As part of malaria characterization study in the South-Tongu district of Ghana, the current study was conducted to explore relationships between malaria, schistosomiasis, soil transmitted helminths and malnutrition in riparian community settings that had hitherto encountered episodes of mass deworming exercises.Schistosoma haematobium. Blood samples were analyzed for malaria parasites and haemoglobin (Hb) concentrations, respectively. Anthropometric indices were measured. Relationships were determined using generalized linear models.School-age children were enrolled in a cross-sectional study from April through July 2012. Stool and urine samples were examined respectively for helminths and Plasmodium falciparum  and S. haematobium  infections. The associations between significance terms in the multivariate analysis for P. falciparum infections were further assessed to test the significance of the product terms directly i.e., age in years , Hb concentration , and stunted malnutrition . The P. falciparum-associated decrease in mean Hb concentration was 2.82 g/dl  in stunted children, and 0.75 g/dl  in the non-stunted cohort. The anaemia-associated decrease in mean parasitaemia in stunted children was 3500 parasites/µl of blood , and in non-stunted children 2127 parasites/µl of blood . Stunted malnutrition was the strongest predictor of S. haematobium infection  but significant associations as described for P. falciparum infections were absent. The population attributable risk of anaemia due to P. falciparum was 6.3 % (95 % CI 2.5–9.3), 0.9 % (95 % CI 0.4–2.3) for S. haematobium, and 12.5 % (95 % CI 9.11–19.52) for stunted malnutrition.The results show low numbers of asymptomatic Plasmodium falciparum, S. haematobium, intestinal helminths and their co-infections were uncommon in our school-age children. Stunting exacerbated the extent to which malaria was associated with loss in Hb concentration.The online version of this article (doi:10.1186/s13104-016-2025-3) contains supplementary material, which is available to authorized users. The South Tongu District is one of the 25 districts of the Volta region of Ghana drained by the Volta river and its tributaries . In rece2 located on Latitude 5°58′37.2′′ and Longitude 0°38′49.2′′. Rainfall is bimodal with latter rains in September to November [Plasmodium falciparum. Malaria transmission occurs throughout the year with peaks during the two rainy seasons.The study was conducted during the first annual rainy season (April through July) of 2012 in the South-Tongu district, an area with seasonal malaria in Volta region, Ghana , 12. TheNovember . The maiNovember , 15. TheThe target population comprised school-age children ages 6–13 years) without manifestations compatible with malaria in the past 14 days. Children on nutritional supplements were excluded. Four primary schools across the district namely Dabala Evangelical, Tefle Presbyterian Church of Ghana, Sogakope Cuniberto, and the Tsavanya district assembly basic in Agbakope were selected . Ten millilitres of urine samples were examined for S. haematobium eggs using the nucleopore filtration method. The intensity of S. haematobium infections was calculated as the number of ova per 10 ml of urine. For haemoglobin (Hb) measurements and malaria testing, approximately 3 ml of blood samples were collected from each pupil. Thick blood smears were prepared, stained with Giemsa and examined microscopically for detection, identification and quantification of malaria parasites. Parasite densities were determined with absolute white blood cells (WBC) counts as a ratio of P. falciparum counts relative to 200 WBC in thick films. Hb concentrations were determined using the cyanmethaemoglobin method. Briefly, blood aliquots of 0.02mls were put into five milliliters of Drabkin’s solution to make a 1 in 250 dilution. The solutions were well mixed and incubated in the dark for 10 min before Hb estimations (g/dl) in a colorimeter at 540 nm wavelength. Quality control was performed by randomly selecting and microscopically re-examining 10 % of blood, urine and stool preparations by an experienced independent technician blinded to all previous results.Children were provided with plastic containers and requested to bring about 3 g of stool and 20 ml of urine at 10:00 a.m. in the morning. Stool samples were processed within 6 h of collection and examined microscopically within 1 h of preparation. They were examined for P. falciparum, EPG of faeces for intestinal helminths, and eggs per 10 ml of urine for S. haematobium. Children were classified as stunted, underweight or wasted when their HA, WA or WH Z-scores were <−2 below the reference mean, respectively. A child was identified as being malnourished with a score of <−2 in 1 of the HA, WA, or WH assessment [P < 0.05. The proportion of anaemia cases attributable to malnutrition or to specific parasitic infections was estimated as population attributable risk percentages (PAR%) [s) and their coefficient of determination (r2 or rs2). Univariate comparisons between study outcomes and covariates were computed with Chi square tests and unadjusted odds ratios (OR) at 95 % confidence interval (CI). From univariate analyses, variable with a P < 0.05 were analyzed in multivariate logistic regression models to identify independent risk factors. Predictive accuracy of the models was assessed by Hosmer and Lemeshow goodness-of-fit test with P > 0.05 indicating that the model predicts accurately on average. The area under the receiver operating characteristic (ROC) curve >0.7 was used to evaluate the discriminatory capability of models. Multiple linear regression was used to compare stunted and non-stunted children regarding their associations between malaria, hemoglobin concentration, anaemia, P. falciparum parasitaemia, and S. haematobium egg count. Effect size determination and possible interaction of the variables were computed with analysis of covariance (ANCOVA).Data from interviews and parasitological investigations were captured into Microsoft Excel database, and exported into Statistical Package for Social Sciences  for editing and statistical analyses. Missing data were excluded from analysis. Any anaemia was defined as Hb <12 g/dl; severe anaemia, Hb <7 g/dl; moderate anaemia, Hb 7 to <10 g/dl; and mild anaemia, Hb 10–11 g/dl. Based on positive samples only, infection intensities were calculated as parasites per microlitre of blood for sessment , 18. Pois (PAR%) , 20. DesOverall 591 SAC were available for sampling based on parents/guardians informed consent. One-hundred and eighty seven of these were excluded from the study. Reasons for exclusion included absence from school (n = 51), unwillingness to provide blood, urine and stool samples (n = 46) and the refusal of children to assent (n = 74). A total of 404 children including 198 girls and 206 boys in the south-Tongu district of Ghana were enrolled. The mean age of SAC was 8.2 years (range 6–13 years); and about 94 % had received antihelminthic drugs in the previous 3 months.P. falciparum accounted for all infections. Ten children  harboured S. haematobium ova. None of the children was identified with intestinal helminths nor suffered from co-infections with multiple species or different parasites. About 22 % of the children were stunted with a mean HA Z score of −1.90 ± 1.23; whilst 21.5 % were underweight with a mean WH Z score of −0.88 ± 0.57. The prevalence of wasted children was 8.4 % with a mean WA Z score of −2.04 ± 0.9. About 295 (73.0 %) of the children suffered from anaemia—5.7 % (n = 17) of which were severe.Table P. falciparum infected children, the mean parasitaemia was 4641 parasites/µl (95  % CI 1812–8973) of blood. About 24.3 % (n = 9) of the children had low P. falciparum parasitaemia <500 parasites/µl of blood and more than half  had moderate parasitaemia ranging between 500 and 10,000 parasites/µl of blood. A few participants  had high parasite count >10,000 parasites/µl of blood. Of the ten infected by S. haematobium, six had light infections (<50 ova per 10 ml urine) with a mean of 37 ± 12. Four children suffered heavy S. haematobium infections (>50 ova per 10 ml urine) with egg intensity of 103 ± 21. The mean ova count for S. haematobium infections was 63 ± 24.Among P. falciparum infection: older age (11–13 years), being malnourished and stunted, use of repellents as protection against malaria, use of river as source of household water, child’s activity in river, travel in past 4 weeks, and high parent/guardian education. The same variables were significantly associated with S. haematobium infection, with the exception of recent travel. However, no anti-helminthic drug use in past 3 months was also significantly associated with S. haemtobium infection.From Table P. falciparum infection. Also, a unit increase in Hb concentration was determined to be putatively protective against P. falciparum infection . Stunted malnutrition was the strongest predictor of S. haematobium infection . The frequency of a child’s activities per week in river was identified as an independent risk factor (AOR = 1.68 per one activity increase) for S. haematobium infection—with infected children engaging in river activities almost twice as frequent as those uninfected. Reported use of anti-helminthic drug in the past 3 months was found to be 87 % protective against S. haematobium infection.In adjusted models Table , older aP. falciparum infections were further assessed to test the significance of the product terms directly i.e., age, Hb concentration and stunted malnutrition. Figure P. falciparum infections among all study children—the trend of infections declined with increasing age . When data was adjusted for stunting, the frequency of infections showed no tendency to increase or decrease with age . Meanwhile, a significant drift towards lower parasitaemia with increasing age was observed in non-stunted children  . Among the infected children, the stunted group showed higher (P < 0.001) mean parasitaemia level (3218 ± 1184 parasites/µl) than their non-stunted counterparts (2064 ± 1287 parasites/µl). We observed within the stunted cohort a significant correlation between Hb concentration and parasite density ; such association was also present in the non-stunted cohort albeit at a slower rate   in stunted children, and 0.75 g/dl  in the non-stunted cohort. Of the P. falciparum infected children, 93.7 % (n = 15/16) of those stunted were anaemic whiles 90.4 % (n = 19/21) of the group without stunting suffered from anaemia. Among the stunted and non-stunted children, the anaemia-associated decrease in mean parasitaemia were 3500 parasites/µl of blood  and 2127 parasites/µl of blood  respectively , and 12.5 % (95 % CI 9.11–19.52) for stunted malnutrition.Figure S. haematobium infection was 0.9 % (95 % CI 0.4–2.3). When we compared the associated effect of S. haematobium infection and Hb concentration between the two cohorts, S. haematobium-associated decrease in mean Hb concentration was 0.6 g/l  in stunted children; and 1.2 g/l  in those without stunting .The PAR % of anaemia attributable to P. falciparum infections with largely low to moderate level parasitaemia. About half of the affected suffered from stunted malnutrition with a high proportion of moderate to severe anaemia. The prevalence of urinary schistosomiasis was 2.5 %, with low to high ova counts for S. haematobium—the principal manifestations were stunted malnutrition and moderate anaemia. For two reasons, our findings of low malaria levels deserve particular attention. First, P. falciparum accounted for all malaria cases reported here, and the level of infection using comparative methodology was lower than that reported for other malaria-affected children in Ghana [P. falciparum infections were observed between the various community schools. Taken together, the findings should not be considered as an artefact of a single study period but as results which perhaps points to a temporally stable infection rate among SAC in the South-Tongu district. The results is also in keeping with previous mass deworming, malarial prevention and schistosomiasis control exercises in this region [The study showed low numbers of asymptomatic in Ghana , 21, 22,in Ghana  and in min Ghana , 25. Secs region . AlthougP. falciparum infections. Stunted malnutrition was also present after assessing data separately for S. haematobium infections. Stunting is a manifestation of recent and acute under nutrition. Children who are stunted are thought to have increased predisposition to malaria and