P. falciparum malaria in children.ABO blood group antigens are formed by terminal glycosylation of glycoproteins and glycolipid chains present on cell surfaces. Glycosylation modulates all kinds of cell-to-cell interactions and this may be relevant in malaria pathophysiology, in which adhesion has been increasingly implicated in disease severity. This study was done to determine the association between ABO phenotypes and the severity of P. falciparum was done. Statistical tests used were odds ratio and chi square at a significance level of p<0.05.One hundred and twenty one children were assessed at the Department of Child Health, KBTH from May to August 2008. ABO blood groups were determined by agglutination. The haemoglobin measurement was done with the haematology analyzer, Sysmex KX-21N. Malaria parasites were enumerated and the presence of malaria pigment noted. Identification of 24.3% of the 121 children had severe falciparum malaria, and their mean haemoglobin was 4.49 g/dl (SD ±1.69). No significant association was found between the ABO phenotypes and malaria infection (p>0.05). Blood group A was associated with more severe malaria as compared to the blood group O individuals ; blood group AB  and also there was a significant difference in severity of malaria between blood group O and blood group B .P. falciparum malaria than the group O, despite the lack of significant association between ABO blood groups and falciparum malaria.Non-O blood group children are more prone to severe malaria caused by  P. falciparum resetting [ABO blood groups are carbohydrate histo-blood antigens that are also expressed in many tissues and have important roles in modulating protein activities both in infection and in some types of cancer . These aesetting , whereasesetting . There iesetting . The proesetting . The othesetting .P. falciparum [P. falciparum [Malaria is the most important parasitic infection of man, and is associated with a huge burden of morbidity and mortality in many parts of the tropical world. Mortality rates of 10-30% have been reported among children referred to hospitals with severe malaria, although these rates are even higher in rural and remote areas where diagnosis and treatment are not readily available . Over 40lciparum . Severe lciparum .Plasmodium spp. known as malarial toxins direct systemic release of proinflammatory cytokines ) which act on many other cellular systems such as endothelium. Equally important are parasite antigens, which stimulate T cells to directly secrete or induce production of cytokines from other cells. Before P. falciparum infection, many individuals have P. falciparum reactive T cells, often at high frequency. Such parasite-reactive T cells have probably arisen as a result of antigenic cross reactivity between environmental organisms and parasite-derived molecules [It is helpful to consider the multifaceted nature of the interaction between the host immune system and the parasite. Central to this interaction are cytokines that are released by immunocompetent cells in a highly regulated fashion . They paolecules .∼ by monocytes, they have the potential to be involved in disease pathogenesis. Both IFN-∼ and TNF-cl may play roles in dyserythropoietic anemia, and TNF-a may contribute to cerebral malaria as a result of up-regulation of intercellular adhesion molecule-1 (ICAM-1) in cerebral blood vessel endothelium. With respect to TNF-et production, T cells--whether they express y8 [Incubation of blood mononuclear cells with parasitized erythrocytes can drive proliferation of these T cells even when the parasitaemia is as low as one parasite per microliter of blood. Because many of these T cells secrete interferon y (IFN-y) and other cytokines and can facilitate production of TNF-opress y8  or oil8 P. falciparum is unique among malaria species in that infected erythrocytes express an adhesive determinant, termed Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP-1). PfEMP-1 is encoded by the parasite genome and is expressed on the outer surface of infected RBCs. PfEMP-1 binds to a variety of target molecules found on RBCs, platelets, and vascular endothelial cells [The adherence of parasitized RBCs to other cells is central to the pathophysiology of severe malaria syndromes including cerebral malaria, respiratory failure, multi-organ failure and death ,17. Paraal cells ,17. The al cells ,28. CIDRP. falciparum malaria [It has been established that parasitized erythrocytes form rosettes more readily with red blood cells of A, B, or AB groups than with cells of blood group O. This parasite-triggered RBC rosette formation is associated with severity of malaria disease . Rosette malaria . The adh malaria ,17.P. falciparum malaria is influenced by ABO blood group but the extent of association between both is yet to be well defined. P. falciparum, the most dangerous or deadly of the four human malaria parasites, causes about 90% of all malaria deaths in the world today especially in sub-Saharan Africa [P. falciparum malaria has been reported to date, in Ghana. Thus, this study investigated the hypothesis that ABO blood groups are associated with the severity of P. falciparum infection and the results showed that non-O blood group children are more prone to severe malaria caused by P. falciparum than the group O children.There is increasing evidence that n Africa . HoweverThis study was approved by the Ethics Review Committee of the School of Allied Health Sciences, College of Health Sciences; Korle-Bu. Parental/guardian consent for child participation was sought during the study.The study was carried out at the Department of Child Health, Korle-Bu Teaching Hospital (KBTH) from May to August 2008. The KBTH is the largest tertiary hospital in Ghana with 17 departments and 1500 beds. One hundred and twenty one (121) children between 3 months and twelve years were sampled.Plasmodium specie to be falciparum.Patient between 3 months and twelve years old, whose blood film (BF) test was reported as malaria parasites present and thin film showed Plasmodium specie to be either falciparum or malariae.Patient below 3 months old or twelve years and above; whose blood film (BF) test was reported either as no malaria parasite present or malaria parasites present and thin film showed Two ml venous blood from the patient was collected into an EDTA-anticoagulated tube  using a 2ml syringe and needle set. Two drops of blood were placed on a clean glass slide and a thick film and a thin film were prepared on the same slide and allowed to air dry. The thin film was fixed with methanol for one minute. The slides were placed in a staining rack and immersed in a staining trough. Giemsa stain  diluted 1 in 10 with phosphate buffer pH 7.2 was used to stain the film for 10 minutes. The stain was washed off the slides using the buffer, pH 7.2. The back of the slides were wiped dry and placed in a draining rack for the film to air-dry.The parasite density was counted as parasite numbers per microlitre of blood by counting parasites against white blood cells. Using the x100 objective, 200 WBCs were counted systematically, estimating at the same time the numbers of parasites  in each field covered, with two tally counters.Calculation = WBC count/µl of patient X Parasites counted against 200 WBCs/200The above formula was used if after 200 WBCs have been counted, and 10 or more parasites were identified. However, if after 200 WBCs have been counted, and 9 or fewer parasites have been counted, counting continued until 500 WBCs have been counted and the number of parasites was recorded.Plasmodium falciparum malaria disease was determined based on the parasite density calculated as above. Severe parasitaemia: was defined as hyperparasitaemia ; and/or severe anaemia (Haemoglobin concentration of<5g/dl). Moderate parasitaemia: was defined as parasitaemia between 51,000-249,000 parasites/µl and/or Haemoglobin concentration between 5-8g/dl. Mild parasitaemia: was defined as parasitaemia of 1,000-50,000 parasites/µl and/or Haemoglobin concentration>8g/dl.Severity of the Tube 1-1 volume anti-A serum and 1 volume 5% patient's red cell suspension in salineTube 2-1 volume anti-B serum and 1 volume 5% patient's red cell suspension in salineTube 3-1 volume patient's serum and 1 volume 5% A cells in salineTube 4-1 volume patient's serum and 1 volume 5% B cells in salineThe spun tube technique , a liquiThe contents of the tubes were mixed by gently tapping the base of each tube with the finger. The tubes were left at room temperature for 5 minutes and then centrifuged at 150g for a minute. The results were read by gently tapping the base of each tube, looking for agglutination or haemolysis. Red cell reagents  were used to control the antisera (A and B). An autocontrol was also set i.e. patients red cells were added to their own serum to detect the possible presence of autoagglutination. The haemoglobin measurement and leucocyte count were done with an automated haematology analyzer, Sysmex KX-21N. The Sysmex KX-21N employs DC detection method for the leucocyte count and Non-cyanide Sodium Lauryl Sulfate (SLS) method for the haemoglobin measurement.The basic demographic data of the 121 children is presented in 2=5.47, p>0.05). Blood group A was associated with severe malaria when it was compared with individuals with blood group O   . Moreove-96,306) .The ABO blood group system in Ghanaians and other West Africans from earlier studies have given the prevalence of blood group O as 50%, AB about 3%, and the remainder being groups A and B in about equal proportions with B being slightly more than A –34. The P. falciparum malaria infection.Studies conducted show that parasitized erythrocytes form rosettes more readily with red blood cells of either A, B or AB groups than with those belonging to blood group O,,35,37 anThe mechanism by which blood group O confers the somewhat protective effect against severe malaria compared to blood groups A, B, or AB is not totally understood as indicated by studies reviewed . HoweverP. falciparum malaria infection, as observed in Nigeria among children with severe malaria [In this study, there was no significant association between the ABO blood group system and  malaria , 40. It P. falciparum than the group O children. Therefore, identifying ABO phenotypes could help to detect those children at high risk for malaria.This study has shown that non-O blood group children are more prone to severe malaria caused by


Enhanced inflammatory host responses have been attributed as the cellular basis for development of severe malaria as well as sepsis. In contrast to this, filarial infections have been consistently reported to be associated with an immunological hypo-responsive phenotype. This suggests that successful control of filariasis by employing mass drug administration, could potentially contribute to an increase in incidence of sepsis and cerebral malaria in human communities. A case control study was undertaken to address this critical and urgent issue.P. falciparum malaria all originating from Odisha, were tested for prevalence of circulating filarial antigens - a quantitative marker of active filarial infection. Antibodies to four stage specific malarial recombinant proteins were measured by solid phase immunoassays and circulating CD4+CD25high T-cells were quantified by flow cytometry with an objective to study if pre-existing filarial infections influence antibody responses to malarial antigens or the levels of circulating T-regulatory cells in P. falciparum infected patients.Eighty-nine patients with sepsis and one hundred and ninety-six patients with W. bancrofti infections. Plasma TNF-a, RANTES and antibodies to recombinant malarial proteins as well as levels of circulating CD4+ CD25high cells were comparable in malaria patients with or without filarial infections.Prevalence of filarial antigenemia was significantly less in sepsis patients as compared to controls suggesting that pre-existing filariasis could influence development of sepsis. On the other hand, levels of circulating filarial antigen were comparable in severe malaria cases and healthy controls suggesting that development of severe malaria is independent of pre-existing These observations imply that successful control of filariasis could have adverse consequences on public health by increasing the incidence of sepsis, while the incidence of severe malaria may not adversely increase as a consequence of elimination of filariasis. Lymphatic dwelling filarial parasites cause severe morbidity in human hosts and persist for long durations in infected hosts. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) is currently targeting elimination of the disease through annual mass drug administration (MDA) of albendazole with either DEC or ivermectin. This has been widely acclaimed to be one of the successful public health programmes and is expected to block transmission of filariasis in endemic countries by 2020 .Plasmodium-infected erythrocytes and occlusion of microvessels are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, suggesting a unique role for RANTES in generation and maintenance of the malaria-induced inflammatory response [Ascaris lumbricoides infection, suggesting that endemic subjects harboring helminthic infections could become protected against development of cerebral malaria [S. mansoni and P. berghei ANKA infection has been reported to lead to reduced cerebral manifestations [Litomosoides sigmodontis and P. chabaudi, mice free of circulating microfilaria were shown to develop more severe forms of malaria than animals free of microfilaria [Sepsis is one of the major causes of mortality around the world and malaria is considered to be one of the most severe infectious diseases afflicting the world’s most impoverished populations. Severe malaria presents itself with a range of biological dysfunctions, i.e. anemia, respiratory complications, acidosis, renal failure, pulmonary edema, multi-organ failure and cerebral malaria etc. . Developresponse . Low levresponse . Howeverresponse , but in response . One of response . Systemiresponse . This laresponse . These o malaria . Animal stations . More restations , althougstations . For exaofilaria .P. falciparum malaria and quantified circulating filarial antigen (CFA), to test the hypothesis whether pre-existing filarial infections could influence development of severe malaria or sepsis. Insights into this aspect are of critical public health importance in predicting possible outcomes of the ongoing successful filariasis control programme on the incidence of sepsis or severe malaria in human populations.Here we report a case control study in two cohorts of patients, one with clinically proven sepsis and the other with severe , viz., leukocytosis or leucopenia, increased C-reactive protein, increased procalcitonin levels. 2) Severe sepsis (n=24): Sepsis complicated by multi-organ dysfunction. 3) Septic shock (n=29): Severe sepsis with acute circulatory failure characterized by persistent arterial hypotension despite adequate volume administration. Details of sepsis patients are shown in Table Patients with symptoms of sepsis admitted to the Department of Medicine at S.C.B. Medical College were classified into three categories. 1) Sepsis (n=36): Infection, documented or suspected, with signs and symptoms of an inflammatory responseepsis n=2: Sepsis P. falciparum in thick blood smears were recruited for the study. Diagnosis by microcopy was further confirmed by immuno-chromatographic card test. Details of malaria patients are shown in Table P. falciparum infection. Patients classified with severe malaria belonged to one of the following three groups:1) Cerebral malaria , 2) Non cerebral severe malaria  and 3) Multi-organ-dysfuction  [Patients reporting at the out-patient department and/or admitted to the Department of Internal Medicine at S.C.B. Medical College , with a short history of fever associated with unarousable coma or multi organ dysfunction were clinically assessed. Patients with microscopically demonstrable D, n=64) . Thirty-About 5ml of the venous blood was collected in heparin from patients, plasma was separated and frozen at −20°C until further use. 100 ul of whole blood was used for two color staining with PE-cy5 labelled anti-CD4 and FITC labelled anti-CD25 (BD Biosciences), along with appropriate isotype controls. Stained cells were then acquired on a 2-laser/4 channel BD FACS Calibur Flow Cytometer and analysed using CellQuest Pro Software.P. falcipaarum. Sera were tested after 200 fold dilutions and bound antibodies were detected using 1000-fold-diluted HRP-labeled anti-human IgG  and enzyme activity was measured using OPD and absorbance read at 492 nm. The results were expressed as arbitrary ELISA units, using internal laboratory standards.Plasma concentrations of TNF-a and RANTES were estimated using commercial sandwich ELISA kits  according to the manufacturer’s instructions. Circulating Filarial Antigens (CFA) were measured by Trop Bio ELISA test kit  as described earlier by us . AntibodP-values<0.05 were considered to be statistically significant.Statistical analyses were performed by using GraphPad Prism (version 5.01). For analysis of Figure P. falciparum malaria is not influenced by pre-existing filarial infection. However, TNF-a was significantly higher in SM cases in comparison to healthy controls (data not shown). Significantly higher levels of RANTES were observed in NCM patients in comparison to SM patients suggesting their importance during non-complicated malarial infection  was significantly less in patients with sepsis in comparison to healthy endemic controls Table . While 4n Figure E.P. falciparum recombinant proteins were quantified in SM, NCM patients and healthy controls and the data for SSP-2, CSP, Exp-1 and LSA-1 are shown in Figure Specific IgG antibody titers to four different . For this preliminary study CD4+CD25high cells were regarded as T-regulatory cells and the gating strategy to score them is shown in Figure high populations, we also scored CD4+CD25+ T cells and significantly higher levels of both the cell populations were observed in NCM cases compared to SM patients  use in vitro in a non-inflammatory pathway and also blocks development of endotoxemia in mice. LPS failed to activate PBMCs of subjects with active filarial infection as shown by significantly low levels of synthesis and release of inflammatory cytokines in vitro[et al. in Litomosoides sigmodontis, which suggests that adult worms suppress LPS induced endotoxemia [Our finding on significantly low prevalence of sepsis in subjects with filarial antigenemia suggests that development of clinical sepsis (a hyperinflammation state) is prevented by pre-existing chronic filarial infections. This is analogous to another report in the literature in which prevalence of chronic inflammatory diseases such as type1 diabetes was less frequent in subjects who were positive for filarial antigenemia . Recentl in vitro. A somew in vitro. The abootoxemia . Howeverotoxemia . Low preThe above study suggests that incidence of severe malaria may not adversely increase as a consequence of filariasis elimination programme in human communities. Furthermore a significantly low prevalence of sepsis in subjects with filarial antigenemia was observed which suggests that development of clinical sepsis (a hyperinflammation state) is prevented by pre-existing chronic filarial infections.The authors declare that they have no competing interests.MP, ADM and PS carried out the experiments. MP, ADM wrote and drafted the manuscript. BKD supplied the blood samples and performed clinical categorization of patients. AKS contributed to analysis of data. BR conceived the idea, designed the experiment and finalized the manuscript. All authors read and approved the manuscript.


Plasmodium species may still be qualified to donate blood for transfusion purposes.The incidence of transfusion-transmitted malaria is very low in non-endemic countries due to strict donor selection. The optimal strategy to mitigate the risk of transfusion-transmitted malaria in non-endemic countries without unnecessary exclusion of blood donations is, however, still debated and asymptomatic carriers of Plasmodium malariae infection. The patient had never been abroad, but nine weeks before, she had received red blood cell transfusion for anaemia. The presumptive diagnosis of transfusion-transmitted quartan malaria was made and subsequently confirmed by retrospective PCR analysis of donor blood samples. The donor was a 36-year-old Dutch male who started donating blood in May 2006. His travel history outside Europe included a trip to Kenya, Tanzania and Zanzibar in 2005, to Thailand in 2006 and to Costa Rica in 2007. He only used malaria prophylaxis during his travel to Africa. The donor did not show any abnormalities upon physical examination in 2011, while laboratory examination demonstrated a thrombocytopenia of 126 × 109/L as the sole abnormal finding since 2007. Thick blood smear analysis and the Plasmodium PCR confirmed an ongoing subclinical P. malariae infection. Chloroquine therapy was started, after which the infection cleared and thrombocyte count normalized. Fourteen other recipients who received red blood cells from the involved donor were traced. None of them developed malaria symptoms.In April 2011, a 59-year-old Dutch woman with spiking fevers for four days was diagnosed with a P. malariae infections in non-immune travellers may occur without symptoms and persist subclinically for years. In addition, this case shows that these infections pose a threat to transfusion safety when subclinically infected persons donate blood after their return in a non-endemic malaria region.This case demonstrates that Plasmodium species.Since thrombocytopenia was the only abnormality associated with the subclinical malaria infection in the donor, this case illustrates that an unexplained low platelet count after a visit to malaria-endemic countries may be an indicator for asymptomatic malaria even when caused by non-falciparum  Transfusion-transmitted malaria (TTM) was first described in 1911 , suggestPlasmodium spp. may still be accepted for blood donation, and therefore the possibility of TTM is not completely excluded  non-endemic countries ,10. In sxcluded  and thrombocytopenia (71 × 109/L). In the peripheral blood smear malaria parasites were seen which were subsequently identified as P. malariae by morphology and PCR. The parasitaemia was 2%. The patient was treated with chloroquine (25 mg/kg) and recovered completely.A 59-year-old Dutch woman was seen at the outpatient clinic of the Leiden University Medical Centre (LUMC) on April 14The patient had never been abroad, neither had she recently been near an international airport. During her admission in February 2011, she had received one unit of packed red blood cells immediately after surgery. Hence, the presumptive diagnosis of transfusion-transmitted quartan malaria was made.Plasmodium-specific semi-quantitative real-time PCR showed a weak, though positive signal for P. malariae , confirming the persistent, asymptomatic P. malariae infection in the donor  163 U/L. Standard malaria diagnostics by a malaria rapid test, thin and thick blood smears as well as quantitative buffy coat (QBC) analysis were negative. However, upon his second visit in November 2011, a single malaria parasite was detected after meticulous investigations of over five thick and five thin blood smears , confirming an ongoing subclinical P. malariae infection. The high Ct-values (37-38) observed in the subsequent blood specimens of the donor are close to the detection level of these tests and correspond to extremely low parasite numbers in the order of 1-100 parasites per mL.Physical examination showed no abnormalities and the spleen was not enlarged (11.5 cm) on abdominal ultrasound examination. Laboratory examination demonstrated a thrombocytopenia of 126 × 10Plasma and thrombocytes from the donor were never used for transfusion, but red blood cells were transfused into 15 recipients. None of the other 14 recipients developed malaria symptoms after transfusion. When the look-back procedure was performed, already 11 recipients had died of reasons other than malaria. The remaining three recipients were invited for a thorough malaria screening. One recipient was tested negative for malaria by both serology and real-time PCR. The two other recipients had no clinical signs of malaria and declined the invitation.9/L) in February and May 2007. The blood bank staff then advised the donor to consult his general practitioner. To exclude other causes of thrombocytopenia apart from malaria, bone marrow morphology was examined, which demonstrated no abnormalities. In addition, no platelet antibodies were detected and the donor had normal thrombopoietin levels . All results together with the longstanding thrombocytopenia and travel history demonstrated that the donor must have had a subclinical P. malariae infection for at least four years and that the infection was most probably acquired during his travel through Kenya, Tanzania and Zanzibar in 2005 or during his visit to Thailand in 2006. He was treated orally with chloroquine 600 mg followed by a dose of 300 mg after six, 24 and 48 hours, respectively. On follow-up, his platelet count quickly normalized , used by the Dutch blood bank as well as by several other blood transfusion services in non-endemic countries to shorten deferral periods for ex-residents of malaria endemic areas and for individuals with a history of malaria, did not detect this asymptomatic malaria infection in the donor. This test was performed retrospectively  and real-time PCR analysis Table , did detP. malariae infections can occur in non-immune persons after a visit to malaria-endemic areas and thus pose a continued threat to transfusion safety. Since thrombocytopenia was the only abnormality associated with this asymptomatic P. malariae infection in the donor, an unexplained low platelet count may be an indicator for asymptomatic malaria even when caused by non-falciparum Plasmodium species.This case demonstrates that asymptomatic chronic Written informed consent was obtained from the patients for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.EEB, JvH, PG, LVL, ES, LV and PJW contributed to acquisition of the clinical and laboratory data. EEB prepared the draft version of manuscript and all authors contributed to the concept and design of manuscript and approved its final version.


Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question.Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic , present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels .An observational study of 636 individuals was performed in Rondônia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions. Plasmodium clearance, but is also implicated in disease severity P. yoelii infection in mice P. falciparum and/or P. vivax to evaluate how HBV infection influences the malaria burden in a region from the Brazilian Amazon.Malaria continues to be a major health threat worldwide. Most regions highly endemic for malaria are also endemic for other infectious diseases, which may affect the malaria infection Written informed consent was obtained from all participants or their legally responsible guardians, and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The project was approved by the institutional review board of the Faculdade de Medicina, Faculdade São Lucas, Rondônia, Brazil, where the study was performed.P. vivax infection represents up to 80% of the malaria cases in Brazil, and P. falciparum infection accounts for 16.3% P. vivax infection have been reported in Brazil P. falciparum and P. vivax have been detected in epidemiological surveys in some regions of the Brazilian Amazon, indicating that clinical immunity does exist in both autochthonous and migrant populations A field observational study was performed between May 2006 and September 2007 in Rondônia State , Brazilian Amazon. In this region, most malaria cases occur between April and September, with a high risk of infection Both active and passive malaria case detection and diagnosis of HBV infection were performed. These included home visits in areas of high transmission, and study of individuals seeking care at the diagnostic centers of Brazilian National Foundation of Health (FUNASA). Individuals of both sexes, ranging in age from five to seventy years, who had resided in the endemic area for more than six months, were invited to participate. Exclusion criteria were as follows: documented viral hepatitis , chronic alcoholism, human immunodeficiency virus type 1 infection, yellow fever, leptospirosis, cancer and chronic degenerative diseases, sickle cell trait and the use of hepatotoxic or immunosuppressant drugs. Twelve individuals withdrew consent and were excluded from the study. The study participants were interviewed and examined by a physician, and 20 mL of venous blood and thick blood smears were collected. Plasma samples and total blood were stored in liquid nitrogen. Total blood samples were used for molecular diagnosis of malaria and plasma samples analyzed in our laboratory facilities in Salvador, Bahia, Brazil.Plasmodium infection without any presumptive malaria symptoms were considered asymptomatic, whereas cases with positive parasitological tests in the presence of symptoms were classified as symptomatic. Study individuals were then classified into three groups according to Plasmodium infection: non-infected (n = 205) or Plasmodium-infected  and either symptomless (n = 221) or symptomatic (n = 210)  was calculated in positive cases. Nested PCR was performed as described previously n = 210) . Only twhe study .−/anti-HBS−/anti-HBc−), 193 presented markers of previous HBV infection (HBSAg−/anti-HBS+/anti-HBc+), 61 were currently infected (HBSAg+/anti-HBS−/anti-HBc+) and 56 were vaccinated (HBSAg−/anti-HBc−/anti-HBS+). All HBV infected individuals were positive for anti-HBc. No acute HBV infection was detected, as there were no individuals with anti-HBc IgM. Viremia was estimated by real-time PCR (COBAS® TaqMan® HBV assay) of all samples to confirm serological results. We also evaluated HBV-infected individuals for HBeAg and anti-HBe.Diagnosis of HBV infection was performed at the State Central Laboratory (LACEN) of Salvador, Bahia, Brazil, using the AXSYM® automatic ELISA system . All individuals were screened for HBSAg, total anti-HBS, total anti-HBc, anti-HBc IgM, HBeAg and anti-HBe IgG. We found that 326 individuals presented no markers of HBV exposure , alanine amino-transaminase (ALT), total bilirubin, hemoglobin, fibrinogen and C-reactive protein (CRP) were made at the clinical laboratory of Faculdade São Lucas and at the Pharmacy School of the Federal University of Bahia, Brazil. A flow chart of the study is shown in the IL-10, IFN-γ and TNF-α plasma levels were measured using the Cytometric Bead Array - CBA®  according to the manufacturer's protocol, with all samples run in a single assay. The flow cytometric assay was performed and analyzed by a single operator, and standard curves were derived from cytokine standards.Plasmodium parasitemia and HBV viremia in co-infected individuals was checked using the Spearman test. We also plotted a non-linear curve fit to illustrate the general trend of this correlation. For each analysis, P<0.05 was considered statistically significant. The statistical analysis was performed using the software STATA 9.0 . The graphics were plotted using GraphPad Prism 5.0 .In the exploratory analysis of the data, frequency tables were constructed and the Chi-square test was applied to evaluate the association between qualitative variables. One polynomic  logistic regression model was carried out because the response variable  was classified into three groups . The following independent variables were included: HBV infection, malaria episodes, time residing in the area, residents per household, age, gender and plasma cytokine levels of IL-10 or IFN-γ. The threshold values of IL-10 or IFN-γ plasma levels, which discriminate asymptomatic from symptomatic malaria infection with a high likelihood ratio, were estimated using a ROC curve analysis to categorize the individuals according to cytokine levels and to perform the multinomial logistic regression (data not shown). Malaria parasitemia, cytokine plasma levels, and plasma levels of AST, ALT, total bilirubin, fibrinogen, and CRP were compared between groups using the Kruskal-Wallis test with Dunn's multiple comparisons. HBV DNA plasma levels were compared between groups using the Mann-Whitney test. The correlation between Plasmodium infection with regard to all variables studied, except for gender  and for markers of HBV exposure (P = 0.02). As expected, gender did not show any association with the absence of malaria or with asymptomatic infection  were similar to those enrolled with regard to age, time of residence in the endemic area, number of residents per household, and number of previous malaria episodes, but were more likely to be female (P = 0.03) and test negative for nfection .To identify the variables independently associated with asymptomatic malaria, we performed a multinomial logistic model with adjustment for the all variables studied. We included variables previously described as protective factors against malaria We also tested whether the cytokine balance is associated with clinical immunity to malaria by examining the levels of two cytokines that indicate an inflammatory profile P. vivax cases, which represented 84.22% of the cases in our study , which allowed us to continue to analyze P. vivax and P. falciparum cases together.We then re-analyzed the data considering only the ur study . The disfections . The excnfection . Thus, tPlasmodium parasitemia   .Plasmodium presented similar systemic levels of IFN-γ compared with those infected solely with HBV . Helminth infection can affect malaria Plasmodium parasitemia and possibly diminishing parasite burden. The results presented here need to be confirmed in future prospective studies.In conclusion, HBV infection seems to modify the physiology of the host's immune system, stimulating increased inflammatory responses, reducing


RV217/ECHO aims at capturing HIV-1 incident cases during the very earliest stages of acute HIV-1 infection(AHI). Limitations in single genome amplification(SGA) sampling depth have the potential to confound the study of multiplicity of infection and viral evolution during AHI. Here we combined SGA and deep, next-generation sequencing(NGS) to investigate the prevalence of minor viral variants during pre-peak viremia and characterize subsequent viral evolution.A male CSW from Thailand with documented nucleic acid testing(NAT)-conversion and seroconversion, and with measured viremia peak, was studied. Plasma viruses from pre-peak viremia, immediate post-peak viremia, and 6 months post-infection were characterized by SGA and targeted NGS. A library prepared from 1,500 PCR-equivalent copies was analyzed using IonTorrent(LifeTechnologies).Reading coverage was>20K and the experimentally-measured error rate was<0.0028/base.Based on 47 env SGA sequences from pre-peak viremia, the infection was initiated by a single T/F virus (mean env pair-wise nt diversity:0.047%).A second variant, highly similar but distinguishable from the T/F , was detected during immediate post-peak viremia(18/56 SGA sequences). By 6 months post-infection, the viral population was dominated by the second variant along with numerous recombinants.NGS analysis of multiple, independent subgenomic areas with concentrated sequence variation, revealed that the second variant was already established at 1% at pre-peak viremia. Outgrowth of the second variant, along with its derivatives, was confirmed by longitudinal NGS.Supplementing SGA analysis with NGS allowed us to demonstrate the presence of two T/F viruses at pre-peak viremia with substantial frequency differences, in the study participant. Due to its implications, the prevalence of this phenomenon needs to be further explored. The application of NGS to the study of HIV-1 evolution during AHI promises to foster the advancement of vaccine development.


