Plasmodium vivax remains unexplored. Because acute vivax malaria has been related to pro-inflammatory responses, the main hypothesis investigated in this study was that Plasmodium vivax infection is associated with elevated levels of circulating MPs, which may play a role during acute disease in non-immune patients.In the last few years, the study of microparticles (MPs) - submicron vesicles released from cells upon activation or apoptosis - has gained growing interest in the field of inflammation and in infectious diseases. Their role in the human malaria parasite P. vivax infections from an area of unstable malaria transmission in the Brazilian Amazon. The MP phenotype was analysed by flow cytometry using the classical MP marker, annexin, and fluorochrome-labeled monoclonal antibodies against specific cell surface markers. The frequencies of plasma MPs in P. vivax patients (n = 37) were further compared to malaria-unexposed controls (n = 15) and ovarian carcinoma patients (n = 12), a known MPs-inducing disease non-related to malaria.Plasma MPs were analysed among thirty-seven uncomplicated P. vivax patients, as compared to healthy age-matched malaria-unexposed controls. Although platelets, erythrocytes and leukocytes were the main cellular sources of MPs during vivax malaria, platelet derived-MPs (PMPs) increased in a linear fashion with the presence of fever at the time of blood collection  and length of acute symptoms . Finally, the results suggest that plasma levels of PMPs diminish as patient experience more episodes of clinical malaria .The frequencies of plasma circulating MPs were markedly increased in P. vivax infection, and platelet derived-MPs may play a role on the acute inflammatory symptoms of malaria vivax.Abundant circulating MPs are present during acute  Plasmodium vivax is the most widespread Plasmodium species being responsible for up to 390 million clinical cases each year [P. vivax infection was considered a benign and self-limited disease, especially when compared to the burden of Plasmodium falciparum infection. Recent studies from Indonesia, Papua New Guinea and India have, however, highlighted the association between vivax malaria and life-threatening manifestations, such as respiratory distress, severe anaemia, and neurological manifestations with coma [P. vivax infection in a higher status as a global health concern.ach year ,2. For lith coma , placingP. vivax infection concerns its poorly explored pathogenesis. While a large bulk of literature on the immunological and inflammatory patterns of falciparum malaria is available, the pathogenesis of vivax malaria was not submitted to such scrutiny. It is, nevertheless, intriguing that P. vivax infection triggers fever with a lower peripheral parasitaemia [P. vivax infection induces greater host inflammatory responses compared to P. falciparum malaria [An additional neglected effect of the "benign" sitaemia -8. The f malaria -11 with  malaria -13. HencP. falciparum cerebral malaria by transferring platelet antigens to the surface of iRBCs and dramatically enhancing their cytoadherence to endothelial cells within brain micro vessels [P. berghei malaria by inducing potent activation of macrophages [Cellular microparticles (MPs) - submicron (< 1 μm) membrane vesicles released from cells into the blood stream in response to a variety of stimuli, i.e. cell activation, stress, serine proteases, pro-inflammatory cytokines and apoptosis, have emerged as potential inflammatory biomarkers as elevated levels are found in plasma during many diseases of inflammatory etiology . Consequ vessels ,18. MPs rophages . HoweverP. vivax infection is associated with elevated levels of circulating MPs of different cellular origins, and those MPs play a role in P. vivax induced inflammation. For this purpose, the phenotype of circulating MPs in plasma from P. vivax malaria patients in a hypoendemic area of the Brazilian Amazon was assessed. As control, the plasma MP phenotype from healthy malaria-unexposed donors and patients with ovarian carcinoma, a known MPs-inducing disease non-related to malaria, was simultaneously investigated. The results showed that platelet (PMPs), erythrocyte (EMPs) and leukocyte (LMPs) derived-MPs are significantly increased in plasma from P. vivax patients as compared to healthy donors. Interestingly, the frequencies of annexin V/CD41a double-positive MPs (PMPs) were associated with the length of acute illness and the presence of fever at the time of blood collection. The results presented here comprise the first evidence of the presence of MPs during vivax malaria and offer a new tool for the study of the pro-inflammatory patterns of P. vivax infection.The main hypothesis to be investigated here is that Anopheles darlingi and the number of infective bites associated with malaria is estimated to be between 2 to 10 per inhabitant per year [This study was conducted between January and November 2008, in the outpatient clinic of the Tropical Medicine Center of Porto Velho (CEMETRON), the capital of Rondônia State, Brazilian Amazon area. In the Amazon area, malaria is mainly an occupational disease, with population at risk largely represented by adult males. Low rates of transmission occur throughout the year with large dominance of vivax over falciparum malaria [per year .P. vivax, were invited to participate in the study. Selected volunteers were all negative for P. falciparum and/or Plasmodium malariae infection by both microscopic examination and a nested-PCR, carried-out latter. Thirty-seven patients, aging 15 to 66 years, were enrolled in the study after the written informed consent, in accordance with guidelines for human research, as specified by the Brazilian National Council of Health (Resolution 196/96). Antimalarial and supportive therapies were given according to standard protocols.Individuals who sought care at CEMETRON and whose thick blood smear was positive for P. vivax infected-volunteers.Clinical and demographical data were acquired through a standardized questionnaire, and the haematological profile was assessed by automated complete blood count carried-out at CEMETRON's hematology facility. Table In the studied area, subjects were migrants from malaria-free areas. Consequently, their ages do not necessarily correlate with malaria exposure. Cumulative exposure to malaria was, therefore, based on self-reported number of lifetime malaria episodes. As their previous malaria experience showed a variable number of infections, extreme values and the possibility of recall bias were avoided by categorizing patients into three groups: i) five or less previous