other parasitic infection for a variety of reasons, most notably through a reduction in the function of T-lymphocytes, deficiencies in antibody formation, diminished complement formation, and degeneration of thymus and other lymphoid tissues [P. falciparum infection and corresponded to an AOR of 0.71 . Meanwhile, children with stunted malnutrition showed higher parasitaemia levels that negatively correlated with lower Hb concentration. The PAR % of anaemia attributable to P. falciparum remained about twofold lower than that of stunted children who accounted for approximately one-eighth of anaemia cases. The data corroborates with our other results regarding stunted-malnutrition-associated decrease in Hb concentration among malaria affected children (Fig. P. falciparum affected children. It is noteworthy that although these observations with stunting were not apparent in schistosomiasis affected children, a clear evidence that stunting is associated with increased prevalence in malarial infection is beyond the scope of this study.In multivariate analysis, the reported usage of antihelminthic by study respondents was determined to be protective against urinary schistosomiasis. The study also showed that stunted malnutrition was independently associated with increased odds of  tissues , 22, 27.P. falciparum, S. haematobium, Hb concentration and stunted malnutrition is considered in Fig. It is conceivable that malaria may cause stunting. Yet given that stunting becomes manifest only after a prolonged period of nutritional insufficiencies, and that malaria infection is a transitory state, we consider it more likely that host immunity impairment which results from conditions such as Hb deficiency exacerbate host vulnerability to malaria , 22, 27.P. falciparum and S. haematobium. Last, other haematological markers such as serum C-reactive protein and soluble transferrin receptor concentrations were not determined [There are some potential limitations of this study that must be discussed briefly, and for which our data must be interpreted carefully. First, the studies for helminths used only one stool and urine sample from each child; hence, the proportion of children with low-intensity infections could have been misclassified as uninfected , 28. Thetermined . The effP. falciparum infections.The silver lining to this study is the fact that our data shows malnourishment that cause stunting may also exacerbate the extent to which malaria is associated with deficiency in Hb concentration , 22, 27.


Plasmodium falciparum.A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools , a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP.ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.These results suggest natural malaria transmission induces CelTOS-specific The online version of this article (doi:10.1186/s12936-014-0539-5) contains supplementary material, which is available to authorized users. Current malaria control strategies combine identification and elimination of vectors with prompt diagnosis and treatment of infected individuals in endemic populations. Various insecticides have been applied for the control of the mosquito vectors, and chemoprophylaxis is used to prevent blood stage infection associated with the clinical symptoms of malaria. These disease control measures are however being hampered by the resistance of parasites and mosquito vectors to drugs and insecticides respectively . VaccinePlasmodium falciparum cell-traversal protein for ookinetes and sporozoites (CelTOS) is required for motility of the parasite in both the mosquito vector and the human host, and is required for successful malaria infections [in vitro, and induced sterile protection in test animals [ex vivo ELISpot assays using peripheral blood mononuclear cells (PBMCs) from irradiated sporozoite-immunized volunteers [The malaria parasite has a complex life cycle and it is believed that an effective anti-malarial subunit vaccine may need to target antigens in multiple stages of the parasite. The fections . Anti-Ce animals ,8. Moreolunteers . There ax vivo ELISpot assays have previously been shown to be capable of detecting recall IFN-γ responses from adults in Ghana using HLA-matched DR- or class I-restricted peptides derived from the circumsporozoite protein (CSP), thrombospondin-related adhesion protein (TRAP), liver stage antigen-1 (LSA1), liver stage antigen-3 (LSA3) and exported protein-1 (EXP1) [ex vivo ELISpot assays, and found that using positivity criteria defined as at least a two-fold difference comparing test samples and medium controls, and at least a difference of 10 spot forming cells (sfc) per million PBMC between test samples and medium controls, led to 60% reproducibility [ex vivo ELISpot assays, using the positivity criteria based on that study [E1 (EXP1) . MoreoveThis study was conducted according to a human use protocol “Quality Control of Immunological Reagents and Validation of Improvements to Immunological Assays in Support of Malaria Vaccine Trials” approved by Intuitional Review Boards at the Noguchi Memorial Institute for medical Research (NMIMR) and the Naval Medical Research Center (NMRC). NMIMR holds a United States Government Federal Wide Assurance (FWAA00001824) from the Office for Human Research Protections, as does NMRC (FWA00000152). NMRC also holds a Department of Navy Addendum to the FWA for human subject protections. Written informed consent was sought from all study participants who willingly agreed to be part of the study and met the inclusion criteria.2 for a maximum of 20 h before use in ex vivo ELISpot assays.The study was conducted within the University of Ghana, Legon and its surrounding communities in Accra, Ghana. Legon is an urban community that lies within latitude 5.65 (5° 39′ 0 N) and longitude −0.18 (0° -11' 0 W). It is about 10 kilometres north from the capital city, Accra. It is home to the University of Ghana, and a 10 square km area around Legon has an approximate population of 100,000. The average annual rainfall in the study area is below 1000 mm and more than half of this figure is usually recorded between April and June. Malaria transmission follows the pattern of rainfall and most malaria admissions to the health facilities occur between May and August. Malaria transmission in the study area however occurs mainly along the peri-urban fringes that have suitable breeding sites for the mosquito vector. Malaria slide positivity in the study area is usually below 1% for most of the year [Synthetic 15mer peptides that overlap by 11 amino acids and span the full length CelTOS, CSP, AMA1, TRAP, the 42 kDa fragment of MSP1 and truncated C-terminal portion of LSA1, all 3D7 strain, were synthesized by Mimotopes, VIC, Australia (>80% purity). CelTOS peptides were grouped into four CelTOS peptide pools  or combined into one pool (CelTTp) . Subsequently a pairwise post hoc test for differences in proportions was performed when statistically significant differences were observed in proportions of positive responders to the various antigens. Statistical analysis and graphics were done with Graph Pad prism  and the R statistical package . A p value less than 0.05 was considered statistically significant.Activities were calculated as spot forming cells per million PBMCs (sfc/m). The assay was considered positive if there was (1) at least a doubling of sfc/m in test wells relative to control wells, and (2) a difference of at least 10 spots between test and control wells, based on our previous studies [A total of 45 healthy Ghanaian adults were screened and 35 volunteers who met eligibility requirements and gave informed consent participated in the study. Volunteers were between 24 and 43 years with an average age of 29 years. All female volunteers tested negative for pregnancy and all volunteers were negative for malaria parasitaemia by light microscopy and malaria RDT. All volunteers made positive IFN-γ responses to Con A or CEF or both  to the CelTOS 15mer peptide pools CelTp1, CelTp2, CelTp3 and CelTp4 is shown in Figure IFN-γ responses to peptide pools representing the six tested malaria antigens CelTOS, CSP, AMA1, LSA1, TRAP and MSP1 are shown in Figure Comparison of responder frequencies to peptide pools from the six malaria antigens tested showed statistical significance in differences between at least two of the six peptide pool responses . Post-Hoc pairwise comparison revealed that the proportion of positive responses against CelTTp was not significantly different from that against any of the other five antigen pools . However, very little is known about naturally induced immune responses to CelTOS in a population with exposure to P. falciparum. A previous study with volunteers aged one to 30 years and living in a low malaria transmission area of southern Ghana showed very low levels of naturally induced CelTOS-specific antibodies [P. falciparum parasites, which was previously confirmed by seropositivity to erythrocytic stage antigens [The development of an effective anti-malarial vaccine is an important public health priority as it will add to the currently available disease control tools. Subunit vaccine development requires the identification of conserved immunodominant antigens from multiple stages of the parasite in order to ensure broad protection and curtail disease transmission. CelTOS is a pre-erythrocytic malaria vaccine candidate antigen that has been shown to induce potent antibody and T cell responses and prevent the establishment of blood stage infection in mice -8. CelTOlunteers . Moreove berghei ,7. CelTO berghei ,13, and tibodies . The aimantigens . Howeverantigens .Four pools of 15mer peptides that together span the entire amino acid sequence of CelTOS were tested against PBMCs from 35 study volunteers. Fourteen percent 14%) of volunteers made positive responses to the four CelTOS peptide pools, and all positive responses were to the first three peptide pools  with no response to CelTp4  have been mapped to the C-terminal region of the protein sequence that has been predicted to have immunodominant B cell epitopes [Ex vivo ELISpot analysis of mouse PBMCs showed a concentration of immunogenic T cell epitopes at the C terminal end of CelTOS, although murine HLA-restricted CD4+ and CD8+ epitopes were predicted by bioinformatics tools to be distributed over the entire antigen in the same study [These results show that peptide pools from the N-terminal end of CelTOS  induced responses whereas the C-terminal peptide pool (CelTp4) did not (Figure epitopes  and may me study . It is pme study , there ime study .In these studies the proportion of positive volunteers was greatest to the blood stage antigen MSP1 but statistically, only the difference between responders to MSP1 on the one hand and CSP and TRAP on the other hand was significant Figure , Table 2ex vivo IFN-γ responses as HLA-matched single peptides. In that earlier study the highest frequency of responses was to LSA1 [In a previous study, short Class I-restricted and DR-restricted long peptides representing CSP, TRAP, and LSA1 were used as stimulants and these matched the HLA of volunteers in Ghana ; in tota to LSA1  and MSP1P. falciparum. The proportion of volunteers who responded to the CelTOS antigen was not significantly different from the proportions that responded to other established malaria vaccine candidate antigens. Immune responses to CelTOS may block the development of pre-erythrocytic parasites and have the potential to protect malaria endemic populations by reducing or preventing infection of liver cells by sporozoites, thereby reducing disease severity. Since natural transmission and immunization with irradiated sporozoites induce cellular IFN-γ responses to CelTOS, these findings support further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine. It has been shown previously that two vaccine candidate antigens, CSP and AMA1, combined in a DNA-prime, adenovirus-boost vaccine regimen induced complete protection that was primarily mediated by CD8+ T cell IFN-γ responses in 4/15 volunteers to malaria challenge [In summary, CelTOS-specific T cell responses were detected in five out of 35 study volunteers who have a history of exposure to hallenge ,24. It iNatural malaria transmission in an endemic area in Ghana induces cellular IFN-γ responses against CelTOS in adults that were similar to responses against other pre-erythrocytic antigens CSP, TRAP and LSA1. These results support further development of CelTOS as a malaria vaccine, either alone or in combination with other antigens.