Previous studies demonstrated that fewer mosquitoes enter houses which are screened or have closed eaves. There is little evidence about the effect on malaria infection in humans that changes in house construction may have. This study examines the impact of protective housing improvements on malaria infection on Bioko Island.P. falciparum. Prevalence of infection in those living in houses with open eaves was 23.0% compared to 18.8% for those living in houses with closed eaves . The prevalence of infection for children in screened houses was 9.1% versus 20.1% for those living in unscreened houses . The proportion of houses with closed eaves increased from 66.0% in 2009 to 74.3% in 2012 (test for trend p = 0.01). The proportion of screened houses remained unchanged over time at 1.3%.Data from the annual malaria indicator surveys between 2009 and 2012 were used to assess trends in housing characteristics and their effect on RDT confirmed malaria infection in household members. Odds ratios were adjusted for socio-economic status of the household.22726 children between the ages of 2 and 14 years were tested for As a malaria control intervention, house modification has the advantages that it is not affected by the growing threat of insecticide resistance; it protects all household members equally and at all times while indoors; and it offers protection against a number of vector borne diseases. The study provides evidence in support of efforts to regulate or encourage housing improvements which impede vector access into residences as part of an integrated vector control approach to complement existing measures which have been only partially successful in reducing malaria transmission in some parts of Bioko. Preve vectors . Due to  vectors .Perhaps the most obvious way of preventing mosquitoes from entering a house is to screen the entry points with a mesh. A trial of house screening in The Gambia showed that screening lead to a reduction in the number of mosquitoes entering houses and in the prevalence of anaemia in children, but did not show a reduction in malaria infection prevalence . Other sThis study examines the effect of closed eaves and house screening on malaria infection on Bioko by examining data collected from malaria indicator surveys during the second 5 year phase of the Bioko Island Malaria Control Project.Anopheles funestus and An. gambiae respectively [Bioko Island is part of Equatorial Guinea and lies 32 kilometres off the coast of Cameroon. The population of Bioko is approximately 250 000. Malaria is endemic, with continuous year round transmission. Before the launch of the Bioko Island Malaria Control Project (BIMCP), malaria transmission was high, with annual entomological inoculation rates (EIR) of over 750 and 250 infectious bites per person per year by ectively , and a mectively .TM, Bayer) from 2005 to 2012. A satisfactory level of coverage has consistently been achieved [The BIMCP, funded by the Government of Equatorial Guinea and a consortium of private donors led by Marathon Oil Corporation, started an intensive malaria control strategy in 2004. The third five year phase of the project will begin in 2014. IRS was started in 2004, with a first round of deltamethrin followed by biannual rounds of the carbamate insecticide Bendiocarb  have either wood or cement walls. Plasmodium falciparum using RDT , subject to informed written consent from a caregiver. Children testing positive for parasitaemia, with haemoglobin <11 g/dL or who were febrile, were referred to a local field clinic for appropriate treatment . In accordance with malaria indicator surveys in high endemicity settings [Household malaria indicator surveys have taken place annually on Bioko since 2004 –17,19–21settings , testingThe average number of occupants per household, the average number of rooms, the average number of people per room, the proportion of houses with wooden walls, the proportion of houses with dirt floors, the proportion with closed eaves and the proportions in the 2 categories of screening were tabulated for each survey and overall. Either linear regression (in the case of number people and rooms), logistic regression , or ordered logistic regression, (in the case of categories screening), with year of survey as a linear independent variable was used to assess trends over time.Household socio-economic status (SES) was calculated using the first principal component score based on variables related to household size, asset ownership, livestock ownership and household utilities. The SES scores for each survey year were converted to quintiles for analysis. Analysis of the association between household characteristics and SES was carried out in the same way as for time trends described above.P. falciparum Apical Membrane Antigen-1 (AMA-1) by enzyme linked immunosorbent assay (ELISA), for calculating sero-prevalence by age and for estimating the serological conversion rate (SCR) for each sentinel site [In 2008, filter paper blood samples were collected from 7387 individuals of all age groups, for detection of antibodies to nel site . SCR hasnel site ,25. In tP.falciparum infection in 2–14 year olds. Crude odds ratios were calculated, adjusted for survey year only. Adjusted odds ratios were calculated, adjusting for the following factors previously shown to be associated with malaria prevalence in Bioko [Multivariable logistic regression was used to investigate the relationship between household characteristics and RDT confirmed in Bioko : whetherIn all analyses standard errors were adjusted for the survey design by taking into account the between site variation in prevalence using the svy set of commands in Stata ,27. The Ethics approval for the study was granted by the Equatorial Guinea Ministry of Health and Social Welfare and the ethics committee of the London School of Hygiene and Tropical Medicine . Informed written consent was given by each participant or, in the case of children, a responsible adult. In the case of participants being unable to read, the text was read and explained to them, and consent was confirmed by an independent witness identified on the consent form.p = 0.01) but there was no evidence of a change in the proportion of houses screened (p< 0.01) but the average size of the household did not, meaning that the average household occupancy decreased from 1.36 to 1.01 persons per room (p< 0.01). The number of households surveyed in 2009, 2010, 2011 and 2012 respectively was 2245, 2938, 2819 and 2791. The proportion of houses with closed eaves increased over time from 66.0% to 76.4% (screened . The avep = 0.12). Dirt floors were more common in rural sites  as were wooden walls . Houses in urban sites were more likely to have closed eaves . Screening was significantly more common in urban houses (p < 0.01)., both partial (16.8% vs 7.3%) and total (1.5% vs 0.8%).Urban and rural households had a similar average number of residents, 5.1 and 5.0 respectively. Rural houses had a slightly larger number of rooms on average (5.2 vs 4.9) but this difference was not significant (p< 0.01). Households with higher SES tended to have more rooms (p < 0.01), more inhabitants (p < 0.01) and fewer occupants per room (p< 0.01). Higher SES was associated with fewer wooden walls (p< 0.01) and dirt floors (p< 0.01). All household characteristics showed a strong association with SES . Closed P. falciparum malaria infection in the 2009, 2010, 2011 and 2012 surveys was 5107, 6470, 5422 and 5727 respectively (data not tabulated). The prevalence of infection in each year was 20.4%, 25.5%, 20.2% and 13.7% respectively, with overall mean of 20.1%. The prevalence of malaria infection in 2-14 year olds was similar in rural and urban . The numbers of children who were reported to have slept under untreated and treated nets the night before the survey were 3059 (13.5%) and 3177 (14.0%) respectively. The number of children who lived in houses that were reported to have been sprayed within the last 6 months was 15023 (66.1%).The number of 2-14 year olds tested for p < 0.01), but those in partially screened houses did not have lower levels of infection than those in unscreened houses (p = 0.03). Both these effects remained after adjusting for confounders. Infection prevalence was associated with the density of people living in a house in the unadjusted analysis (p = 0.01) but after adjusting for confounders there was no evidence of an effect (p = 0.14). The proportion of houses with wooden walls was 58.5 %; of the remainder, all but 1% had cement walls. Open eaves were more common in wooden houses than in cement houses . Neither dirt floors nor wooden walls were associated with malaria prevalence (p = 0.50 and p = 0.23 respectively).The 287 children who lived in totally screened houses had a substantially lower prevalence of malaria infection than those who lived in unscreened houses (9.1% vs 20.1%, d houses . Closed Njie et al [An. gambie s.l. entered houses with closed eaves. There has, however, been little evidence on whether this reduction in mosquitoes entering translates into lower malaria infection in those sleeping in the house. A study [This study shows that children living in houses with open eaves on Bioko Island were at a higher risk of malaria infection than those living in houses with closed eaves even after adjusting for the SES of the household. Many studies have shown that mosquitoes are more likely to enter a house with open eaves ,10–13; f A study  in the E A study . Therefo A study  have shoPrevalence of malaria infection in children living in houses reported as fully screened was substantially lower than in those living in unscreened houses. There was, however, no evidence of a difference for those living in partially screened houses compared to unscreened houses. This suggests that screening must be complete, or nearly complete, to be effective. A randomised trial in the Gambia showed screening to be effective at preventing mosquitoes entering a house and in reducing the level of anaemia in children, but did not show a reduction in prevalence of infection . There iAlthough only 1.3% of the houses surveyed over the 4 years were fully screened, this was a large study involving over 10 000 houses and 20 000 children, of whom 287 lived in screened houses and showing strong statistical evidence for an effect, even after adjusting for important confounders. This effect was consistent across different years, with odds ratios of 0.59, 0.29, 0.49 and 0.42 for infection in houses with complete screening compared to no screening in the four survey years respectively. The odds ratios for association between open eaves and malaria infection were similarly consistent over the four survey years, being 1.33, 1.34, 1.23 and 1.25 respectively.The emergence and spread of resistance in malaria vectors to all 4 classes of insecticides currently used for malaria vector control is recognised as a serious threat for the effectiveness of insecticide based vector control methods and hence for malaria control and elimination . One of The protective effect of closed eaves and window screening remained after adjusting for LLIN use and IRS coverage. This suggests that there may be a benefit to these interventions in addition to the standard malaria prevention tools.It has been shown in previous studies that mosquitoes are more attracted to houses with high occupancy . In thisA limitation of this study is that it uses observational data and houses were not randomised to the interventions. Although many observed confounding variables were adjusted for in this analysis there could still be residual confounding. In particular there could be aspects of socio-economic status not accounted for in the asset based score used in this analysis. There is a possibility of some mis-recording of data for house screening; a small number of non-randomly selected houses reported as screened were revisited and some had solid shutters over the windows which the inhabitants reported were kept permanently closed rather than a mesh over the windows. Although the effect of closed eaves and screening on mosquito abundance in houses is well known, it is a limitation of this study that no entomological data were available to corroborate the effect on risk of infection in humans. The proportion of houses with closed eaves on Bioko is high and increasing, but the proportion with screening has remained very small. The rigorous analysis of data from four annual surveys presented in this study provides robust evidence of tangible protection against malarial infection resulting from relatively minor house improvements. The high transmission that persists in parts of the island even after nearly a decade of comprehensive interventions, and the additive protective contribution that housing improvements can make to reduce the prevalence of infection, suggest that strategies to regulate and encourage the adoption of protective housing improvements should be seriously considered for inclusion in the overall malaria control programme. As malaria control ultimately progresses towards elimination, socio-economic factors, including housing quality, will have an increasingly important role to play. Malaria control programmes should consider targeted housing improvements as a sustainable additional intervention to reduce transmission. To generate stronger evidence of the efficacy of this intervention for policy makers and funders generally, we would urge that randomised trials with epidemiological, entomological and costing outcomes be carried out as soon as possible.


Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, infects humans and can cause fatal malaria. It is difficult to diagnose by microscopy because of morphological similarity to Plasmodium malariae. Nested PCR assay is the most accurate method to distinguish P. knowlesi from other Plasmodium species but is not cost effective in resource-poor settings. Rapid diagnostic tests (RDTs) are recommended for settings where malaria is prevalent. In this study, the effectiveness of three RDTs in detecting P. knowlesi from fresh and frozen patient blood samples was evaluated.P. knowlesi, ten P. vivax and two P. falciparum) diagnosed by microscopy were recruited in Sarawak, Malaysian Borneo during a 16-month period. Patient blood samples were used to determine parasitaemia by microscopy, confirm the Plasmodium species present by PCR and evaluate three RDTs: OptiMAL-IT, BinaxNOW® Malaria and Paramax-3. The RDTs were also evaluated using frozen blood samples from 41 knowlesi malaria patients.Forty malaria patients  of fresh and 24%  of frozen samples. The Paramax-3 RDT tested positive for P. vivax with PCR-confirmed P. knowlesi samples with sensitivities of 40%  with fresh and 32%  with frozen samples. All RDTs correctly identified P. falciparum- and P. vivax-positive controls with parasitaemias above 2,000 parasites/μl blood.OptiMAL-IT was the most sensitive RDT, with a sensitivity of 71%  for fresh and 73%  for frozen knowlesi samples. However, it yielded predominantly falciparum-positive results due to cross-reactivity of the Plasmodium in P. knowlesi-infected blood samples with poor sensitivity and specificity. Patients with P. knowlesi could be misdiagnosed as P. falciparum with OptiMAL-IT, P. vivax with Paramax-3 and more correctly as non-P. vivax/non-P. falciparum with BinaxNOW® Malaria. There is a need for a sensitive and specific RDT for malaria diagnosis in settings where P. knowlesi infections predominate.The RDTs detected  Plasmodium  were known to cause malaria in humans. However, a fifth species, Plasmodium knowlesi, has been identified as a cause of human malaria in almost all countries in Southeast Asia  and extreviewed . In Malafections -9.Plasmodium species infections are typically diagnosed by microscopic examination of stained blood films, but there are limitations in sensitivity and specificity [Plasmodium species. Molecular methods are the most sensitive detection method for malaria and can distinguish P. knowlesi from the morphologically similar P. malariae[cificity . Nested  malariae,12. HoweP. falciparum, were developed for use in remote areas with limited facilities. A large number of RDTs are available for malaria diagnosis, including OptiMAL-IT produced by DiaMed, CA, USA [P. falciparum lactate dehydrogenase (pLDH) and a pan-malaria antibody (19G4) that identifies pLDH from P. vivax, P. malariae and P. ovale as well as P. falciparum. BinaxNOW® Malaria is the only test approved by the US FDA and contains an antibody that detects P. falciparum-specific histidine rich protein 2 (HRP-2) and a pan-malaria antibody that detects Plasmodium aldolase, a glycolytic enzyme produced by all species of the parasite. The Paramax-3 test strip contains three antibody test lines as well as a control line: an antibody detecting P. falciparum HRP-2, similar to the BinaxNOW® Malaria test, a pan-malaria pLDH antibody, and an antibody specific for P. vivax LDH. None of the tests include P. knowlesi-specific reagents.Malaria antigen-based rapid diagnostic tests (RDTs), mainly for the detection of  CA, USA ,14, Bina CA, USA ,16, and  CA, USA ,18. The P. knowlesi under experimental conditions. An imported case of human P. knowlesi from Sarawak to the Netherlands with 2% parasitaemia  gave positive results for both the P. falciparum-specific LDH and pan-malarial LDH test lines, suggesting cross-reactivity between P. knowlesi LDH and the monoclonal antibody used in the test to detect P. falciparum LDH [et al.[et al. using P. knowlesi-infected Japanese macaque blood samples with OptiMAL-IT [et al. using a P. knowlesi-positive blood sample from a patient with 0.2% parasitaemia [OptiMAL-IT, and another RDT under development , have bearum LDH . This crH [et al., was alstiMAL-IT  and by Ositaemia .P. knowlesi in several case reports with varying results [P. knowlesi malaria imported into the Netherlands, BinaxNOW® Malaria but not OptiMAL-IT gave a positive result for non-P. falciparum malaria [P. knowlesi-infected patient with 0.2% parasitaemia [P. knowlesi malaria patients with parasitaemia of 0.0005 and 0.1% [BinaxNOW® Malaria has been used to detect  results ,22-25. I malaria . Similar malaria . Howeverand 0.1% ,25, suggP. knowlesi samples. The objective of this study was to evaluate three RDTs, OptiMAL-IT, BinaxNOW® Malaria, and Paramax-3, in the detection of P. knowlesi infection from both fresh and frozen blood samples from knowlesi malaria patients.None of the aformentioned studies has evaluated the Paramax-3 test with All consecutive malaria-positive patients, diagnosed by microscopy, were recruited following acquisition of informed consent at Sarikei Polyclinic and Sarikei Hospital in the town of Sarikei, Sarawak, Malaysian Borneo. The study was approved by the Malaysian Ministry of Health’s Medical Research and Ethics Committee.P. knowlesi and three P. vivax) from Sarikei Polyclinic and 30  from Sarikei Hospital. No mixed species infections were identified. Venous blood samples collected from these patients were used to make thick and thin blood films for verification of parasitaemia by microscopy, blood spots on filter paper for malaria species identification by nested PCR assays, and to evaluate RDTs.A total of 40 malaria-positive blood samples were collected between March 2010 and July 2011: ten (seven P. knowlesi (N = 41), P. falciparum (N = 3) and P. vivax (N = 1), admitted to Sarikei and Sibu Hospitals in the two years prior to the start of this study and from those recruited during a previous study at Kapit Hospital [The three RDTs were also evaluated using 44 frozen whole-blood samples collected from malaria patients with PCR-confirmed Hospital . Whole-bThick blood films from samples acquired at Sarikei Polyclinic and Hospital were allowed to dry overnight, then stained with 3% Giemsa for 45 minutes. Thin blood films were fixed with methanol and stained with 10% Giemsa for 30 minutes. Parasitaemia was later determined by an experienced microscopist. Parasitaemia was calculated as the number of parasites per μl of blood for each sample by using each patient’s actual white blood cell (WBC) count, and by counting up to 500 WBCs in thick blood films and the corresponding number of malaria parasites.Plasmodium genus-specific primers rPLU1 and rPLU5 for the Nest 1 amplification, which targets the small sub-unit ribosomal RNA gene of the Plasmodium genus [DNA was extracted from the blood spots on filter paper using the Instagene method . All samum genus . ReactioP. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. For the species-specific primer pairs for P. falciparum, P. vivax, P. malariae and P. ovale  the annealing temperature was 58°C, and for the P. knowlesi-specific primer pair (kn1f and kn3r) the annealing temperature was 62°C. The Nest 2 products were analysed using agarose gel (2.5%) electrophoresis and Sybr green® staining (1x concentration for 30 minutes), viewed via UV transilluminator, photographed for documentation and recorded.In order to identify the malaria species present in each sample, Nest 1 amplification products were screened using Nest 2 species-specific primers, as described previously ,26,27 foThe three antigen-based RDTs, OptiMAL-IT, BinaxNOW® Malaria, and Paramax-3, were used according to the manufacturers’ instructions. Fresh blood samples from malaria patients were processed within three hours of collection, and immediately when using thawed frozen blood samples. Thawed samples were processed as for fresh blood. OptiMAL-IT requires 10 μl of blood and takes 20 minutes to complete, BinaxNOW® Malaria requires 15 μl of blood and takes 10–15 minutes to complete, and Paramax-3 requires 5 μl of blood and takes 15 minutes to complete. Tests were interpreted as successful when the control band was positive.The sensitivity of each RDT was calculated with PCR results as the reference standard. The 95% confidence interval (CI) for each test was calculated based on the test sensitivity and number of tests performed using 1.96 as the standard normal deviate (using the formula: 95% CI = Sensitivity ± 1.96 × standard error of the test) .P. knowlesi, two P. falciparum and ten P. vivax), along with 44 frozen whole-blood malaria samples . All RDTs were successful in that the control line was positive on all tests performed.RDTs were evaluated against 40 microscopy-positive fresh blood samples  and 41 of each RDT were performed using frozen P. knowlesi samples .In total, 28 OptiMAL-IT and BinaxNOW® Malaria and 25 Paramax-3 tests were performed on P. knowlesi samples cross-reacted with the P. falciparum LDH test reagent in 18 of the 20 fresh samples identified. Only two of the fresh P. knowlesi samples were identified as non-P. falciparum malaria using OptiMAL-IT.OptiMAL-IT was the most sensitive of the three RDTs evaluated, with a sensitivity of 71%  and 73%  for fresh and frozen knowlesi malaria samples, respectively  of fresh samples with parasitaemia >5,000 parasites/μl , all positive results for P. knowlesi cases were correctly identified as non-P. falciparum malaria infections.BinaxNOW® Malaria showed the lowest sensitivity, detecting only 29%  of fresh and 24%  of frozen samples. This test was negative for all ten P. knowlesi samples, respectively. Of the ten Paramax-3 tests that yielded positive results with fresh blood samples, one indicated a P. falciparum infection, one indicated a non-P. falciparum, non-P. vivax result and the remaining eight gave results indicating P. vivax infections  and 32%  for fresh and frozen Plasmodium vivax (N = 10) and P. falciparum (N = 2) samples from Sarikei Hospital and Polyclinic were collected for testing with RDTs as positive controls. All three RDTs gave positive results accurate for the species present when the parasitaemias were above 2,000 parasites/μl blood . The RDTs did not detect samples with P. vivax (N = 2) or P. falciparum (N = 1) parasitaemia less than 2,000 parasites/μl. All frozen P. vivax (N = 1) and P. falciparum (N = 2) positive controls yielded positive RDT results appropriate for the species. The parasitaemia for these samples was relatively high .P. knowlesi-infected blood samples. However, the test was not specific and the majority of P. knowlesi samples were identified as P. falciparum by this test due to antibody cross-reactivity, as noted in previous studies [P. falciparum antibody for P. falciparum-HRP-2 and P. knowlesi samples. In this study, all positive results attained using this test with P. knowlesi-confirmed samples correctly indicated a non-P. falciparum malaria infection. The Paramax-3 test showed low sensitivity and cross-reactivity between the P. vivax LDH-detecting antibody and P. knowlesi. This observation has been noted in several other single case reports