malaria episodes (PME); ii) 6 to10 PME; and ii) more than 10 PME. Venous blood was obtained from patients on admission, and in those with fever (above 37.5°C) at the time of blood collection. In five patients it was possible to take additional samples, during convalescence, on day 7 and 21 after treatment. Plasma samples from fifteen age-matched malaria-unexposed donors were used as baseline control. As MPs positive samples plasma from 12 patients, aging 45 to 65 years, diagnosed with ovarian carcinoma, as it has been shown that cancer cells from this highly lethal gynaecological malignancy can release MPs .g for 15 min, and plasma was then cooled to -20°C before storage at -80°C. Samples were further centrifuged at 13,000 x g for 3 min to obtain platelet-free plasma. The latter was diluted 1:3 in citrated PBS containing heparin and centrifuged at 14,000 x g for 90 min at 15°C. The resultant MP pellet was then resuspended in 1× annexin V binding buffer .MPs were prepared as described elsewhere ,19. Brie+ events gated within the R2 region were further assessed for immunolabeling with FITC-conjugated mAbs against the cell markers CD41a (platelets), CD144 , CD235a (erythrocytes), CD45 (leukocytes) and CD14 (monocytes) or the correspondent mouse IgG FITC-conjugated isotype control mABs. The samples were analysed in a Flow Cytometry FACSCalibur . Over 100,000 events were acquired on each sample, to reach at least 2,000 events within the MPs gate.Unless otherwise stated, all reagents and mAbs used in the Flow cytometry experiments were from BD Biosciences. MPs isolated from plasma were gated (R1) based on their forward (FSC) and side (SSC) scatter distribution as compared to the distribution of synthetic 0.7 - 0.9 μm SPHERO™ Amino Fluorescent Particles  Figure . Taking To confirm if the absolute numbers of plasma MPs were different between malaria patients and healthy donors, as well as to determinate the numbers of plasma MPs per microliter (MPs/μl), the cytometer was set to operate at a high flow rate setting for 60 seconds for each sample. The number of MPs/μl of plasma was calculated as described elsewhere : MPs/μl Data were analysed using Graph pad Instat 4.0 and SPSS 15.0 statistical software. Differences in the means of the frequencies (double staining for annexin V and cell markers) between groups were analysed using two-tailed student's t test or Mann-Whitney when data did not fit a Gaussian distribution. The log-transformation of data was applied for situations where variances normality assumptions failed followed by linear regression to investigate the association between the clinical parameters and MPs levels. Pairwise correlations were evaluated with Pearson's correlation coefficient ρ. Multiple linear regression models with stepwise backward deletion were built to describe independent associations between covariates  and the presence of PMPs. A P value < 0.05 was considered to be statistically significant. For illustration purpose , erythrocyte (EMPs) (p = 0.0023) and leukocyte derived-microparticles (LMPs) (p = 0.0087) were significantly increased in plasma samples from malaria patients as compared to healthy donors. The increased levels of PMPs, EMPs and LMPs were confirmed when the absolute number of MPs per μl of plasma was calculated . These results suggest that platelets, erythrocytes and leukocytes are the main source of plasma circulating MPs during acute P. vivax malaria and its presence may be associated with inflammation.The frequency of platelet (PMPs) p < 0.000, erythroP. vivax infection were analysed, including demographic, clinical, haematological and parasitological parameters. No association was found between levels of MPs and haematological  or parasitological data. Of note, positive linear trends were observed between the presence of fever at the time of blood collection and the frequency of PMPs in plasma    Figure . Signifi) Figure . More sp) Figure . No sign) Figure .Although manifestation of acute malaria such as fever can be influenced by different factors including host age and immunological background - evaluated here by previous malaria experience - multiple linear regression models confirmed the independent association between fever and microparticles, as well as length of symptoms and microparticles (p < 0.001).P. vivax patients, plasma MPs levels were assessed 7 and 21 days after anti-malarial chemotherapy. The levels of plasma MPs remain unchanged at 7 days post infection; however, as shown in Figure In five P. vivax associated with severe and fatal disease, little is known about its pathogenesis. This study provides the first evidence that microparticles (MPs) from leukocytes, platelets, and erythrocytes are significantly increased in plasma from acute P. vivax malaria patients. These results are consistent with the observation that active, non-cytokine, factors are present in paroxysm plasma from non-immune patients with acute P. vivax infections [P. vivax paroxysms. However, at this time, it is not possible to conclude on the role of MPs during paroxysm, because it remains as undefined complex phenomenon, which involves several components, including host-derived cytokines [Despite increasing evidence of multidrug-resistant fections ,24. As sP. vivax malaria is not unexpected. It rather suggests that different cell types are activated and participate in the myriad of inflammatory processes induced by P. vivax parasites/infection. In fact, these data are in concordance with a recently published study reporting that children infected with P. falciparum presents high levels of circulating MPs, including those from platelet, erythrocytes, endothelial cells and leukocytes [P. vivax, and by others with P. falciparum [As virtually all cell types can release MPs, the presence of MPs of different origins circulating during acute ukocytes . In theslciparum , which sP. vivax infection is not clear, but they are of particular interests as both platelets [P. falciparum. Moreover, MPs have been shown to be mediators of the inflammatory process that occur during infection with Plasmodium berghei in mice [In the present study, elevated levels of platelet-derived microparticles (PMPs) were associated with clinical manifestation of disease, such as fever and prolonged time with malaria symptoms. At this time, the significance of this finding in latelets  and PMPslatelets  have bee in mice . Recentl in mice  providesP. falciparum infection, PMPs levels significantly correlated with coma depth and thrombocytopenia [Although evidence for a role of microparticles in human malaria diseases at present is still only limited, new