Haemophilus influenzae (Hib), diphtheria (DT), hepatitis B (Hep B) and tetanus toxoid (TT).Parasitic infections are prevalent among pregnant women in sub-Saharan Africa. We investigated whether prenatal exposure to malaria and/or helminths affects the pattern of infant immune responses to standard vaccinations against Schistosoma haematobium worm antigen (SWAP), and filaria antigen (BMA) were also assessed. Three immunophenotype categories were compared: i) tolerant ; ii) sensitized ; or iii) unexposed .450 Kenyan women were tested for malaria, schistosomiasis, lymphatic filariasis (LF), and intestinal helminths during pregnancy. After three standard vaccinations at 6, 10 and 14 weeks, their newborns were followed biannually to age 36 months and tested for absolute levels of IgG against Hib, DT, Hep B, and TT at each time point. Newborns’ cord blood (CB) lymphocyte responses to malaria blood-stage antigens, soluble Overall, 78.9% of mothers were infected with LF (44.7%), schistosomiasis (32.4%), malaria (27.6%) or hookworm (33.8%). Antenatal maternal malaria, LF, and hookworm were independently associated with significantly lower Hib-specific IgG. Presence of multiple maternal infections was associated with lower infant IgG levels against Hib and DT antigens post-vaccination. Post-vaccination IgG levels were also significantly associated with immunophenotype: malaria-tolerized infants had reduced response to DT, whereas filaria-tolerized infants showed reduced response to Hib.There is an impaired ability to develop IgG antibody responses to key protective antigens of Hib and diphtheria in infants of mothers infected with malaria and/or helminths during pregnancy. These findings highlight the importance of control and prevention of parasitic infections among pregnant women. Haemophilus influenzae (Hib) and diphtheria (DT) antigens post-vaccination from birth to 30 months of age. This study found that the response to DT was reduced in malaria-tolerized infants, and the response to Hib was impaired in filarial-tolerized infants; by contrast, the Schistosoma-tolerized group showed no effect. Deworming campaigns must be directed towards pregnant mothers, infants, and young children to improve response to vaccination.Parasitic infections are prevalent among pregnant women in sub-Saharan Africa. Prenatal exposure to parasitic infections can generate several potential effects on fetal immune responses and affect functional antibody generation during subsequent vaccination. There is a paucity of data on the detrimental effects of chronic parasitic infections during pregnancy on the response to vaccine from birth to childhood. This paper highlights the overwhelming presence of helminth infection and malaria in pregnant women in rural Kenya. The study shows that the presence of single and multiple antenatal parasitic infections is associated with impaired infant IgG levels against  Haemophilus influenzae type b (Hib), and typhoid vaccine efficacy in the presence of malaria infection [Vaccine-preventable diseases (VPD) continue to kill an estimated one to two million children each year, comprising about 14% of global mortality in children under 5 years of age . Vaccinanfection , 17.sensitized”, with cord blood lymphocytes (CBL) producing IFNγ, IL-2, IL-4, and IL-13 to parasite antigen challenge) and those that have a suppressed response  [H. Influenzae type B, diphtheria toxoid, tetanus toxoid, and hepatitis B virus vaccination.The immune consequences of parasitic infections can be reflected in the unborn children of infected mothers. Prenatal exposure to parasitic infections can generate a number of effects on fetal immune responses, and can affect functional response to post-partum vaccination, as we and others have shown for BCG –21. Overallenge) , 32. TheApproval for the study was obtained from the Kenya Medical Research Institute National Ethical Review Committee and from the Institutional Review Board for Human Studies at University Hospitals of Cleveland Case Medical Center. Mothers provided written informed consent for their own participation and that of their infants.Healthy pregnant women and their offspring born at the Msambweni District Hospital on the south coast of Kenya were enrolled in this mother-child cohort study. Mothers underwent a detailed questionnaire that queried their education level, spouse’s occupation, and household income. Women enrolled in the study were given malaria prophylaxis consisting of two single doses of sulfadoxine–pyrimethamine (SP) at the beginning of the second and third trimester, respectively, of pregnancy, and a single dose of albendazole (400mg) in accordance with recommendations from the Kenya Ministry of Health. Mothers and children were also examined and tested for parasitic infections at times of any intercurrent acute illnesses during the follow-up period, and treated appropriately.Pregnant women provided venous blood, urine, and stool at their first antenatal clinic visit and again at delivery. For the mother-infant pairs, maternal venous blood, placental intervillous blood, and umbilical cord blood were collected at delivery, as previously described . Infant Plasmodium falciparum (Pf) by real time quantitative PCR (RTQPCR) [in utero if one or more of the blood smear preparations or RTQPCR results were positive from antenatal, placental, or cord blood testing. A newborn was considered “not exposed” when both diagnostic tests were negative on all specimens. Stool and urine were also obtained from infants at every 6 month visit and examined for the presence of intestinal helminths and S. haematobium ova as described previously [S. haematobium was also assessed by ELISA detection of SWAP-specific (soluble worm antigen of S. haematobium) IgG4 antibodies in plasma samples collected [Wuchereria bancrofti infection was detected by assay for circulating microfilaria antigen in plasma samples with the use of a commercial Og4C3 antigen detection assay  and also assessed by ELISA detection of BMA-specific  IgG4 antibodies [Maternal venous blood, intervillous placental blood, cord blood, and infant venous blood were examined for malaria infection status by light microscopy. DNA was extracted from blood and tested for (RTQPCR) . A newboeviously , 20. Infollected , 20. Wuctibodies , 20.Brugia malayi antigen, (BMA), was used as the antigen marker for anti-filarial immune response, as this antigen is preserved across species and can be used to detect exposure to W. bancrofti, the filarial worm endemic to Kenya [CBMC were isolated from fresh cord blood and were cultured in the presence of malaria antigens for recall responses to malaria as described . For schto Kenya , 20. Allto Kenya .adw subtype, Fitzgerald Industries, Concord, MA, USA) or 1 µg/ml H. influenza type b oligosaccharide-human serum albumin conjugate (ATCC: NR-12268) and incubated overnight at 4ºC. After blocking and washing, diluted plasma samples were added to the plates and incubated for 1 h at room temperature. The plates were washed and alkaline phosphatase-conjugated anti-human IgG  was added at 1:1000 for 1 h at 37°C. Substrate orthophenoylenediamine (o-p-NN) was added after the final wash. The reaction was stopped by adding 5% EDTA, and absorbance was read at 405 nm with an ELISA reader. For all assays, standard sera obtained from NIBSC  were used to create standard curves for determination of the subjects’ anti-antigen IgG concentrations.Response to vaccination was determined by standard ELISAs for IgG levels against tetanus toxoid (TT), diphtheria toxoid (DT), Hepatitis B virus (Hep B), and Hib (using its polyribitol phosphate (PRP) antigen) –36. BrieTo assess potential mechanisms of altered vaccine responses in some offspring of women with parasite infection, we hypothesized that prenatal exposure to one or more parasite antigens would generate a tolerogenic immunophenotype, such that upon exposure to vaccines in early childhood, a bystander effect might alter the ability to generate a robust immune response. Children were classified as either sensitized, tolerized, or unexposed to the individual parasite pathogens studied.Schistosoma, and lymphatic filarial infection status and on their detectable Th1/Th2-type cord blood (CB) recall responses to each of these respective parasite’s antigens. The tolerant classes were defined as: i) tolerant to malaria if children had mothers with malaria  but lacked detectable Th1/Th2-type CB recall responses to malaria blood stage antigens; ii) tolerant to Schistosoma if mothers had schistosomiasis, (SWAP IgG4 and/or S. haematobiumegg positive) but lacked detectable Th1/Th2-type CB recall responses to SWAP antigens, and iii) tolerant to LF if mothers had evidence of LF, (BMA IgG4 and/or Og4C3 antigen positive) but lacked detectable Th1/Th2-type CB recall responses to BMA antigens. Sensitized infants were defined as sensitized to malaria if cord blood lymphocytes had detectable Th1/Th2-type recall responses to malaria, sensitized to Schistosoma if there was a SWAP-specific recall response in CB, and sensitized to LF if there was a BMA-specific recall response in CB; the mothers may or may not have been tested positive for the respective infection during delivery. SWAP and BMA were used at 25 µg/ml and 10 µg/ml respectively; these concentrations of antigens failed to induce an immune response in North American controls. Unexposed classes were defined as children who had mothers P. falciparum-negative, anti-SWAP-negative, and who also had a lack of CB lymphocyte responses individually to malaria, Schistosoma or LF antigens.This classification was based on their mother’s malaria, a priori based on biological knowledge and included characteristics determined at enrollment: maternal age, parity, sex of infant, education and income level of the mothers, and maternal dose of tetanus (for IgG response to TT only), and characteristics collected during the study (infant parasitic infections at their follow-up visits). Due to very low infant infection rates of schistosomiasis, LF and hookworm, only infant malaria infection was included in the model when studying maternal malaria infections. Similar models were used to investigate the effect of sensitization and tolerization on infants’ IgG responses to DT, Hep B, Hib and TT during the first 30 months of life. SAS Version 9.2 was used for all analyses. Statistically significant differences were assessed using P < 0∙05 as criterion.Demographic characteristics of participating mothers were summarized using descriptive statistics. Pearson chi-squared test was used to compare maternal infection rates among different demographic groups, including age, education, household income, ethnic group and parity. To investigate the association between maternal infections and infants’ IgG responses to DT, Hep B, Hib and TT during the first 30 months of life, mixed-effects model was used to model infants’ IgG responses over time. A random intercept and slope were included to account for between-subject variance in vaccine response over time. A quadratic function of age time was also included as a fixed effect accounting for the natural curvature of infant vaccine response during infancy and early childhood. To avoid over-fitting models, covariates were selected An overview of the enrollment and follow up of participants is presented in S. haematobium, and soil-transmitted helminths (STH). The level of maternal education was inversely related to rates of STH (P<0.0001). Mothers from low-income households had more schistosomiasis than mothers from high-income households (P = 0∙003). There was no significant difference in infection rates according to mother’s gravidity or ethnicity.The demographic characteristics of the participating mothers, along with their rates of malaria, schistosomiasis, lymphatic filariasis and soil-transmitted-helminth infections are shown in P. falciparum malaria, lymphatic filariasis (LF), schistosomiasis and/or intestinal helminths  malaria alone, ii) malaria plus one helminth and iii) malaria plus two or more helminths. As shown in increased anti-DT IgG levels in malaria-sensitized infants at 6 and 12 months  had levels of anti-Hib IgG that were significantly reduced at 12, 18, and 24 months of age  or without (black line) the selected prenatal maternal infection; Row A: maternal malaria; Row B: maternal filariasis; Row C: maternal schistosomiasis; Row D: maternal hookworm.(DOCX)Click here for additional data file.S2 FigAdjusted mean anti-Hepatitis B and anti-tetanus toxoid antibody levels from 6 to 30 months of age, according to the multiplicity of the mother’s pre-natal infections. Row A: No maternal infection (black line), one maternal parasitic infection (blue line), two maternal parasitic infections (red line), three or more maternal parasitic infections (grey line). Row B. No maternal infection (black line), maternal malaria infection alone (blue line), maternal malaria infection and one helminth (red line), maternal malaria and two or more helminth infections (grey line). Row C: No maternal infection (black line), single maternal helminth and no malaria (blue line), two helminth infections and no malaria (red line), and three or more helminth infections and no malaria (grey line).(DOCX)Click here for additional data file.S3 Figin utero (black line), exposed-sensitized (green line), or exposed-tolerized (red line), based on cord blood anti-parasite reactivity at birth; Row A: maternal malaria; Row B: maternal filariasis; Row C: maternal schistosomiasis.Adjusted mean antibody responses to Hepatitis B and tetanus toxoid antigens in infants who were unexposed to parasite infections (DOCX)Click here for additional data file.