using different tests which also contain a P. vivax LDH-detecting antibody [Among the RDTs tested in this study, the OptiMAL-IT test was the most sensitive for  studies -22. Binaantibody ,23,24.P. knowlesi in blood samples with all three RDTs assessed in this study was significantly lower than that reported for other Plasmodium species. For example, the sensitivity of detection of P. falciparum using OptiMAL-IT has been reported as 95.3% (100% for >500 parasites/μl and 72% for 50 parasites/μl) and 96% for P. vivax malaria infections [P. falciparum has been reported as 95.3%  and 68.9% for P. vivax malaria infections [P. falciparum and P. vivax malaria infections is reported as 100% in an in-house study of 251 samples [P. vivax or P. falciparum infections in fresh blood samples with parasitaemias less than 2,000 parasites/μl.The sensitivity of detecting fections . For Binfections . For Par samples . Althougversus frozen samples. Freeze-thawing and the storage of blood at low temperatures can accelerate deterioration of antigen activity, although it is also possible that target antigens are more accessible in freeze-thawed samples and may actually improve the sensitivity of RDTs [One limitation of this study is the relatively low numbers of fresh samples tested. To strengthen the results from fresh samples, frozen blood samples were also included. The sensitivity of detection of knowlesi malaria infections with the RDTs tested were fairly similar using fresh  of RDTs .Plasmodium LDH and Pf-specific HRP-2, and ParaHIT™, which detects pan-Plasmodium aldolase and Pf-specific HRP-2 [P. knowlesi patient samples were studied, only 34 of whom were enrolled in the study prior to treatment, while the remainder were referred from district hospitals where they had already received anti-malarial treatment [P. knowlesi samples; 29% with the ParaHIT™ test [A recently published paper from Sabah, Malaysian Borneo presents the use of two different RDTs, First Response™, which detects pan-ic HRP-2 . A totalreatment . The finIT™ test , and 29%Plasmodium species infections, especially P. knowlesi. The development of loop-mediated isothermal amplification (LAMP) assays combine the sensitivity and specificity of PCR with low cost, low technology and rapid results. LAMP-based tests for malaria diagnosis that include reagents specific for P. knowlesi are under development and may be useful for resorce-poor settings [RDTs cost between 10 and 15 Malaysian Ringgit (US$3.17-4.80) per test when purchased at a dispensary in Malaysia. Although when purchased in bulk for malaria control programs this cost tends to be significantly reduced, microscopy is still the most affordable diagnostic tool and costs just one Ringgit (US$0.30) per patient to screen for malaria. The cost of nested PCR assay is comparable to RDTs per patient sample, and although PCR assay is significantly more sensitive and specific than microscopy, this technique requires specialized equipment, electricity supply and training, and is not suitable for resource-poor settings. RDTs confer the advantages of speed , minimal training and ease of use, and do not require electricity or any specific hardware. However, currently available RDTs lack sensitivity and specificity compared to microscopy and PCR-based methods for all settings ,32.et al., unpublished data). As such, it would have cost between MYR 1,080 and 1,620 (US$342-832) to perform RDTs for these 108 query malaria patients and only two were positive.In areas with relatively low malaria prevalence such as Sarawak, the cost of RDTs, even if sensitive and specific, would likely outweigh the benefit. To understand this in practical terms, consider, for example, the case of Julau Health Clinic, which is a small, rural health clinic in Sarawak surveyed as part of the current study. The prevalence of malaria at this clinic during a five-month study period in which 108 febrile patients whose clinical presentations were suggestive of malaria were screened using nested PCR assay was 0.2% (Foster P. knowlesi. Also, P. knowlesi cross-reacted with P. falciparum and P. vivax LDH antibodies used in two of the three commercially available RDTs tested, resulting in misdiagnosis of malaria species in an area where human P. knowlesi infections are prevalent. Since not all species of malaria warrant the same level of medical care, misidentification can result in mismanagement, especially when the potentially severe knowlesi malaria is misdiagnosed as vivax malaria. Because P. knowlesi is morphologically similar to P. malariae and P. falciparum, it is also misdiagnosed by microscopy [P. knowlesi occurs until the sensitivity, specificity and costs are comparable.This study confirms that the RDTs evaluated are not adequately sensitive for use in the diagnosis of croscopy ,23,24. HP. knowlesi by the three RDTs evaluated is low compared with microscopy. Cross-reactivity is common between P. knowlesi-infected blood and both the P. falciparum-detecting antibody used in the OptiMAL-IT test and the P. vivax-detecting antibody used in the Paramax-3 test. As such, a patient with knowlesi malaria may be diagnosed as P. falciparum by OptiMAL-IT, as P. vivax by Paramax-3 and as non-P.vivax and non-P. falciparum by BinaxNOW® Malaria. Until more sensitive RDTs are developed that can distinguish P. knowlesi from P. falciparum and P. vivax, serious consideration should be taken before using RDTs for the diagnosis of malaria in settings where P. knowlesi is the predominant species.The sensitivity of detection of The authors declare that they have no competing interests.BS, JCS, SK and DF were involved in study conception and design, procurement of RDTs and writing the manuscript. DF was involved in data collection and analysis, and drafting the manuscript. PPC assisted with data collection and analysis and DSAM assisted with laboratory data analysis. BS was involved in training of personnel and oversaw the project throughout. All the authors have read and approved the manuscript.


P. falciparum malaria, the most virulent species continues to be a major health problem in sub-Saharan Africa. This study was carried out to establish the prevalence of pregnancy-associated malaria and its associated consequences including maternal anaemia and low birthweight (LBW) deliveries and placental malaria among pregnant women in a sub-urban area in coastal Ghana.Malaria and anaemia have adverse effects in pregnant women and on the birth weight of infants in malaria endemic areas. A facility-based investigation was carried out among 320 pregnant women seeking antenatal care in a hospital in suburban coastal Ghana. Information on the use of Insecticide Treated Nets (ITNs) and Intermittent Preventive Treatment in pregnancy (IPTp) were collected using a structured questionnaire at enrolment. Venous blood was collected for microscopy and screening for Glucose 6-phosphate dehydrogenase (G6PD) deficiency. Haemoglobin concentration was obtained from an automatic blood analyzer. Placental smears and birth weight measurements were taken at delivery.Plasmodium falciparum parasitaemia was 5%. The mean haemoglobin (Hb) level at registration was 11.44g/dL (95% CI 11.29 – 11.80). Placental blood parasitaemia and low birthweight were 2.5% and 3% respectively. ITN possession was 31.6% with 5.4% usage. The IPTp coverage was 55%.The prevalence of The prevalence of malaria and anaemia among the pregnant women were low at enrolment. Placental blood parasitaemia and LBW at delivery were also low. These are clear indications of the high coverage of the IPTp. Increase in ITN use will further improve birthweight outcomes and reduce placental malaria. Malaria, which is transmitted by female anopheles mosquitoes, results in peripheral blood parasitaemia which may be asymptomatic in adults living in malaria endemic areas. Among pregnant women, however, peripheral blood parasites may accumulate in the placenta resulting in malaria-associated pregnancy (PAM) with adverse consequences such as maternal anaemia and low birthweight infants . InterveMalaria is the main determinant of anaemia among women who are pregnant for the first time in endemic regions  whereas Low birthweight (LBW) is a significant risk attributable to malaria in pregnancy. It is estimated that in malaria endemic areas 19% of infant LBW is due to malaria and also that 6% of infant deaths are due to LBW caused by malaria. These estimates imply that around 100,000 infant deaths each year could be due to LBW caused by malaria during pregnancy in malaria endemic areas in Africa .The infaMost studies on the risk of anaemia in pregnancy have examined associations with micronutrient deficiencies , 10 or wPlasmodium falciparum accounts for 98% of all infections reported in the hospital.The study was conducted at the Alpha Medical Centre (AMC) in Madina town. Madina is situated in the Ga-East District of the Greater Accra Region about 13.5 kilometres from Accra, the capital of Ghana. It is densely populated with an estimated 2007 population of 112,888 from the 2000 census report [The number of pregnancies expected in the study area was 20,000 within the year, with an expected prevalence of pregnancy malaria of about 20% in Southern Ghana . The minPregnant women living in Madina and its environs making initial attendance to seek antenatal care at the maternity unit of the AMC for the current pregnancy who had not received IPTp and planned to deliver at the AMC were enrolled for the study from July to August 2008 and followed to delivery. The infants were also followed at monthly intervals at the Child Healthcare Clinics for six months.Ethical clearance was received from the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, Legon. All participants provided informed consent prior to data collection.Demographic survey details as well as information on knowledge, possession and use of Insecticide Treated Nets (ITNs) were collected using a structured questionnaire. Venous blood was collected from each participant into a 5ml ethylene diamine tetra-acetic acid (K3EDTA) vacutainer tubes . Each tube was labelled with the participant's unique identification number and was stored in a cold box, which was immediately transported to the laboratories of the NMIMR. Haematological, parasitological and clinical chemistry analyses including the Glucose-6-phosphate dehydrogenase (G6PD) deficiency test were run for each blood sample. An automatic blood analyzer  was used to obtain the haemoglobin concentration.P. Falciparum malaria parasitaemia, two sets of thick and thin blood films per blood sample were prepared on glass slides and allowed to dry on a rack. The thin films were fixed in absolute methanol and stained with 2% giemsa  for ten minutes. The slides were then washed, left to dry and later examined in immersion oil under the microscope  at X6 eyepiece and X100 oil immersion objective lens [To determine asexual ive lens .The numbInfants were weighed at delivery using a previously calibrated scale at the labour ward and weights recorded on record sheets provided with the mother's unique identification number. The infants born to study participants were followed at monthly intervals for six