evidence is accumulating rapidly to support this theory. In Cerebral Malaria, the most severe complications of ytopenia . ConsequP. falciparum PMPs facilitate cytoadherence of iRBCs to brain endothelial [P. vivax malaria as suggested by Anstey et al [As in othelial , it is rey et al . Howeverey et al , it is cey et al ,29-31. FA potential limitation of the statistics analysis performed here was that multiple comparison were realized, aiming to find possible associations between MPs and clinical/biological parameters; however, it should be take in consideration that these significant findings were hypothesis-generating, and, consequently, its represent a realistic interpretation of the facts. In addition, multiple linear regression models confirmed the independent association between fever and length of symptoms with platelets-derived microparticles.P. vivax malaria. The role of leukocytes in malaria pathogenesis is still not fully understood; nevertheless, it has been shown that these cells are the main source of pro-inflammatory cytokines, especially TNFα which is highly pyrogenic [P. vivax and P. falciparum infections, malariotherapy studies have shown greater destruction of non-infected RBCs per infected erythrocyte in P. vivax as compared to P. falciparum malaria [P. vivax infection does not enable to conclude on the value of this variable as determinant of severity [An interesting finding was the presence of leukocyte and erythrocyte derived-MPs during acute yrogenic . The preyrogenic . Althoug malaria . Accordiseverity .P. vivax malaria, the data shown here raise important questions on the role of cell derived MPs in malaria pathogenesis revealing a fruitful area for investigation. Thus, the potential of circulating cell derived-MPs as non-invasive inflammatory marker of P. vivax morbidity is likely.While it is not possible at this time to characterize the mechanism by which MPs might be involved in the pathogenesis of P. vivax patients platelets derived-MPs were significantly correlated with fever and length of symptoms. Elucidation of the microparticles composition and the mechanisms involved in exertion of their effects could contribute to additional intervention strategies against malaria.This paper describes the initial attempts made at characterization of the MP phenotype in vivax malaria. It is shown here that the plasma levels of platelet, erythrocyte and leukocyte-derived MPs are increased during acute uncomplicated vivax malaria. In The authors declare that they have no competing interests.FFMC and BSF were involved in all stages of this study. CJF coordinated the field study. ATC and CFAB gave substantial constructive advice in the initial design of the project. ALSF and SCOP selected clinically the ovary carcinoma patients. LHC accepts direct responsibility for the conception and coordination of the study. All authors have read and approved the final version of the manuscript.P. vivax patients, before and after antimalarial treatmentQuantification of platelet, erythrocyte and leukocyte derived-MPs levels in plasma from . Plasma levels of platelet, erythrocyte and leukocyte derived-MPs were calculated as described in Methods; results are expressed as MPs/μl. The absolute numbers of plasma MPs was compared among healthy donors and P. vivax malaria patients. Statistical analysis was performed using the Mann-Whitney two-tailed test. A p value < 0.05 was considered significant.Supplementary Figure.Click here for file


Salmonella Typhi-associated infection and was discovered to be heterozygous for haemoglobin E. Despite treatment with appropriate antibiotics, the patient's condition did not improve, prompting further investigation, which revealed malaria due to Plasmodium falciparum. Dhaka is considered a malaria-free zone, and the patient denied recent travel outside Dhaka. Subsequently, the patient recovered fully on antimalarial therapy.A 12-year old girl from Uttar Badda, Dhaka, Bangladesh, was admitted to the Dhaka Hospital of ICDDR,B, with a 23-day history of fever and diarrhoea. After admission, she was treated for culture-proven  Plasmodium falciparum cases has been increasing at an alarming rate since 1999 and reached 83% in 2008  1:160, TH (antibody against flagellar antigen) 1:320. Peripheral blood film was reported as normocytic and normochromic; the Hb electrophoresis report revealed haemoglobin A to be 71.5%, haemoglobin E 25.3%, and haemoglobin A2 3%. Chest x-ray findings were normal, and ultra-sonogram of the whole abdomen showed mild hepato-splenomegaly. At that time, a diagnosis of typhoid fever with haemoglobin E trait was made. She was treated with ceftriaxone for 11 days, followed by azithromycin for seven days as she did not respond. Despite this, her temperature failed to settle. Antibiotics were stopped for 48 hours, and blood and urine cultures were sent again on the 21st hospital day. No growth of organisms was reported. ICT for kala-azar was negative. A blood-film showed trophozoites of P. falciparum. So, after three weeks of admission, a diagnosis of malaria due to P. falciparum was made. She was treated with oral quinine for seven days and became afebrile on the second day after starting treatment. She was discharged five days after starting quinine, completing the full course of treatment at home.Investigation revealed a haemoglobin of 9.5 g/L, total peripheral white blood cell count of 13,360/cmm, of which 67% were neutrophils, 27.5% lymphocytes, and 4.9% monocytes, and platelet count of 89,000/cmm. Blood culture grew P. falciparum is the most widespread and dangerous of the four Plasmodium species and can lead to fatal cerebral malaria if left untreated. In this case, it is interesting that our patient suffered from continuous fever due to P. falciparum-associated malaria for 44 days without treatment and did not yet deteriorate. It is evident from previous studies that P. falciparum grows slowly within both homozygous and heterozygous forms of haemoglobin E red blood cells (RBCs), which inhibits parasitaemia  are available for malaria, with over 90% sensitivity and specificity whereas there is a chance of missing malarial parasites in microscopy, especially when the parasite count is low and when the microscopists are not used to seeing malaria. It would also be interesting to gather information from the main diagnostic centres and blood-banks in Dhaka that routinely perform RDTs for malaria to investigate the possibility that Dhaka is no longer malaria-free.Our patient was infected by malaria due to