Introduction. Asymptomatic malaria is prevalent in highly endemic areas of Africa and is new challenge for malaria prevention and control strategies. Objective. To determine the prevalence of asymptomatic malaria and associated risk factors among school children in Sanja Town, northwest Ethiopia.  Methods. A cross-sectional study was conducted from February to March 2013, on 385 school children selected using stratified proportionate systematic sampling technique. Pretested questionnaire was used to collect sociodemographic data and associated risk factors. Giemsa-stained thin and thick blood films were examined for detection, identification, and quantification of malaria parasites. Data were entered and analyzed using SPSS 20.0 statistical software. Multivariate logistic regression was done for assessing associated risk factors and proportions for categorical variables were compared using chi-square test. P values less than 0.05 were taken as statistically significant. Results. The prevalence of asymptomatic malaria was 6.8% (n = 26). The majority of parasitemic study participants had low parasite density 65.5% (17/26). Level of grade, age, bed net usage, and frequent exposure to malaria infection were associated with risk of asymptomatic malaria. Conclusion. Asymptomatic malaria was low in this study area and is associated with level of grade, age, bed net usage, and frequent exposure to malaria infection.  Malaria is one of the most widespread human parasitic diseases ranking first in terms of its socioeconomic and public health importance in tropical and subtropical region of the world, especially in sub-Saharan African and Southeast Asian countries –4.Various estimates have been made to measure the global burden of malaria. In 2010, the World Health Organization estimates more than 216 million cases of malaria and 655.000 deaths occur every year worldwide, with 106 countries at risk of malaria infection, among which 91% of deaths occurred in sub-Saharan Africa, 6% in Southeast Asia, and 3% in Eastern Mediterranean Region [Malaria is also major public health problem and obstacle to socioeconomic development in Ethiopia . About 7 Plasmodium parasites lead to the development of partial immunity for protection against further complication and, consequently, the creation of asymptomatic carrier [The major clinical features of malaria include severe anemia, cerebral malaria, hypoxia, and placental infection during pregnancy . Whereas carrier . Asympto carrier . Also, t carrier .Most of the studies of malaria depend on clinical manifestations, severity, and complication because it is the principal cause of malaria-related deaths. Researchers and clinicians have established diagnostic criteria based on the clinical manifestations upon disease onset, which have aided in forming an integrated approach to improving the management and treatment of severe malaria .Diagnosing asymptomatic malaria is not straightforward due to the obvious lack of clinical manifestations and often low level of parasites . AsymptoIn Ethiopia, the current malaria control methods largely focus on early detection of parasite on suspected individual and treatment, insecticide-treated bed nets (ITN) and indoor residual spraying. Since individuals with asymptomatic parasitaemia will not be identified by early detection and treatment programs, they may continue to serve as a source of infection for vector mosquitoes, complicating control measures . Plasmodium reservoir is maintained, with individuals who are not treated because they are not diagnosed, since they are asymptomatic. On the other hand, the diagnosing of such cases becomes difficult because of the low level of parasitemia; therefore, the present study was aimed at determining the magnitude of asymptomatic malaria and associated risk factors among school children of Sanja General Elementary School in Sanja Town, northwest Ethiopia.Asymptomatic malaria is a new challenge for national strategic plan for malaria prevention and control (2011–2015): a situation in which a humanA cross-sectional study was conducted from February 1 to March 30, 2013, at Sanja General Elementary School children aged 6 to 15 years in Sanja Town, northwest Ethiopia.The study was carried out in Sanja General Elementary School. The town Sanja is located 792 km far from the capital city Addis Ababa on the roadside to Gondar-Humera. Sanja has an altitude of 1800 m above sea level, with annual rainfall range from 800 to 1800 mm and annual temperature range from 25°C to 42°C. There are two elementary, one junior, and one high school. One health center gives service for the dwellers of the town and the surrounding areas. There is a river known as Sanja that flows throughout the year. Sanja General Elementary School is located on the west of the main road. A total of 2079  students were enrolled in the school for 2012/13 academic years. Those students, enrolled in the school during the study period, were used as study population.n = (z2p(1 − p)d2)) considering 95% confidence interval and 50% prevalence. Based on this assumption 385 study participants were selected from 2079 students. To select the study subjects, the students were first stratified according to their educational level (Grade 1 to Grade 8). Allocation of student was proportional to the number of students in each grade. Finally, the study subjects were selected using systematic sampling by using class roster as the sampling frame. Every fifth study subject was selected for the study.The sample size was determined using statistical formula  parasitological tests in district laboratory practice in tropical countries, part 2) .A person with no recent history of symptoms and/or signs of malaria who shows laboratory confirmation of parasitemia .To ensure good and reliable result, the following care was taken. Quality control of microscopy involves ensuring that good-quality Giemsa stain is used and staining procedures adhere to recognized methods. In addition, ten percent of the total slide was randomly selected and reexamined at the end by experienced laboratory technologist, who is blind to the first examination result. Double entry of data was done to maintain quality of the data, and before starting analysis, data was checked for completeness and being filled out clearly.P values less than 0.05 were taken as statistically significant.Data were entered and analyzed using SPSS 20.0  software. Descriptive statistics was used to give a clear picture of background variables. The frequency distribution of both dependent and independent variables was worked out. Multivariate logistic regression was done for assessing associated risk factors and proportions for categorical variables were compared using chi-square test. Ethical clearance was obtained from research and ethics review committee of School of Biomedical and Laboratory Sciences, University of Gondar. Before starting the actual data collection, permission was obtained from school director. Additionally, after explaining the importance of the study, an informed written consent was obtained from study participant's parent/guardian. An assent was also taken from individual school children. Those students who were positive for malaria infection were treated according to the national protocol.A total of 385 individuals were included in the study. Their mean age was 12.7 years (range 6 to 15 years) with standard deviation of 2.3. Among these, 132/385 (34.3%) were males and the rest 253/385 (65.7%) were females. Of the total 385 school children, 312 (81%) were in the age range of 11–15 years  and 6/385 (1.6%), respectively. Of the 385 school children, 13/132 (9.8%) and 13/253 (5.1%) of males and females had asymptomatic malaria, respectively. The distribution of asymptomatic malaria among each age group showed that 11/73% (15.1%) of 6–10 years and 15/312 (4.8%) of the 11–15 years were infected. Age significantly associated with both P. falciparum and P. vivax  were positive fornd 6/385 .6%, respμL; there was a significant difference in the level of parasitemia among age groups , but there was no significant difference between the sexes  had less than 500 parasites/he sexes .P > 0.05)  more likely to develop asymptomatic malaria. Similarly there was significant difference between exposure rates to malaria infection; more exposed group was 2.12 times  more likely to have asymptomatic malaria than those who were less exposed. Age and grade significantly associated with asymptomatic malaria with the risk of 1.3 times higher in lower aged and graded groups, while sex, residence, mother/guardian education status, years of stay in the study area and surroundings, previous illness of malaria, and treatment for symptomatic malaria were not associated with asymptomatic malaria ( > 0.05) . Plasmodium asymptomatically and submicroscopic asymptomatic infections below the limit for microscopic detection that can only be detected using molecular techniques [Malaria in areas of unstable transmission usually follows seasonal patterns of transmission as mosquito populations fluctuate, with the prevalence of parasitaemia at a minimum in the cooler dry season. In this time asymptomatic infections of malaria are critical, as this reservoir is likely responsible for sustaining the parasite population from one transmission season to the next . Besideschniques . Plasmodium infection was observed 26/385 (6.8%) in this geographical setting; this might be the effect of climate change on highland malaria transmission which is strongly dependent on the temperature, because temperature is known to influence transmission intensity through its effects on the population growth of the mosquito vector and on pathogen development within the vector [High prevalence of asymptomatice vector . This fie vector –27. The e vector , 29 and e vector , and thee vector . In addie vector . The dia P. falciparum was 76.9%; this is almost similar to conducted in Indonesia 80.2% [ P. vivax in this study was 23.1%, which is more or less comparable to 19.8% prevalence reported in Indonesia [Among malaria positive study subjects, the prevalence ofia 80.2%  and but ia 80.2% . Howeveria 80.2%  and Kenyia 80.2% . On the Regarding the level of parasitemia, result from this study showed that the majority of parasitemic study participants had low parasite density. This may be due to the low diagnostic sensitivity of routine microscopic examination. The level of parasitemia has significant association with older aged group; this might be due to repeated exposure of malaria infection.P < 0.05). Similar result was also reported in native Amazonian populations [P < 0.05). This finding is comparable with the study done in Burkina Faso [In this study, we had also evaluated the risk factor associated with asymptomatic malaria. An association was observed between asymptomatic malaria and the age of the children where the prevalence decreased from 15.1% among children aged 6–10 years to 4.8% among children aged 11–15 years  considering different age groups and malaria transmission level to scale up the elimination and eradication program of malaria in the country .