months. The infants’ body temperature was determined at each visit.Eligible women received Intermittent Preventive Treatment (IPTp) for malaria from hospital staff by direct observed therapy (DOT) at monthly intervals. Information about IPTp administration was initially entered by the hospital staff into the ANC attendance record booklets and was transferred onto the record forms provided for the study.The mean values for various parameters such as haemoglobin levels, and gestation were compared for the various categories of the pregnant women using standard statistical methods. Pair wise and three-way comparisons of mean Hb, proportion of low birthweight and prevalence of parasitaemia and parasite densities were done by Students't-test, ANOVA, and Fishers’ test. A value p< 0.05 was considered significant.The characteristics of the pregnant women are shown as A significant proportion (46.6%) of participants lived outside Madina but within a 20 kilometer radius. Gestation at registration or first booking ranged from 2 weeks to as late as 39 weeks with a mean of 18.5 weeks (95% CI: 17.12 – 19.05). The prevalence of peripheral blood asexual P. falciparum parasitaemia on enrolment was 5% (n=15).The mean haemoglobin level at registration for all the women was 11.44 g/dL (95% CI 11.29 – 11.80). Thirty-two percent of women had mild to moderate anaemia based on Hb<11g/dL as a cut-off. The lowest Hb recorded was 8.4g/dL among two women  The meanThree percent of babies were of low birth weight. There was no significant association between the weight of babies and the number if IPTp doses received. None of the women who delivered infants with LBW had peripheral parasitaemia or anaemia at the time of first ANC registration but were among those who did not receive any IPTp during pregnancy. Microscopic examinations of the placental smears showed a 2.5% (n=3) prevalence of placental malaria at delivery. There was no association between placental parasitaemia and the incidence of LBW.Knowledge of bed nets was high (97.3%). Sixty one percent of the women had ever slept in a net, however only 5% had slept under an ITN the night preceding the interview. ITN coverage was 31.6%. Fifty-five percent of women received IPT during pregnancy. Of those who received IPTp, 38% received three doses .The present study examined the prevalence of pregnancy associated malaria and its associated consequences including maternal anaemia and the incidence of low birth weight of infants and placental malaria among a group of pregnant women in a low malaria transmission setting in coastal Ghana. The prevalence of malaria parasitaemia on enrolment was much lower than what is reported in other studies in Ghana , 11.Although, the absence of peripheral blood malaria parasites might reflect low rates of transmission of malaria in the study area (as indicated by relatively high Hb levels) or wide spread use of antimalarials as a result of the improved education on malaria during pregnancy in Ghana, in general, it is plausible that many women may have had submicroscopic peripheral blood parasites undetected by microscopy but detectable by the more sensitive PCR method , 17. ThePregnant women with anaemia may have an increased risk for poor pregnancy outcomes, particularly if they are anaemic in the first trimester. Although women in the present study had a relatively low prevalence of anaemia compared with other studies in Ghana , 11 almoLow birth weight attributable to malaria is associated with infant mortality. Only 3% of infants in this study had low birth weight. IPTp has been shown to improve birth weight , 26 hencAlthough malaria transmission in Madina is low to moderate, use of ITN was low despite high awareness levels. Low utilization of insecticide-treated nets was also observed in a malaria endemic area in Burkina Faso  where usSome limitations must be acknowledged. First, helminthiasis and nutritional deficiencies are important causes of maternal anaemia in pregnancy. These factors were not investigated in the present study. Second, we were unable to examine the association between malaria parasitaemia and maternal anaemia and birth weight due to the small number of women who tested positive for P. falciparum infection. Third, although malaria transmission is seasonal with a peak during the rainy season (July to September), the very low prevalence of malaria among the pregnant women limited our ability to assess seasonality effects.The malaria prevalence on enrolment among the pregnant women was very low and was mostly asymptomatic. None of the women who delivered infants with LBW had peripheral parasitaemia or anaemia at the time of first ANC registration in their second to third trimesters. A high coverage of IPTp showed a reduced risk in maternal anaemia and LBW. Post partum studies need to be carried out on women who take IPTp. Although three of the women in the present study with G6PD deficiency had malaria infection at first presentation at ANC, the number was too low to make any meaningful conclusion about its protective effect. High knowledge of ITNs has not translated into use and even the few women who had ITNs were not sleeping in them. The non-use of ITNs has implications for malaria infection. The absence of clinical symptoms in the infants does not rule out infection especially with the low rate of use of ITNs.


Fulani ethnic group individuals are less susceptible than sympatric Mossi ethnic group, in term of malaria infection severity, and differ in antibody production against malaria antigens. The differences in susceptibility to malaria between Fulani and Mossi ethnic groups are thought to be regulated by different genetic backgrounds and offer the opportunity to compare haematological parameters, Tregs and γδT cell profiles in seasonal and stable malaria transmission settings in Burkina Faso. The study was conducted at two different time points i.e. during the high and low malaria transmission period.Two cross-sectional surveys were undertaken in adults above 20 years belonging either to the Fulani or the Mossi ethnic groups 1) at the peak of the malaria transmission season and 2) during the middle of the low malaria transmission season. Full blood counts, proportions of Tregs and γδ T cells were measured at both time-points.P. falciparum infection rates and parasite load. Differential white blood cell counts showed that the absolute lymphocyte counts were higher in the Mossi than in the Fulani ethnic group at both time points. While the proportion of CD4+CD25high was higher in the Fulani ethnic group at the peak of malaria transmission season (p = 0.03), no clear pattern emerged for T regulatory cells expressing FoxP3+ and CD127low. However CD3+γδ+ subpopulations were found to be higher in the Fulani compared to the Mossi ethnic group, and this difference was statistically significant at both time-points (p = 0.004 at low transmission season and p = 0.04 at peak of transmission).As previously shown the Fulani and Mossi ethnic groups showed a consistent difference in Our findings on regulatory T cell phenotypes suggest an interesting role for immune regulatory mechanisms in response to malaria. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities. The pathogenesis of malaria is complex and most likely entails immunologic and non immunologic mechanisms . The malPlasmodium falciparum infection rates, malaria morbidity, prevalence and levels of antibodies to various P. falciparum antigens between Mossi and Fulani ethnic groups, where the Fulani ethnic group individuals are clearly less parasitized, and less affected by the disease [β, TGFβRs, CTLA4, and FOXP3 genes in Fulani compared with Mossi ethnic group or European donors not exposed to malaria [+ CD25+ Tregs are induced rapidly in humans following P. falciparum infection and are associated with a burst of the transforming growth factor-β (TGF-β) production, decreased parasite-specific immune responses, and higher rates of parasite growth. IL-10 from CD4+CD25-Foxp3-CD127- adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection [P. falciparum malaria exhibit a marked increase in the number of peripheral blood γδ T cells, which remain elevated for more than a month after treatment [P. falciparum in vitro [in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that required granulysin [P. falciparum malaria haematologic disorders which are the most common complications, play a major role in these fatal complications. These changes involve red blood cells, leukocytes, and haemostasis [Considering clinical protection against malaria in resident populations of endemic areas, previous studies have shown consistent differences in  disease ,3,8-10.  disease . The dif malaria . CD4+ CDnfection . Similarnfection . It has reatment . Subsequin vitro -18 and tanulysin . γδT celanulysin . γδ T ceanulysin . Human ganulysin  suggestianulysin . A similanulysin . As bloomostasis . HaematoIn this study we have assessed haematological parameters, Tregs and γδ T cell profiles in the Mossi and Fulani ethnic groups living in a seasonal and stable malaria transmission setting of Burkina Faso at two different time points during the malaria transmission period in order to evaluate seasonal variation of haematological parameters and lymphocytes subsets (Tregs and γδ T cells) distribution and their impact on malaria susceptibility.Anopheles gambiae, An. arabiensis and An. funestus. Two chromosomal forms of An. gambiae and An. funestus are sympatric in the study area [Details descriptions of study area have been reported elsewhere ,3. The sudy area . The MosAdults aged more than 20 years from the Mossi and Fulani ethnic groups were recruited under a protocol approved by the Ethical Committee for Biomedical Research of the Ministry of Health, Burkina Faso and by the Ethics Committee of World Health Organization (Geneva). The study was conducted in compliance with International Conference on Harmonisation's Good Clinical Practice principles, the Declaration of Helsinki and the regulatory requirements of Burkina Faso. Individual written informed consent was obtained from all participants. Individuals were eligible for inclusion in the study if they were found to be healthy at a general medical examination and gave written informed consent. Exclusion criteria included: i) Pregnant women confirmed positive by the home pregnancy test (HPT), ii) any confirmed or suspected condition of immunosuppressive diseases including HIV (no screening test was been done for this purpose) by the physician; iii) chronic administration (defined as more than 14 days) of immunosuppressant or other immune-modifying drugs at the beginning of the study ; iv) any chronic diseases  suspected by the physician to cause any supplementary risk to the volunteer; v) any other circumstances and condition suspected by the physician to be a risk for the volunteer health.This study was a cohort study that involved Fulani ethnic group  and the Mossi ethnic group . Two cross-sectional surveys were carried out, one during the malaria high transmission season 2007 (October) and one during the low transmission season in 2008 (May) in both ethnic groups. A list of potential adult volunteers has been generated based on the general census database. After explaining the study procedures, only those adults willing to participate were invited to attend to the study scheduled visits. For each adult who fulfilled eligibility criteria, a medical history was recorded, axillary temperature measured, physical examination done. Blood samples were obtained for malaria thick/thin smears, 4 ml in EDTA tube for haematological full blood count and 12 ml in Heparinated tubes for immunological parameter measurements.Thick and thin blood films prepared from finger prick blood samples, were air-dried and stained with 5% Giemsa. Asexual and sexual parasites were counted separately and species differentiated. Malaria parasites were counted against 200 white blood cells (WBC). A slide was declared negative only after reading against 2000 WBC without observation of a malaria parasite. The parasite count was converted to a parasite density per μL of blood. A full blood count was performed using a semi-automated haematology analyzer (Pentra 60).