Plasmodium falciparum and lactate deshydrogenase from all Plasmodium species.The rapid diagnostic tests for malaria (RDT) constitute a fast and opportune alternative for non-complicated malaria diagnosis in areas where microscopy is not available. The objective of this study was to validate a RDT named Parascreen™ under field conditions in Iquitos, department of Loreto, Peru. Parascreen™ is a RDT that detects the histidine-rich protein 2 (HRP2) antigen from Parascreen™ was compared with microscopy performed by experts (EM) and polymerase chain reaction (PCR) using the following indicators: sensitivity (Se), specificity (Sp), positive (PV+) and negative predictive values (PV-), positive (LR+) and negative likehood ratio (LR-).P. falciparum malaria, Parascreen™ in comparison with EM, had Se: 53.5%, Sp: 98.7%, PV+: 66.7%, PV-: 97.8%, LR+: 42.27 and LR-: 0.47; and for non-P. falciparum malaria, Se: 77.1%, Sp: 97.6%, PV+: 91.4%, PV-: 92.7%, LR+: 32.0 and LR-: 0.22. The comparison of Parascreen™ with PCR showed, for P. falciparum malaria, Se: 81.8%, Sp: 99.1%, PV+: 75%, PV-: 99.4, LR+: 87.27 and LR-: 0.18; and for non-P. falciparum malaria Se: 76.1%, Sp: 99.2%, PV+: 97.1%, PV-: 92.0%, LR+: 92.51 and LR-: 0.24.332 patients with suspected non-complicated malaria who attended to the MOH health centres were enrolled between October and December 2006. For Plasmodium species, in addition to the genetic characteristics of the parasites in the area, must be considered before applying any RDT, especially after the finding of P. falciparum malaria parasites lacking pfhrp2 gene in this region.The study results indicate that Parascreen™ is not a valid and acceptable test for malaria diagnosis under the field conditions found in the Peruvian Amazon. The relative proportion of  Plasmodium falciparum and Plasmodium vivax remains an important public health problem, especially in the Amazon region where more than 70% of the cases of the country are reported .Table P. falciparum and P. vivax infections. For P. falciparum, the indicators were: Se: 81.8%, Sp: 99.1%, PV+: 75%, PV-: 99.4, LR+: 87.27 and LR-: 0.18. For non-P. falciparum the following indicators were observed: Se: 76.1%, Sp: 99.2%, PV+: 97.1%, PV-: 92.0%, LR+: 92.51 and LR-: 0.24.When PCR was used as gold standard, Parascreen™ showed a better sensitivity for detection of P. vivax infections at parasite densities below 99 parasites/μl, which increased for the detection of parasite densities between 99 and 500 parasites/μl (sensitivity of 60%). For P. falciparum infections, parasite densities below 1,000 parasites/μl were detected with less sensitivity (25%) than parasite densities > 1,000 parasites/μl (sensitivity between 66.7% and 75.0%) (Table Parascreen™ had a low sensitivity (12.5%) for detection of %) Table .Plasmodium species  and parasitaemia of the infections.This study, conducted in Iquitos, Department of Loreto, showed an unacceptable performance of Parascreen™ for malaria diagnosis under the field conditions found in the Peruvian Amazon. Even though the RDT was of simple training and use for the local health staff, it showed variable indicators when compared with EM and PCR, depending on the P. falciparum infection was lower than that obtained with OptiMAL™ which detects Plasmodium lactate deshydrogenase. HRP2-based RDTs such as Parascreen™ have also been evaluated in various studies in South American countries [P. falciparum and P. vivax infections observed in this study were better than those reported with AMRAD fast test ICT P.f/P v in Peru [P.f/P.v in Colombia, but lower than those observed with NOW-Malaria-ICT RDT in an evaluation study in Colombia [In this study, Parascreen™ sensitivity for malaria infection was lower than the 100% sensitivity obtained in a validity assessment done by Zephyr Biomedical System . In studountries -28. The  in Peru , and simColombia  and in vColombia ,13,26.P. falciparum and non-P. falciparum infections; confirming that patients with positive tests would have a high probability to be infected.The likelihood ratio (Lr) is independent of the prevalence of the disease in the population, and can be used to determine the accuracy of positive or negative test results . In thisP. falciparum infections [On the contrary, the observed likelihood ratios of a negative Parascreen™ test (Lr-), with values above 0.1, indicated a high risk of error when excluding malaria diagnosis in patients with negative tests. Similar findings were found in an evaluation study of OptiMAL™ in febrile travelers returning from malaria endemic countries, especially with fections .More generally, it should be noted that PCR could be more relevant as a reference method for the evaluation of non-microscope procedures like RDTs ,14,31, bRDTs generally achieve a sensitivity of > 90% at densities above 100 parasites per μL of blood, and the sensitivity decreases markedly below that level of parasite density.P. falciparum infections in this study could explain the low Parascreen™ sensitivity for P. falciparum infections, as well as the sensitivity discrepancies when using EM and/or PCR as gold standards. Other possible factors, could consist in the genetic diversity of the parasite antigen HRP-2 [pfhrp2 gene in this region [P. falciparum isolates lacking the pfhrp2 gene in this region was still unknown and unpredictable, but now this recent discovery has to be taken into account when interpreting results of RDT evaluation studies in general.The low number of en HRP-2 , the exis region ,33 and ts region . When thP. vivax infections and ≥1,000 parasites/μl for P. falciparum infections. In general, Parascreen™ sensitivity was low for Plasmodium infections, especially for fatal infections like P. falciparum. This RDT was not acceptable for malaria diagnosis under the field conditions found in the Peruvian Amazon, even though it was easy to learn and to use by the local health staff. The choice of a RDT for Amazonian countries such as Peru, should consider the local malaria incidence and predominance of Plasmodium species, as well as the genetic characteristics of the parasites, especially the existence in this region of P. falciparum field isolates lacking the pfhrp2 gene.The present study showed that Parascreen™ sensitivity was comparable to EM and PCR at parasitaemia levels ≥100 parasites/μl in The authors declare that they have no competing interests.AR, TG, ALL-C, JE and HG participated in designing the study. TG directed the field study. JB performed laboratory work. TG, AR and JB verified data. AR and JB analyzed data and participated in the manuscript preparation. DG was involved at all stages of this study and led the manuscript preparation. All authors have read and approved the final manuscript.