Malaria is a public health problem in Tanzania affecting all age groups. It is known that school children are the age group most commonly infected with malaria parasites. Their infections are usually asymptomatic, go unnoticed and thus never get treated, result in anaemia, reduced ability to concentrate and learn in school and if fallen sick may lead to school absenteeism. Effective malaria control requires frequent evaluation of effectiveness of different malaria interventions.Plasmodium parasite detection by malaria rapid diagnostic test (mRDT) and haemoglobin level assessment by Easy Touch® GHb system machine. A questionnaire was administered to assess use of insecticide-treated nets (ITNs) and anti-malarial drugs.A cross-sectional study design involving 317 out of 350 school children aged 6–13 years from five primary schools within municipality was conducted. Multistage cluster sampling and simple random sampling methods were used to obtain primary school and study participants, respectively. Finger-prick blood samples were collected for The prevalence of asymptomatic malaria was 5.4 % (95 % CI 3.3–8.6 %) and anaemia was 10.1 % (95 % CI 7.2–13.9 %). School children aged 6–9 years were more affected by malaria than those aged 10–13 years. The proportion of ITNs used was 90.6 % (95 % CI 86.3–93.9 %) while that of artemisinin combination therapy (ACT) was 71.9 % (95 % CI 66.2–77.1 %).Findings show existence of asymptomatic malaria and walking anaemia among primary school children in Morogoro municipality. The majority of school children reported use of ITNs and ACT for malaria control. These findings provide a rationale for using schools and school children to assess effectiveness of malaria interventions. Plasmodium that affect humans: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [P. falciparum infections are asymptomatic. The asymptomatic carriers do not seek treatment for their infection and, therefore, add up to a reservoir of parasites available for transmission by Anopheles mosquitoes [Malaria infection is caused by five species of parasite of the genus knowlesi . In malasquitoes .Malaria is a public health problem in Tanzania and the major cause of morbidity and mortality, accounting for about 30 % of all hospital admissions and 15 % of all hospital deaths . AsymptoThe use of artemisinin-based combination therapy (ACT) in combination with insecticide-treated nets (ITNs), long-lasting, insecticidal nets (LLINs), and indoor residual spraying (IRS) is perhaps the most aggressive methods for reducing the malaria burden in endemic regions . An obseStudies revealed that ITNs used for protection against mosquito bites have proven to be a practical, highly effective and cost-effective intervention against malaria. A decline in malaria in sub-Saharan Africa is attributed to malaria control measures, predominantly to the use of ITNs, IRS and ACT, which have been implemented on a wide scale .Malaria control efforts in Africa have been intensified in young children; this has resulted in exposure-dependent immunity and increases the risk of malaria transmission to school-aged children and above. But little is known about the burden of malaria in African school children and evidence suggests that malaria causes up to 50 % of all deaths in this age group .This study was conducted in Morogoro municipality, which is located at altitude 500–600 m above sea level and between longitude 37º–39ºE and latitude 6º–5ºS. The municipality consists of the town of Morogoro, one of the oldest towns in Tanzania, located about 223 km southeast of the capital city Dodoma, and 195 km west from the commercial city of Dar es Salaam, and has a population of 315,866. It is the capital of the Morogoro region and enjoys a mixture of warm and cool temperatures ranging between 27 and 33.7 °C in the dry/warm season and 14.2 and 21.7 °C in cold/wet season.The study was conducted in 5 out of 60 public primary schools within the municipality. The unit of analysis was primary school children both male and female aged 6–13 years randomly selected within the five primary schools.Multistage cluster sampling method was used to obtain the required primary schools and their respective classes. First stage: all 60 public primary schools in the municipality were divided into five clusters, namely (1) schools around mount Uluguru; (2) schools around swampy areas; (3) schools located in the town centre; (4) schools in peri-urban areas; and, (5) schools in squatter areas. From the five clusters, five primary schools were randomly selected, namely: Mlimani, Jitegemee, Uhuru, Kingolwira, and Mafisa A. The second stage was to select one representative class (from standard 1–6) for each selected school. Then simple random sampling technique was used to recruit 70 children in each school who were available on the sampling day. The selected children were given a written informed consent form for their parents or guardians to read and sign if they agreed their children could participate in the study. For those parents who could not read, the children were instructed to read for them and also to tell their parents to finger print in the signature area.A structured and pre-tested questionnaire was used to collect information on demographic characteristics, knowledge about the transmission and prevention of malaria, ownership and utilization of ITNs, and use of ACT of all 317 out of 350 eligible participants. To obtain an asymptomatic individual, a thorough medical history was taken of all 317 school children by asking if a child had the following features: history of fever within the last 24 h, headache and generalized joint pain plus measuring axillary body temperature <37.5 °C by thermometer. Fortunately all 317 school children showed no clinical signs and symptoms of malaria. The interviews were conducted in Swahili in a separate classroom from that used for haemoglobin and malaria examination. A finger-prick blood sample was collected after cleaning the finger surface using a sterile cotton swab saturated with 70 % isopropyl alcohol.P. falciparum histidine-rich protein II (PfHRP-II) and other Plasmodium species   type SD 05FK60.Presence of malaria parasites among asymptomatic school children was ascertained during the study survey by using multi-species malaria rapid diagnostic test  results.Data entry, storage and analysis were done by the principal investigator (PI) using SPSS version 15 software. The double entry was performed for raw data to minimize possible errors during data entry. Frequencies and cross tabulation were calculated to obtain proportions and Chi squared tests for each of the study variables. Chi square test and logistic regression analyses were done to obtain the association and relationship between independent and dependent variables. P value of <0.05 at 95 % CI was used to test the statistical significances between the dependent and independent variables. Epi Info version 3.5.4 was used to calculate the 95 % CI of proportions. Results are presented in the Tables and Figures.Ethical clearance was sought from Muhimbili University of Health and Allied Sciences before commencing the study. The Institution Review Board (IRB) reviewed the proposal of this research study and advised accordingly. After receiving research ethical clearance, other permission was requested from Executive Municipal Director (EMD) through Municipal Education Officer to allow the PI to conduct the research study on their school children. Informed consent was requested from each primary school head teacher after receiving a permission letter from Municipal Education Officer, and finally a written informed consent form was given to the selected school children. The selected school children were requested to take the written informed consent to their parents or guardians and request them to read and sign if they allowed their children to participate in the study. All selected children were instructed to bring back the signed informed consent forms. Parents/guardians who did not want their children to participate in the study were free to refuse participation. If a parent or guardian chose not to allow their children to participate in the survey, the child’s name was removed from the participant list. During data collection procedures, participants who were diagnosed with anaemia and any other health-related problems were referred to the nearest health facility for further medical management.A total of 317 out of 350 school children were recruited in the study after receiving the signed informed consent from their parents, but parents of 33 out of 350 primary school children refused to allow them to participate in this study. The mean age was 9.85 years (standard error of the mean (SE) = 0.106, standard deviation (SD) = 1.891).The overall prevalence of asymptomatic malaria on RDT was 5.4 % (95 % CI 3.3–8.6 %) and 7.6 % (95 % CI 5.0–11.2 %) were positive