® 1077-1, St Louis, USA). The isolated PBMC were washed and adjusted to a concentration of 20 × 106 cells/ml in a freezing solution (20% DMSO and 80% FBS) and after 18-24 hours of temporary storage at-80°C freezer, the cells were then transferred to liquid nitrogen until analysis.Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous whole blood by gradient centrifugation on Ficoll-Hypaque (Histopaque 1077: Sigma2 for intracellular detection of cytokines. Mitogens as PMA (10 ng/ml) and Ionomycin (1 μM) and at the same time protein inhibitor Brefeldin A (10 μg/ml) was added. An amount of 1 × 106 PBMCs were use for staining procedure according to manufacturer's instructions and about 105 CD4+ were acquired. Acquisitions were done in a three-colour Flow cytometer Cyflow SL with a blue LASER (488 nm) . Data acquisition and FCS files analysis were done on FloMax Software. Table + Tregs as CD4, CD25 and intranuclear FoxP3 phenotype T cells subset based on the notion that CD4+CD25+Foxp3+ subsets exhibit suppressive functions in vitro [Thawed PBMCs were stained for surface and intracellular markers. Fluorochrome-linked monoclonal antibodies were: anti-CD3 PE , anti-CD4 PC5 , anti-CD25 FITC , anti-CD127 PE , anti-TCR Panγδ PC5 , anti-TCR Vδ2 FITC , anti-FoxP3 PE with fixation and Permeabilization kit , anti-IFNγ PE , anti-IL10 PE . Isotype-controls fluorochromes conjugated were used for positive and unspecific fluorescence threshold setting. PBMCs were stimulated for 3 h at 37°C, 5%COin vitro .All data were reported in Microsoft Office Excel 2003 files. Statistical analysis was performed in Stata 9 Software.t-test where indicated. For all statistical tests, p < 0.05 was considered significant.Differences in parasitemia, cytokine levels, and bioluminescence between both ethnic groups and seasons were analyzed using the Student's P. falciparum indexes were higher in the Mossi ethnic groups during both transmission periods and the difference was statistically significant (p = 0.02) during the low transmission and p < 0.01 during the peak of malaria transmission. P.falciparum positive density in Mossi ethnic group were 733 (417-1050) and in Fulani ethnic group 405 (0-947).During the first cross-sectional survey conducted at the peak of the malaria high transmission period 133 adults above 20 years were enrolled (73 Fulani and 60 Mossi). At the second cross-sectional survey, 86 volunteers were re-bled (44 Fulani and 42 Mossi). The sex ratio and the mean ages were similar between both ethnic groups during the surveys (Table p = 0.01), red blood cells counts were similar in both ethnic group during the two time points studied. White blood counts were higher in the Mossi than the Fulani ethnic group and this difference was statistically significant during the malaria high transmission season (p = 0.001) but not during the malaria low transmission season (p = 0.10). Differential white blood cell counts showed that the absolute lymphocytes counts were higher in Mossi than in Fulani ethnic group during both transmission periods (Table p < 0.0001), while it was similar in both ethnic groups during the low transmission season. Eosinophils count was higher during the low transmission season in the Mossi ethnic group but did not differ during the high malaria transmission season.Except for haemoglobin levels, which were higher in Mossi ethnic group during the peak of malaria transmission  with a p = 0.03 . Similar findings were observed for absolute numbers (Data not presented). Then we performed a second panel with also surface markers CD4, CD25 and CD127 assuming that CD25+CD127low subset are proportional to Tregs population . In term of proportion of CD4+CD25+FoxP3+ T cells, no differences between Fulani and Mossi ethnic group at low transmission  and high transmission  seasons were found  compared to Mossi (5.23%) ethnic group mainly at the high transmission season, the difference was not statistically significant (p = 0.07)  and Vδ2 panel. Data on γδ T cells subsets are summarized in Table +Panγδ+ (TCRγδ+) in Fulani ethnic group were higher compare to Mossi ethnic group (with respectively 13.23% and 5.59%). In Mossi ethnic group we noted that the CD3+Panγδ+ proportion remains stable through the season, whereas in Fulani ethnic group the proportion of these cells was double of that measured in Mossi ethnic group at the low transmission season and this difference was highly significant (p = 0.004) . Although the percentages of Vδ2+ predominant among circulating γδ T cells subsets were similar in Fulani compared to Mossi ethnic group during both surveys the differences did not reach statistical significance (p = 0.12 and p = 0.48 respectively). In vitro stimulation of PBMCs for IFNγ secretion by γδ T cells showed that Vδ2+ IFNγ producing cells proportion , no difference was seen may be due to relative low parasite density. There was no evidence for an association between infected and non infected volunteers (Data no shown). This is not consistent with previous findings where WBC and low lymphocytes counts were associated to malaria infection ,31,32. AWe also found that haemoglobin level, absolute monocyte counts were higher in the Mossi ethnic group with a statistical significance during the high malaria transmission season although the medical examination showed that all the volunteers were apparently healthy. This could be due to differences in the immune status of the study subjects related to the level of malaria endemicity, or it could be due to a possible difference in genetic background, physiological feature which may cause differences in cells activation .+CD25+FoxP3+ subsets were similar in Fulani and Mossi ethnic groups at the low and high malaria transmission period. These data suggest that the generation of CD4+CD25+FOXP3+ T cells is not compromised in Fulani ethnic group compared to Mossi ethnic group but they do not indicate whether these cells are endowed with full suppressive activity. A previous study in the same area by Torcia et al., reported that immune modulating genes  were less expressed in CD4+CD25+ cells of the Fulani ethnic group compared to Mossi ethnic group [+ cells in suppressing cell proliferation induced by malaria antigens.Regulatory T cells are reported to be key mediators of peripheral immune homeostasis, and control tolerance to self antigens ,37, and ic group  as measuet al. in the same ethnic groups during the malaria high transmission period. In that study microarrays techniques were used, using RNA from CD4+CD25+ cells, and they could show that immune modulating genes  were less expressed in the Fulani ethnic group. The low activities of Tregs have also been emphasized by the lower rate serum level of TGFβ and higher concentration of proinflammatory chemokines CXCL10 and CCL2 in Fulani ethnic group. In addition depletion of CD25+ Tregs the proliferative response of Fulani ethnic group to malaria specific antigens while this response was significantly increased in Mossi ethnic group [An interesting data emerging from this phenotypical analysis is that the frequency of CD4+CD25+secreting IL10 is higher in the Fulani ethnic group compared to Mossi ethnic group. This IL10 producing subset of regulatory T cells with CD4+CD25+FOXP3 immune phenotype has been reported in murine models of malaria infection . A possiic group . The disic group ,44.+ T cells is observed in many infectious diseases. The increase in γδ T Cells during the first decade of life is largely limited to the Vγ9/Vδ2+ population, causing a shift towards the Vγ9 dominance seen in Caucasian adults [+ cells in the African compared to the Caucasian donors but also due to Vδ2- subset. In conclusion, the high proportion of TCR γδ Cells among peripheral CD3+ cells is mainly due to expansion of others Vδ+ subsets. The phenotyping of γδ T cells subsets has revealed that the proportion TCR γδ+ population was significantly higher in Fulani ethnic group than in Mossi ethnic group. None of the subpopulations (Vδ2+ or Vδ2-) showed a particular pattern in both ethnic groups. A similar trend was observed for the IFNγ+ secreting subsets. Although we were not able to establish a relationship between the parasite load and the frequency of TCRγδ+ because of low parasite density and prevalence in our study population, the cytotoxicity role attributed to TCRγδ+ against P. falciparum [+ cells support the hypothesis that the protection against malaria of Fulani ethnic group might be at least partially mediated by some T cells subset.Expansion of Vγ9/Vδ2n adults . The inclciparum -19 mightOur findings on regulatory T cells phenotypes further on IL 10 producing cells suggest an interesting role of immune regulatory mechanisms in malaria response. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities.The authors declare that they have no competing interests.GSS participated in study design, samples collection, lab work, data analysis and manuscript preparation. TRW participated in study design, samples collection, lab analysis, data analysis and manuscript preparation. ALO participated in study design, lab analysis, data analysis and manuscript preparation. AD participated in lab analysis, data analysis and manuscript preparation. AO participated in study design, clinical collection and manuscript preparation. JBY participated in clinical collection and manuscript preparation. EO participated in clinical collection and manuscript preparation. FV participated in study design and manuscript preparation. CB participated in study design, data analysis and manuscript preparation. MTB participated in study design, data analysis and manuscript preparation. DM participated in study design, data analysis and manuscript preparation. Ado study design, data analysis and manuscript preparation. MGT participated in study design helped to draft the manuscript. YT participated in study design, data analysis and manuscript preparation. SBS participated in study design, data analysis and manuscript preparation. IN participated in study design, data analysis and manuscript preparation and overall study supervision. All authors read and approved the final manuscript.