HIV and malaria have similar global distributions. Annually, 500 million are infected and 1 million die because of malaria. 33 million have HIV and 2 million die from it each year. Minor effects of one infection on the disease course or outcome for the other would significantly impact public health because of the sheer number of people at risk for coinfection. While early population-based studies showed no difference in outcomes between HIV-positive and HIV-negative individuals with malaria, more recent work suggests that those with HIV have more frequent episodes of symptomatic malaria and that malaria increases HIV plasma viral load and decreases CD4+ T cells. HIV and malaria each interact with the host's immune system, resulting in a complex activation of immune cells, and subsequent dysregulated production of cytokines and antibodies. Further investigation of these interactions is needed to better define effects of coinfection. HIV and malaria have similar global distributions, with the majority of those affected living in sub-Saharan Africa, the Indian subcontinent, and Southeast Asia. Given the overlap of their geographic distribution and resultant rates of coinfection, interactions between the two diseases pose major public health problems. Together they accounted for over 3 million deaths in 2007 , 2, and Plasmodium and is transmitted by Anopheles mosquitoes. It is endemic in most tropical and subtropical regions of the world. Of the four Plasmodium species that infect humans, P. falciparum is the most virulent and is responsible for the majority of morbidity and mortality due to malaria. Worldwide, 1.2 billion people are at risk for malaria infection, resulting in 500 million infections and more than 1 million deaths each year. The majority of these deaths occur in young children in sub-Saharan Africa, where one in every five childhood deaths is due to malaria [Malaria is caused by the protozoan parasite  malaria , with re malaria .Areas of the world with high rates of malaria also carry a heavy burden of HIV. There are 33 million people living with HIV worldwide, with 22.5 million in sub-Saharan Africa alone. This results in an estimated overall prevalence of 5% in sub-Saharan Africa, with some countries reporting prevalence rates of greater than 25%. While new HIV infections in adults and children have decreased since 2005, there were an estimated 2.5 million children living with HIV in 2007, nearly 90% of whom are in sub-Saharan Africa. It is estimated that 2.1 million deaths in 2007 were due to HIV, of which 1.6 million occurred in sub-Saharan Africa, making HIV/AIDS the number one cause of mortality in that region [Falciparum malaria has a spectrum of clinical presentations, ranging from asymptomatic parasitemia in patients with immunity to severe anemia, cerebral malaria, multiorgan failure, or death.Anemia is most frequently seen in young children and pregnant women  and can P. falciparum can also result in severe anemia, low birth weight, and maternal death during pregnancy [Cerebral malaria and other end-organ damage is mediated through interactions between infected red blood cells and host receptors on the blood vessel wall, resulting in adherence and sequestration of infected red blood cells in the postcapillary venules, obstruction of blood flow, and subsequent tissue damage . Patientregnancy , 12.HIV infects and depletes CD4+ T lymphocytes, putting patients at risk for opportunistic infection and malignancy, the major causes of death due to HIV and AIDS. However, it also has effects on the systemic inflammatory response, causing activation and/or apoptosis in a variety of immune cells as well as elevated levels of proinflammatory cytokines and chemokines in plasma and lymph nodes. This immune activation, rather than being a reflection of antiviral immunity, is associated with HIV-1 disease progression . It is aWhile no population-based studies are available to determine the number of patients affected by coinfection and the impact of coinfection on a population, a mathematical model applied to previously published data was used to estimate the impact of HIV-1 on malaria in sub-Saharan Africa. It estimated that an additional 3 million cases of malaria and 65 000 additional malaria-related deaths annually are due to the impact of HIV . Rates wCohort studies have shown that malaria infection causes an increase in plasma HIV viral load, even during asymptomatic parasitemia. HIV viral load returned to baseline eight weeks after acute malaria infection . It has While early studies found no association between HIV and malaria disease severity in either adults or children , 19 moreIn HIV-uninfected women, risk for symptomatic or placental malaria decreases with each subsequent pregnancy. In HIV-infected women this gravidity-specific pattern is altered, such that multigravidae women carry the same risk of disease as primigravidae women . PregnanIn a cohort study in Kenya, HIV-coinfected women had higher placental parasite densities and higher rates of antenatal malaria transmission than did HIV-uninfected women . MaternaP. falciparum adhesin to chondroitin sulfate A on human placental cells increased HIV-1 replication in those cells, possibly via TNF-alpha stimulation [Cohort studies in Cameroon show that malaria infection during pregnancy may increase the risk of mother-to-child transmission of HIV , 30. Onemulation . Proinflammatory (Th1) cytokines such as TNF-alpha are thought to play an important role in malaria pathogenesis and in cerebral malaria in particular, as they increase surface expression of adhesion molecules on endothelial cells, promoting parasite attachment. Activated CD4+ T lymphocytes stimulate macrophages to produce TNF-alpha, which leads to cerebral malaria in mouse models .In humans, clusters of cytokines may help discriminate between mild, severe, and cerebral malaria. In an adult population in a malaria-endemic region of India, high levels of IL-12, IL-5, and IL-6 discriminated severe forms of malaria from mild malaria. Levels of IL-1beta, IL-12, and IFN-gamma helped to discriminate cerebral malaria from severe malaria, with high IL-1beta levels being associated with cerebral malaria, and high IL-12 and IFN-gamma levels being associated with severe malaria . In a peAdaptive immunity to malaria is thought to confer protection against febrile parasitemia but does not prevent parasitemia. This is due in part to the development of antibodies against proteins on the infected erythrocyte surface, notably VSA .P. falciparum-infected erythrocytes adhere to human dendritic cells, inhibit their maturation, and subsequently reduce their capacity to stimulate T cells [P. chabaudi found that infected erythrocytes induce maturation of dendritic cells, stimulate IL-12 and IFN-gamma production, and cause CD4+ T