as already demonstrated for P. vivax  that caused the 2009 influenza pandemic. It contained many genetic re-assortments with common swine influenza virus genes that resulted greater pathogenicity to humans -based diagnostic techniques  that canP. vivax and P. malariae in South America and Africa, can compromise malaria control and eradication efforts. This needs to be recognized by the public health authorities responsible for malaria control. In a broader context, the agencies responsible for health and environmental planning in the tropics need to be aware of the present and likely future changes in levels of exposure to zoonotic malaria and develop appropriate mitigating and preventive strategies.Zoonoses, particularly the existence of sylvatic reservoirs of The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.