to microscopy as quality control. Females were more affected with asymptomatic malaria than males (OR = 1.76). School children aged 6–9 years were slightly more affected with malaria than those aged 10–13 years (OR = 0.93). In this study, it was shown that primary schools located in peri-urban areas (Kingolwira primary school) had more children with asymptomatic malaria (13 %) than those located in other parts of the municipality and their observed differences were statistically significant (P = 0.012) as shown in Table The association between asymptomatic malaria parasitaemia and demographic characteristics (sex and age groups) of primary school children was done using Chi square test (P < 0.05 at 95 % CI). From this study, being a male child seemed to be a protective factor for malaria infection . Females were slightly more affected with malaria parasites although it was not statistically significant (P = 0.293). Age group had no association with malaria infection  and not statistically significant (P = 0.742). Logistic regression was performed; no variables were statistically significantly associated with asymptomatic malaria infection.The overall point prevalence of anaemia (haemoglobin level <11 g/dl) was 10.1 % (95 % CI 7.1–14.1 %). The mean haemoglobin level was 13.4 g/dl (95 % CI 13.181–13.677 %) with minimum level of 5.0 g/dl and maximum level of 21.5 g/dl.2 = 6.582, P = 0.01).Among 317 school children, four (1.3 %) had severe anaemia (haemoglobin level <7 g/dl), 13 (4.1 %) had moderate anaemia (haemoglobin level 7–9.9 g/dl) and 15 (4.7 %) had mild anaemia (haemoglobin level 10–10.9 g/dl) as shown in Fig. In the study participants, a total of 254 (80.1 %)  school children reported to have an ITN and to sleep under it, but 63 (19.9 %) school children did not have an ITN (N = 317). Those who reported to have ITNs in their home (N = 254), were asked about their use: 207 (81.5 %)  sleep under an ITN every night, 21 (8.3 %) sleep under an ITN when fallen sick, and 26 (10.2 %) sleep under an ITN a few nights.To minimize recall bias, children were asked about the use of an ITN the night before the interview: 230 (90.6 %)  of school children reported sleeping under an ITN the night before the interview and 24 (9.4 %) reported not sleeping under an ITN the night before the interview. Males were observed to use ITNs more than females; their proportions were 92.4 and 89.3 %, respectively (P = 0.403). Moreover, school children in age group ten to 13 years were observed to use ITNs more (91.7 %) compared to the age group 6–9 years (P = 0.461) as shown in Table Primary school children were asked if they remembered suffering from malaria. Responses enabled the question about use of anti-malarial medicines. A total of 281 (88.6 %) of primary school children remembered suffering from malaria at different times of the year; 36 (11.4 %) did not remember if they had suffered from malaria (N = 317). From 281 primary school children who had suffered from malaria, 202 (71.9 %) (95 % CI 66.2–77.1 %) used ACT, 35 (12.5 %) used sulfadoxine–pyrimethamine, 13 (4.6 %) used quinine, two (0.7 %) used chloroquine and 29 (10.3 %) used other medications such as paracetamol and amoxicillin  on mRDT and microscopy, respectively, among school children in Morogoro Municipality. These findings are similar to other reports in urban cities of sub-Saharan Africa: Brazzaville 4.0 % and 3.6–7.5 % in Dakar . A natioThis study revealed that primary school children aged 6–9 years were more affected with asymptomatic malaria than the other age group. Female school children were found to be more affected than male school children. Similar finding have been documented in a study done in Nyamatongo Ward, northwestern Tanzania , malariaDemographic characteristics, such as age and sex, were analysed to find the association with asymptomatic parasitaemia. Males were less affected with asymptomatic malaria than females (OR < 1) although it was statistically not significant (P = 0.293). Age groups seemed to have no association with asymptomatic malaria (OR = 1) but statistically not significant (P = 0.742). Binary logistic regression was performed to control for any possible confounder such as ITN use in the association. Age group 6–9 years had 1.2 times more chance  to be asymptomatic malaria carrier than age group 10–13 years. Males continue to be less affected with asymptomatic malaria than females . Similar findings were obtained in Nyamatongo Ward, northwestern Tanzania which reported that males were less affected with asymptomatic malaria [Anaemia is a public health problem affecting people in both developed and developing countries with bad consequences on human health as well as social and economic development. It is a critical health concern because of its effects on growth, energy levels and immune mechanism. The overall prevalence of anaemia obtained from this study was 10.1 %  and was statistically significant (P = 0.01) to current malaria infection history (1–6 months prior). This prevalence was similar to the one reported from Kisarawe, Tanzania by Manangwa  but was low compared to the prevalence among school children in Tanga region, Tanzania, which was 79.6 % .However, there was a contrasting result in this study: the observed anaemia was not statistically significant with the prevalence of asymptomatic malaria obtained . This means that malaria is not the only cause of anaemia among school children, other causes such as nutritional anaemia, hookworm and schistosomiasis infections should be considered; a similar conclusion was reported in Tanzania’s demographic health survey . In thisIn this study, it was revealed that 80.1 %  of primary school children reported having ITNs in their bedrooms; this proportion is low compared to the general Morogoro region proportion of 91 % ITN coverage. The reason for observed difference of ITN coverage was clearly explained in Tanzania’s HIV/AIDS malaria indicator survey 2011–2012, that rural households are more likely than urban households to own an ITN . This study has the highest ITN coverage in primary school children compared to a study in Kenya which reported 44.2 % (95 % CI 42.7–45.6 %) ITN use .Frequency of use of ITNs differs among those who reported to have ITNs in their bedrooms. A majority, 81.5 %  reported using an ITN every night and the remaining few reported using an ITN when fallen sick or on a few nights. A good indicator of ITN use was in those who reported sleeping under an ITN the night before data collection day. The proportion of primary school children who slept under an ITN the night before data collection was 90.6 % (95 % CI 86.3–93.9 %), thus meeting the Roll Back Malaria/WHO artnership target that ITN coverage and use in the target community should be ≥ 80 % . This prThe proportion of ACT use observed in this study was 71.9 % (95 % CI 66.2–77.1 %) among primary school children who reported having suffered from malaria infection. This proportion is low for the Roll Back Malaria/WHO partnership target that 80 % of people confirmed to have uncomplicated malaria be treated with ACT within 24 h . A similP. falciparum has developed resistance to commonly used anti-malarials, such as chloroquine and sulfadoxine–pyrimethamine, continued use of them will lead to major challenges to malaria control strategies. Recommendations to use ACT in treatment of uncomplicated malaria should be adhered to [Another finding from this study showed that school children who currently suffered from malaria infection were 1.4 times more likely to use ACT than those who suffered malaria infection more than 2 months previously. Their relationship to ACT use was statistically not significant because some individuals still use sulfadoxine–pyrimethamine (12.5 %) and monotherapy anti-malarial drugs, such as quinine (4.6 %) and chloroquine (0.7 %). Since hered to .The significantly low prevalence of asymptomatic malaria in this study can be due to the reported high proportion of ITN and ACT use among primary school children in Morogoro municipality. Even if there was no statistical significance, school children age 6–9 years and females were the most affected groups with asymptomatic malaria than other groups . The observed prevalence of anaemia was not associated with the diagnosed asymptomatic cases . This means that malaria is not the only cause of anaemia; other factors such as hookworm, schistosomiasis and nutritional anaemia should be considered. Severe anaemia cases were also among the findings of this study even though its prevalence was low; having severe anaemia is life threatening, therefore, further study should be carried out to find the predictors of anaemia among primary school children in Morogoro municipality. School malaria surveys, if well arranged, can be complimentary to current malaria indicator surveys in Tanzania, which survey children under 5 years and pregnant women.


Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria , suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation , whereas markers of total vivax biomass correlated only with systemic inflammation . Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden biomass greatest in severe disease and capable of mediating systemic inflammatory pathology. The lack of association between total parasite biomass and endothelial activation is consistent with accumulation in parts of the circulation devoid of endothelium. Endothelial activation, associated with circulating parasites, and systemic inflammation may contribute to pathology in vivax malaria, with microvascular dysfunction likely contributing to impaired tissue perfusion. P. vivax-pLDH (PvLDH) as proxies of total parasite biomass and found that, as in falciparum malaria, the total biomass of vivax parasites is underestimated by counting parasites circulating in peripheral blood, suggesting a hidden burden of vivax parasites. Markers of total vivax biomass were strongly associated with illness-severity and inflammatory cytokines, suggesting that this hidden burden is capable of contributing to generalised inflammation and hence severe disease. Number of peripheral vivax parasites, but not total biomass, correlated with activation of endothelial cells, suggesting that the hidden vivax-infected red cells may accumulate in parts of organs without endothelium, such as the slow-circulation of the spleen or non-blood-vessel parts of the bone marrow. Severe vivax malaria was associated with increased endothelial activation and impaired microvascular function, suggesting that these processes also contribute to impaired blood flow and disease.How vivax parasites cause severe malaria is not known. In contrast to falciparum parasites, the number of vivax parasites circulating in peripheral blood is low, and there is thought to be little sequestration of parasitized red cells within endothelium-lined small blood vessels in vital organs. Total parasite burden (circulating plus hidden) and activation and dysfunction of the endothelial cells lining blood vessels all contribute to severe disease in falciparum malaria, but have not been evaluated in severe vivax malaria. We measured parasite lactate dehydrogenase (pLDH) and  P. falciparum accounts for a majority of severe and fatal malaria cases worldwide, P. vivax is a major cause of morbidity outside of Africa, causing an estimated 70–390 million malaria cases per year P. vivax is now recognized as capable of causing severe and fatal disease While P. falciparum are rarely seen in peripheral blood P. falciparum histidine rich protein-2 (HRP2). Pathogenesis and disease severity in falciparum malaria are both biomass-related: in contrast to peripheral parasitemia In falciparum malaria, severe and fatal disease is characterised by cytoadherence of parasitized red blood cells (RBCs) to activated and dysfunctional endothelium, leading to parasite sequestration with microvascular obstruction P. vivax infection than in falciparum malaria P. vivaxendothelial microvascular sequestration in vivax malaria has led to the assumption that peripheral parasitemia reflects total parasite biomass. However, this assumption has been questioned P. vivax known to be under-represented in peripheral blood P. vivax-infected RBCs in the spleen or bone marrow has been hypothesised P. vivax-pLDH (PvLDH), produced by viable or recently killed parasites, may be used to estimate total parasite biomass. While plasma pLDH has been shown to demonstrate only moderate correlation with peripheral parasitemia in vivax malaria P. vivax parasite concentration ex vivo, including all parasite stages, suggesting that pLDH reflects total P. vivax parasite biomass P. vivax schizonts and trophozoites than by ring-form parasites P. vivax stages in peripheral blood Cytoadherence of infected RBCs to endothelial cells is 10-fold less in P. vivax has been associated with greater endothelial activation than P. falciparum, with Ang-2 concentrations higher in non-severe vivax compared to non-severe falciparum malaria In falciparum malaria, clinical severity and mortality are also independently associated with impaired microvascular function We tested the hypotheses that in vivax malaria, peripheral parasitemia would underestimate total parasite biomass, and that markers of total parasite biomass would be related to systemic inflammation and clinical severity. We also determined the relationship between vivax disease severity and endothelial activation and microvascular function, with patients with non-severe and severe falciparum malaria included for comparison. We found that total vivax biomass was underestimated by peripheral parasitemia, and was associated with both systemic inflammation and disease severity in vivax malaria. Severe vivax malaria was associated with increased endothelial activation and WPB exocytosis and with impaired microvascular function, comparable to that seen in severe falciparum malaria.Streptococcus pneumoniae in one A total of 192 malaria patients and 74 healthy controls were enrolled. Malaria patients included 62 with vivax malaria  and 130 with falciparum malaria . The clinical and epidemiological features of 43 patients with vivax malaria (including 7 with severe disease) and 122 patients with falciparum malaria (including 13 with severe disease) have been previously reported In patients with vivax malaria, the median peripheral parasitemia was 2.4-fold higher in severe compared to non-severe disease , while the median PvLDH was 3.7-fold higher  and pLDH 6.9-fold higher  . After rTo estimate whether the “hidden parasite biomass” was larger in severe compared to non-severe malaria, we calculated the ratio of plasma pLDH and plasma PvLDH concentration to peripheral parasite density in vivax malaria, and the ratio of plasma HRP2 concentration to peripheral parasite density in falciparum malaria. Among patients with vivax malaria the ratios of PvLDH and pLDH to parasite density (log) were 3.7-fold and 5.7-fold higher, respectively, in severe compared to non-severe disease. The corresponding fold-increase in the ratio of plasma HRP2 to parasite count (log) in patients with severe compared to non-severe falciparum malaria was 5.4.Among patients with vivax malaria pLDH and PvLDH were strongly correlated . However there was only a modest correlation between PvLDH and parasitemia , and between pLDH and parasitemia , comparable to the correlation between parasitemia and plasma HRP2 among patients with falciparum malaria  .Among patients with vivax malaria who had detectable pLDH and PvLDH levels on enrolment and in whom longitudinal measurements could be performed (until day 3 or until undetectable), levels were undetectable by day 3 in 16/25 (64%) and 22/31 (71%) patients respectively.ex vivo assay conditions, P. vivax parasites exist as mature schizonts for an estimated 5% of their 48-hour life-cycle P. vivax parasites at ring  and trophozoite stage . Among patients with severe and non-severe vivax malaria, there were no differences between the median percentage of rings or trophozoites in peripheral blood.In Median concentrations of Ang-2 and von Willebrand Factor (vWF), the two major products of endothelial WPB release, were both increased in patients with severe and non-severe vivax malaria compared to controls , with Ang-2 nearly twice as high in severe compared to non-severe disease (P<0.0001) . The medThe median concentrations of the vWF-cleaving protease, ADAMTS13, and its activity, were lower among patients with severe and non-severe vivax malaria compared to controls , with median ADAMTS13 activity also lower in severe compared to non-severe vivax malaria (P = 0.027).Median concentrations of IL-6 and IL-10 were higher in severe and non-severe vivax malaria compared to controls .In patients with vivax malaria, peripheral parasitemia correlated with the endothelial WPB products, VWF  and Ang-2 , as well as the endothelial adhesion receptor E-selectin , and was inversely correlated with activity of the VWF-cleaving protease ADAMTS13  . ImportaWhile not associated with markers of endothelial activation, PvLDH was correlated with the systemic inflammatory markers IL-6  and IL-10 , and, in contrast to peripheral parasitemia, was inversely correlated with baseline platelet count  and with platelet nadir .Among patients with falciparum malaria, biomarkers of systemic inflammation correlated with both peripheral parasitemia and the total parasite biomass marker, plasma HRP2 . In addiIn patients with vivax malaria, Ang-2, in addition to correlating with parasitemia, correlated with the adhesion receptors E-selectin  and ICAM-1 , and inversely withADAMTS13 antigen  and activity levels , with all associations remaining significant after adjusting for parasitemia.ADAMTS13 antigen and activity levels correlated with platelet count , with this association remaining significant after adjusting for parasitemia. IL-6, elevated in vivax malaria, is known to be a major inhibitor of ADAMTS13 activity Microvascular reactivity , but not non-severe disease . In severe disease ADAMTS13 antigen and activity levels correlated with microvascular reactivity , although no association occurred in non-severe disease . No association was found between microvascular reactivity and other variables in vivax malaria, including parasitemia or biomass markers.In patients with falciparum malaria, microvascular reactivity was inversely correlated with parasitemia , and positively correlated with platelet count . Among patients with severe and non-severe falciparum malaria, no association occurred between microvascular reactivity and lactate, or between microvascular reactivity and ADAMTS13 antigen or activity. In both species there was no association between microvascular reactivity and age.Among patients with vivax malaria, 6 patients with severe and 16 with non-severe disease had NIRS repeated on day 3, with no change from baseline noted in either group (median microvascular reactivity on day 3  =  5.59 units/sec and 7.39 units/sec respectively). In severe falciparum, microvascular reactivity improved from 4.88 units/sec at enrolment to 6.51 units/sec on day 3 (P = 0.039), although no difference was noted among 37 patients with non-severe falciparum malaria P. vivax-infected RBCs. The proportions of total parasite biomass detectable in peripheral blood in uncomplicated and severe disease were of comparable magnitude in vivax and in falciparum malaria. The association between P. vivax peripheral parasitemia and total parasite biomass is modest, with total parasite biomass more strongly associated with disease severity compared to peripheral parasitemia. The smaller proportion of total P. vivax biomass detectable in peripheral blood in severe compared to non-severe disease suggests that hidden biomass is greater in severe vivax malaria and contributes to pathogenesis of disease. The association of total vivax parasite biomass with systemic inflammation suggests that the hidden biomass is capable of mediating systemic inflammatory pathology. In contrast to peripheral P. vivax parasitemia, total parasite biomass did not correlate with endothelial activation, and we therefore speculate that accumulation of P. vivax-infected RBCs may occur in parts of the circulation devoid of endothelium.Peripheral parasitemia underestimates measures of total parasite biomass in vivax malaria and circulating schizonts are under-represented in peripheral blood, suggesting tissue accumulation of a hidden biomass of P. vivax is not thought to sequester in the endothelium-lined microvasculature to the same degree as P. falciparum, limited histopathological reports show intact P. vivax-infected RBCs in the bone marrow P. vivax-infected RBCs cytoadhere with less avidity to endothelial cells P. vivax-infected reticulocytes were found in the splenic cords P. vivax-infected reticulocytes in the slow open microcirculation of the spleen, possibly through reticulocyte adherence to non-endothelial resident cells P. vivax may avoid macrophage clearance In falciparum malaria, peripheral parasitemia underestimates the total parasite biomass due to sequestration of parasitized RBCs within endothelium-lined microvasculature. While P. vivax does occur and is greatest in severe vivax malaria, and we speculate it may occur in a location devoid of endothelium, such as the slow circulation of the spleen. We found no correlation between the P. vivax biomarkers of total biomass, pLDH and PvLDH, and either of the two products of endothelial WPB release, Ang-2 or VWF, or the endothelial adhesion receptor E-selectin. In contrast, peripheral parasitemia correlated with endothelial activation  in both vivax and falciparum malaria, and with total biomass (HRP2) in falciparum malaria. These findings suggest that endothelial cells may be activated by P. vivax-infected RBCs circulating through the peripheral microvasculature, but not by the hidden P. vivax biomass. Accumulation