lymphocyte proliferation [P. yoelii. Overstimulation of dendritic cells by toll-like receptors makes them refractory to further activation in P. yoelii-infected mice [The effect of malaria on dendritic cells, key players in both the early stages of adaptive immunity and innate immunity, is not clear. Urban et al. found that  T cells . Howeverferation . While tted mice . As malaProinflammatory cytokines play an important role both in control and pathogenesis of HIV infection. During infection, viral particles are taken up by antigen presenting cells (APCs), which are then recognized by CD4+ T lymphocytes, causing activation and release of IL-2 and IFN-gamma. These proinflammatory cytokines in turn stimulate CD8+ T lymphocytes, which control viremia. It is not known if this inflammatory response seen in HIV has an effect on the adherence and sequestration seen in malaria. However, HIV upregulates adhesion molecules on endothelial cells, which may compound the adherence and sequestration seen in malaria .HIV also dysregulates pathways of cytokine expression, such that production of the proinflammatory cytokines IL-12 and IFN-gamma is decreased and expression of the anti-inflammatory cytokine IL-10 is increased . As HIV The impaired innate immune response in patients with AIDS may in part account for the increased rates of symptomatic malaria seen in cohort studies. However, the decreased production of IL-12 and IFN-gamma seen in HIV is confounding, as high levels of these proinflammatory cytokines are associated with severe malaria in clinical studies , and preIncreased expression of IL-10 also appears to play a role in loss of adaptive immunity. IL-10 impairs T helper type 1 (Th1) responses . DendritP. falciparum in children under the age of five. Characterized by coma and/or seizures, it is associated with sequestration of parasitized red blood cells in the brain microvasculature. Occasionally brain edema and elevated intracranial pressure are seen.Cerebral malaria is a major cause of death due to Postmortem samples from children who died from cerebral malaria show activation of endothelial cells (with upregulation of ICAM-1) and macrophages , and disruption of endothelial intercellular junctions  in vessels containing sequestered parasitized red blood cells. No leakage of plasma proteins  into brain parenchyma was seen, suggesting that the blood-brain barrier remains intact. However, there were elevations in cerebrospinal fluid albumin taken prior to death, which may indicate blood-brain barrier permeability .In vitro studies have backed up some of these findings. Gillrie et al. showed that parasite sonicates but not intact malaria-infected red blood cells disrupt the endothelial  barrier, revealed by discontinuous immunofluorescent staining of endothelial junction proteins, formation of interendothelial gaps in monolayers, and loss in total protein content of claudin 5 and redistribution of ZO-1 .P. falciparum samples from patients with uncomplicated malaria showed increased mRNA levels of occludin, vinculin, and ZO-1. Those cultured with samples from severe malaria had no change in mRNA levels, and HUVECs cultured with P. falciparum from patients with cerebral malaria had decreased mRNA levels of occludin, vinculin, and ZO-1 [Additionally it appears that severity of malaria infection is associated with differential expression of tight junction proteins. Quantitative PCR of human umbilical vascular endothelial cells (HUVECs) cultured with and ZO-1 . This daP. falciparum, and brain endothelial cells from patients dying of cerebral malaria and from those with malaria who died of other causes also showed increased levels of tissue factor [Not only are endothelial junction proteins affected, but also endothelial cells themselves seem to be influenced by infected erythrocytes, producing inflammatory mediators in response to malaria infection. Endothelial cells produce tissue factor when cultured with e factor . Tissue e factor .falciparum malaria. In P. falciparum isolates from patients with complicated malaria, binding of infected red blood cells to human lung microvascular endothelial cells was observed and was primarily mediated through ICAM-1 and chondroitin sulfate (CSA) [P. berghei ANKA were protected from mortality compared with C57BL/6 controls [Endothelial cells also upregulate adhesion molecules in the setting of te (CSA) . Mouse mcontrols . Additiocontrols .P. falciparum-infected red blood cells to human brain endothelium, suggesting that both are important for cytoadherence [P. chabaudi has been used to study malaria sequestration in mice, and P. chabaudi AS-infected red blood cells adhered to purified CD36 in vitro [Another adhesion molecule that is likely involved in cerebral malaria pathogenesis is CD36, a receptor with a wide tissue distribution that is also found on endothelial cells. Monoclonal antibodies to CD36 and ICAM-1 partially inhibited the binding of dherence . The rodin vitro . This isin vitro .Unlike malaria, HIV is known to invade and infect the brain parenchyma, causing HIV-associated encephalitis and/or HIV-associated dementia. HIV may infect astrocytes at low levels, and it can activate endothelial cells, but it does not infect endothelial cells. HIV is thought to enter brain parenchyma through the regulated transmigration of HIV-infected mononuclear cells across the blood-brain barrier, mediated by surface receptors on endothelial cells such as ICAM-1. Once across the barrier, HIV-infected cells recruit microglia and astrocytes, allowing for subsequent infection of these cells and spread of HIV within the CNS.As HIV replicates within the CNS, it produces inflammatory mediators, including the chemokine CCL2/MCP-1 . This chPlasmodium and indirect effects related to cytoadherence and phagocytosis.Aspartic proteases play key roles in HIV and are thus important therapeutic drug targets. Aspartic proteases are also important in malaria parasites (as plasmepsins), and antiretroviral protease inhibitors seem to have both direct effects on P. falciparum grown in vitro at concentrations achieved in vivo [P. chabaudi AS had a delay in patency and attenuation of parasitemia when given oral ritonanvir/saquinavir or ritonavir/lopinavir [P. berghei, a rodent strain of malaria, saquinavir and lopinavir inhibited development of extra-erythrocytic liver stages in vitro. In vivo mouse studies using the rodent strain P. yoelii showed a reduction in liver parasite burden when lopinavir/ritonavir was administered [The antiretroviral protease inhibitors saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, and atazanavir directly inhibit erythrocytic stages of  in vivo , 57, andopinavir . Some annistered .The antiretroviral protease inhibitors ritonavir and saquinavir affect CD36-mediated cytoadherence, thought to play a role in cerebral malaria and other end-organ damage in severe malaria. The drugs decrease CD36 surface concentrations on C32 epithelial cells in culture, which was associated with a decrease in cytoadherence of parasitized erythrocytes . No effeThe antimalarial drug chloroquine has effects on HIV, inhibiting the production of infectious viral particles by impairing virus glycosylation. Chloroquine also has synergistic effects on HIV suppression with the protease inhibitors indinavir, ritonavir, and saquinavir at concentrations achieved with prophylaxis dosing . In vitrAn additional drug-related interaction between malaria and HIV involves the use of trimethoprim-sulfamethoxazole prophylaxis in HIV-infected patients. In rural Uganda, HIV-infected participants given trimethoprim-sulfamethoxazole had a 76% decrease in rates of malaria compared with when they were not receiving trimethoprim-sulfamethoxazole, and participants who received antiretroviral therapy plus trimethoprim-sulfamethoxazole had a 92% decrease in malaria rates . It is iMalaria and HIV affect millions of people across overlapping geographic distributions, and risk of transmission of both malaria and HIV may be increased due to coinfection. It has been observed that HIV-infected people in areas of malaria transmission have more frequent episodes of symptomatic parasitemia  and highWhile some interactions between malaria and HIV are known, these interactions have not been extensively studied from an epidemiological perspective. Many aspects of the relationship between malaria and HIV remain unanswered. While malaria is a major cause of morbidity and mortality in children in sub-Saharan Africa , little While it is known that pregnant women with HIV have higher rates of symptomatic malaria , placentIt is also unclear if HIV and malaria coinfection have an impact on treatment or clinical outcomes. Does HIV have an effect on resistance to antimalarial drugs, and are there more effective drugs to treat malaria in those who are also infected with HIV? Are there interactions between antiretrovirals used for HIV and antimalarial drugs currently in use, such as artemesin-derivatives? Also important to study are the effects of malaria on HIV prognosis: do the increases in viral load and decreases in CD4+ T lymphocytes seen during acute malaria infection have long term consequences on HIV progression to AIDS? These questions deserve investigative attention.P. falciparum have a synergistic relationship, as both provoke an inflammatory response from immune cells, with subsequent effects on endothelial activation and blood-brain barrier permeability. Investigative methods that adequately mimic these pertinent clinical scenarios are needed. Thus, efforts to coculture malaria and HIV, and to observe the effects of each microbe during coculture, should be pursued. Examining the interactions between malaria and HIV in the setting of immune cells, endothelial models, and models of the blood-brain barrier would shed light on the effects on immune dysregulation, endothelial activation, and blood-brain barrier permeability.In addition to the need for population-based studies of coinfection, microbe-microbe interactions deserve further research efforts as well. It is likely that HIV and JR-CSF model, which has been used to study blood-brain barrier integrity [Effective animal models of coinfection are also needed to look at systemic inflammatory responses and effects on end-organ damage, especially pertaining to cerebral malaria. While no animal model can fully mimic the effects of HIV and malaria coinfection in humans, it is impractical to invasively study dual infection in humans in a controlled, monitored setting. Established murine models of cerebral malaria rely on altered endothelial cell function and the immune response, both of which play a pivotal role in human disease . A pertintegrity . IntegraAs mentioned previously, 500 million cases of malaria occur each year, causing 1 million deaths [


Across tropical Africa the bulk of malaria-related morbidity and mortality is particularly high during childhood. Classical malariometric surveys have relied on assessing malaria infection prevalence. The last comprehensive evaluation of the malaria situation in Mozambique was carried out during the 1950s. This study aims to characterize the malaria transmission intensities and to estimate the disease burden that may help guide control programme.Between February 2002 and April 2003, a house-to-house survey, was carried out in 24 districts randomly selected. A total of 8,816 children aged below 10 years old were enrolled. Finger prick and blood collection were performed to prepare thick and thin films for malaria parasite species identification, density and haemoglobin concentration. Axillary temperature was also measured. Prevalence of infection, parasite density and anaemia were estimated for age groups category in each region/stratum. Comparisons between proportions were made using Chi-square test or Fisher exact. Relationship between age groups, region/stratum and parasite prevalence, density was determined using linear regression. All survey mean estimations were adjusted for sampling weights, clustering and stratification.Plasmodium falciparum and geometric mean parasite density was 1,211 parasites/μl . Gametocytes prevalence, only for P. falciparum was 5.6% . The burden was highest in the northern regions and in the coastal stratum. Parasite infection and geometric mean parasite density peaked during the second year of life and thereafter decreased with increasing age. Mean haemoglobin concentrations was 9.9 g/dl (95% CI 9.5 – 10.2). Anaemia prevalence was 69.8% (6.257/8.816) and among anaemic children 11.5% (743/6.257) were severely anaemic. Anaemia rose dramatically during the first year of life to peak among children in the 12 – 23 months age group. Highest levels of anaemia were recorded in both northern and central-northern regions 77.9% and 79.4% respectively.Malaria parasite prevalence was 58.9% (5.190/8.816), the majority of blood smears 52.4%  were due to P. falciparum, remains endemic throughout the country. The burden of malaria disease and anaemia-related malaria during childhood constitute a major public health problem and warrant integrated and collaborative interventions towards its control.This survey confirms that malaria especially that caused by  Plasmodium falciparum, remains one of the most important causes of morbidity and mortality among impoverished communities across endemic regions of sub-Saharan Africa . The lowest fever prevalence of 5.9%  was recorded among children in the 5- to 7-year age group. Similarly to malaria parasite density, fever prevalence decreased rapidly with age.P. falciparum infections  accounted for 5.7% . The prevalence of malaria infection associated with fever