of P. vivax in the endothelium-free open circulation of the spleen may account for these findings. The finding that P. vivax peripheral parasitemia and markers of total parasite biomass both correlated with the leukocyte-derived cytokines may reflect the ability of circulating and tissue-accumulated P. vivax to stimulate leukocytes in both peripheral blood and leukocyte-containing organs such as the spleen or bone marrow, and thereby mediating organ dysfunction secondary to systemic inflammation.The findings of our study suggest that tissue accumulation of P. vivax and P. falciparum, and endothelial activation was higher in patients with severe disease. Ang-2 concentrations were twice as high in severe compared to non-severe vivax malaria, and comparable to levels seen in severe falciparum malaria. Ang-2, which causes autocrine endothelial activation, has previously been shown to be markedly elevated in severe falciparum malaria and a consistent predictor of death in both adults Although underestimating total biomass, peripheral parasitemia was still higher in severe compared to non-severe malaria in both Microvascular function was significantly impaired in severe vivax malaria, comparable to the impairment seen in severe falciparum malaria. As previously shown in severe falciparum malaria Plasma ADAMTS13 activity was decreased in vivax malaria, with deficiency associated with both severe disease and with impaired microvascular function. ADAMTS13 is a protease that cleaves ultra-large and prothrombogenic VWF multimers (UL-VWF), and deficiency leads to accumulation of UL-VWF with resultant increase in platelet aggregation and adhesion, and microvascular thrombosis. Accumulation of UL-VWF resulting from ADAMTS13 deficiency is characteristic of the microangiopathic disease thrombotic thrombocytopenic purpura (TTP), and has been reported in patients with both falciparum and vivax malaria ex-vivo concentrations of PvLDH increase progressively during short term culture of P. vivax, indicating that PvLDH is secreted throughout the parasite life-cycle and supporting the use of pLDHand PvLDH as surrogate markers of parasite biomass. Host inflammatory response may contribute in part to the marked under-representation of P. vivax schizonts in peripheral blood, however this would apply similarly to hosts infected with other Plasmodium species with under-representation of schizonts in peripheral blood, such as P. falciparum or P. coatneyi, whose schizonts have been clearly shown to sequester in tissues Our study had several limitations. Firstly, pLDH and PvLDH have not been previously validated as biomass markers in vivax malaria. Other factors may contribute to the plasma concentrations of these markers, such as host metabolic clearance rate, distribution within the host and diffusion rates from host tissue, and natural variation in parasite expression. Release of pLDH solely at the time of schizont rupture may provide a possible alternative explanation for the association between concentrations of pLDH/PvLDH, systemic inflammation and disease severity, with schizogony also associated with an inflammatory response The small number of patients with severe vivax malaria (n = 9) limited our ability to evaluate the association between disease severity and other variables, and to assess for associations of parasite biomass and/or parasitemia that may have occurred only among patients with severe disease. In addition, with multiple comparisons we are unable to exclude the possibility that associations may have occurred by chance. However, the magnitude and direction of the associations with disease severity and with different measures of biomass are consistent, plausible, and are comparable to those in patients with falciparum malaria. Pre-antibiotic blood cultures were not performed in all our patients, and hence it is possible that as in falciparum malaria P. vivax-infected RBCs. While histological studies are needed to confirm this, we propose that a hidden P. vivax biomass is capable of mediating systemic inflammation which may contribute to organ dysfunction. The correlation of markers of total P. vivax biomass with systemic inflammation but not with markers of endothelial activation is consistent with the hypothesis that accumulation of P. vivax-infected red cells may occur in parts of the circulation devoid of endothelium, such as the open circulation of the spleen or extravascular bone marrow. In contrast only peripheral parasitemia was associated with endothelial activation and the Weibel Palade Body products Ang-2 and VWF. The association between clinical severity and endothelial activation, ADAMTS13 deficiency, thrombocytopenia, and impaired microvascular function suggests that thrombotic microangiopathy, systemic inflammation and microvascular dysfunction may contribute to pathogenesis of disease in vivax malaria.In summary, peripheral parasitemia underestimates total parasite biomass in vivax malaria, particularly in severe disease, suggesting tissue accumulation of a hidden biomass of P. vivax endemicity is low P. vivax] or >100,000 [P. falciparum] and/or creatinine >132 µmol/L); significant abnormal bleeding; hypoglycaemia (blood glucose <2.2 mmol/L); metabolic acidosis (bicarbonate <15 mmol/L or lactate >4 mmol/L); acute kidney injury ; hyperparasitemia . Healthy controls were visitors or relatives of malaria patients, with no history of fever in the past 48 hours and with blood film negative for malaria parasites.This study was conducted in Sabah, Malaysia, a region currently in the pre-elimination phase of malaria control and where Standardized history and physical examination were documented. Haematology, biochemistry, acid-base parameters, and lactate  were obtained on admission. Parasite counts were determined by microscopy, and parasite species were identified by PCR Venous blood collection in lithium heparin was centrifuged within 30 minutes of collection and plasma was stored at −70°C. Plasma concentrations of the endothelial activation markers ICAM-1, E-selectin, and Ang-2 were measured using ELISA (R&D Systems), and IL-6 and IL-10 were measured by flow cytometry (BD Cytometric Bead Array). Antigen concentrations of vWF and the vWF-cleaving enzyme, ADAMTS13, were measured in platelet poor plasma using ELISA (America Diagnostica), and ADAMTS 13 activity by fluorescence resonance energy transfer (FRET), as previously described P. falciparum) P. falciparum and P. vivax) and PvLDH (for P. vivax) were measured by ELISA Peripheral blood parasitemia was quantitated per 200 white blood cells by microscopy and expressed as parasites/µL based on automated white cell count. Parasite stage distribution was quantitated on microscopy of peripheral blood. Plasma HRP2 (for P. vivax isolates with a high proportion of ring-stages were thawed and cultured ex-vivo as previously described Text S1), indicating secretion of PvLDH by all parasite stages. In vitro parasitemia remained stable throughout the culture duration.To confirm evidence of PvLDH secretion across the parasite life-cycle, four cryopreserved 2) before and after an ischaemic stress, as previously described 2 recovery slope from release of the vascular cuff until StO2 had reached 85% of the baseline value.Microvascular function was assessed using Near Infrared Spectroscopy  which uses a probe applied to the thenar eminence to noninvasively measure microcirculatory oxygenation  or Peason's (r) correlation coefficients, depending on distribution.Statistical analysis was performed with STATA software . For continuous variables intergroup differences were initially compared using analysis of variance or Kruskal-Wallis tests depending on distribution. Student's T-test or Mann-Whitney tests were used for post-hoc pair-wise comparisons. Categorical variables were compared using χThe study was approved by the Ethics Committees of the Malaysian Ministry of Health and Menzies School of Health Research. Informed written consent was provided by all participating adults, and by the parent or guardian of any participant aged <18 years.S1 TextIn vitro production of PvLDH  and percentage of schizonts , and percentage of rings, trophozoites and schizonts (S1 Table).(DOCX)Click here for additional data file.


Because malaria takes a major toll on human health globally, the focus on how to treat it and reduce the malaria burden is understandable. But I became interested in studying the malaria parasite because of its intracellular niche in the mature red blood cell. This is a specialized host cell designed to carry oxygen to tissues, explaining its high hemoglobin content  and deformability needed to travel through the smallest of capillaries. But these cells are dead ends, without a nucleus and many cellular capacities  that pathogens need from their host cells. Also, the mature red blood cell does not internalize or reorganize its surface membrane, a process critical for host infection by most microbes. No self-respecting virus would infect a red blood cell and most bacteria, parasites, and fungi do not. And yet malaria parasites are large pathogens (~1–2 micron) that infect a relatively small-diameter dead end host cell (~7 micron). You don’t have to be a rocket scientist to infer that the parasite must play an active role in the infection process because the host cell couldn’t—but how did it happen?I decided that secretion from the parasite to the red cell must be very important and my lab would broadly study the parasite’s Golgi structure, because in most cells, this compartment makes important decisions on what cellular cargo is secreted. Moreover, while the parasite needed to make these secretion decisions for destinations bounded by its own surface membrane as well as those beyond in the red cell, there was no evidence of Golgi “stacks” (characteristic of this organelle) in this pathogen.The first three years for my lab in the early 1990s were a hard slog, with a pile of negative results, because most molecular Golgi functions found in other cells are absent in the blood stage malaria parasite. Just as deep anxiety began to set in that this was the wrong question to ask, the onion began to unpeel productively. We discovered that the parasite was not just secreting cargo to the red blood cell; it was also releasing an entire subcompartment of its rather rudimentary Golgi. There has been no looking back since then, because the parasite commits a large proportion of its genetic composition to secreting and remodeling the red cell—a fact that became a certainty after 2002 when the first malaria parasite genome was sequenced. This new understanding didn’t immediately eliminate or eradicate malaria, but it accelerated the linking of fundamental biology and mechanisms of pathogenesis in labs like mine to the discovery of new therapeutic strategies (since the parasite is increasingly drug resistant). It also eventually resulted in the establishment of collaborations with major pharmaceutical companies, e.g., Eli Lilly & Co, and private product partnerships, e.g., the Medicines for Malaria Venture.Our recent work on rare genetic disorders was initiated almost 20 years after we began our studies in malaria. But here again, we started in basic discovery, using mouse models of disease. Driven by the invariably fatal outcome that faces the small community of patients struck by the rare neurological disease, Niemann-Pick, type C (NPC), and armed with genomics, we repurposed existing drugs to improve the treatment of this disease. This effort has led to the establishment of a small pharmaceutical company , the goal of which is to develop treatments for NPC and other neurological disorders, because monogenetic rare disorders, such as NPC, provide powerful portals into the more prevalent multigenic disorders, such as Alzheimer’s and Parkinson’s diseases. This approach may also provide insights into the recent outbreak and spread of Ebola, because the virus uses the NPC1 protein to infect host cells (and deficiency in NPC1 protects against infection).Not every scientific discovery from basic research ensures translation into a therapy. But there have been more therapies realized from basic research than by the emerging trends of directed translational engagement in the absence of evidence-based research. Investment in a broad range of basic research (because it is important to query scientific problems in many ways) enables collective preparedness for new translational challenges that defy political agendas and fearmongering for partisan gain. This outcome is compelling justification for national and international agencies to prioritize unfettered discovery and basic research. Failure to do this will jeopardize future employment, training, and education at the university, college, and high school levels. Therefore, development of a new generation of researchers, trained in a virtuous cycle of rigorous scientific query and hard work, is imperative to sustain our overarching expectations for this upcoming century.