peaked among children during the first year of life and thereafter decreased sharply with increasing age, and the differences among age groups were statistically significant (p < 0.001).Overall the prevalence of fever associated with P. falciparum parasite densities were significantly associated with fever (p < 0.05). According to age group, the risk of fever increased during the first 12 months of life and thereafter decreased significantly with age (p < 0.0001).The risk of being febrile increased with increasing parasite density, particularly from parasite density category equal or higher than 5,000 parasites/μl. High Across the study area there were significant regional variations (p < 0.001). The highest prevalence of malaria infection associated with fever was recorded in the northern region and the lowest was recorded in the southern region.Overall, mean estimation of haemoglobin concentration was 9,9 g/dl , the prevalence of anaemia using the 11.0 g/dl altitude adjusted race-specific WHO cut off was 69.8% and among anaemic children 11.5% had severe anaemia (haemoglobin less than 5 g/dl).There was considerable variation in the prevalence of anaemia among age groups (p < 0.0001). In general, all age groups had low haemoglobin concentration, and consequently high levels of anaemia prevalence. Children during the second year of life had the lowest haemoglobin concentration, thereafter, haemoglobin concentration increased with increased age as is illustrated Figure P. falciparum parasites infection associated, while P. malariae infections were recorded in 4.5%, and mixed infections by P. falciparum and P. malariae accounted for 3.6% among anaemic children.Approximately half of anaemic children had P. falciparum infection was recorded in both northern and the central-northern regions 54.8%  and 58.7% , respectively. Whereas the lowest overall prevalence of 36.8%  and 44.6%  corresponding to central and southern regions.Overall, the prevalence of malaria infection showed variations throughout various regions in the country, decreasing from north-to-south Figure . The higP. falciparum infection, increased during the first 12 months of life from 47.5% and 64.3%, peaking to 63.3% and 67.7%, respectively among the 12 – 23 months age group children, and thereafter decreased progressively with age. Only in the central-northern region, significant differences in age group variations were observed (p = 0.042). Within the same age groups, the peak observed of 41.2% and 44.9% across central and southern regions respectively, was much lower. Thereafter, the prevalence decreased slightly with very little variation among older children. Differences in the distribution between age groups were observed only for the central region (p < 0.0001).In the northern and central-northern regions, the prevalence of P. falciparum infection prevalence from the low lands of the coastal stratum to the highland stratum in both central (p = 0.02) and southern (p = 0.004) regions, while the variations observed across strata in the northern region and in the northern-central region were not statistically significant  was recorded in children across the central-northern region, and young children below 12 months of age harbour the maximum load of parasite density 3,494 parasites/μl, . Comparatively, low mean parasite density of 891 parasites/μl  was recorded among children in central region. The overall mean parasite density in the northern and southern regions was 1,077 parasites/μl  and 1,193 parasites/μl , respectively.Generally, in both northern regions, mean parasite density peaked during the first 12 months of age, while in central and southern regions the peak was recorded later among children in the 12 – 23 months of life. Nonetheless, in all regions, parasite mean densities were markedly age-dependent, and decreased sharply with age (p < 0.0001).P. falciparum gametocytes (p = 0.002). Nevertheless, no significant differences were observed across strata of all regions (p = 0.55). Overall the central and central-northern regions recorded the highest prevalence of gametocytes 7.3%  and 8.2% , respectively. The lowest prevalence was recorded in the southern region 2.0% .There was a significant regional variation on the prevalence of Regions with low prevalence showed an erratic distribution of gametocytes, according to age groups. However, across regions with high gametocytes prevalence, it increased among children less than 12 months of age, and peaking among children in the 12–23 months of age. In the central-northern region the peak was observed earlier during the first 12 months of age. Thereafter, the prevalence of gametocytes decreased considerably with age (p = 0.029).P. malariae parasites was relatively low in the study area, accounting for 3.6%  of malaria infections. Overall, the prevalence of P. malariae infection showed significant regional variations (p = 0.013). The highest was recorded in the central-northern regions 7.4% . While in the central region was recorded the lowest prevalence 1.4% . Across strata, there was a significant decrease of P. malariae infection prevalence from 6.8% in the low lands of the coastal stratum to 1.7% in the highland stratum (p = 0.026).The prevalence of P. malariae infection varied significantly with age (p = 0.02). The peak of infection was recorded among younger children aged between 12 – 23 months old, and thereafter decreased gradually with age. No difference was observed between males and females.In general, the prevalence of Episodes of mixed infections were observed predominantly in the central-northern region 7.0%  and in the northern region 3.1% . The occurrence of mixed infections in both central 0.9%  and southern 0.8% , regions were negligible.Variations in the distribution of mixed infection showed significant differences among age groups. The peak of mixed infections was recorded among children in the 24–59 months age group.Overall, high fever prevalence were recorded in the northern and central-northern regions 12.8%  and 10.8% , while in the central and southern region were recorded the lowest fever prevalence of 6.9%  and 7.2%  respectively. Although the prevalence of fever showed significant regional variations (p = 0.019), declining from north to south following the same pattern of malaria parasites infection distribution, there were no significant differences within strata across the regions. In all regions fever prevalence decreased with age, though a slight increase were observed among children aged 7 years and above, except in the central-northern region.Fever associated with malaria parasites was markedly high in the northern and central-northern regions; 9.1%  and 8.4% , respectively. The central region registered the lowest prevalence of fever associated with parasites 6.9% . In spite of high variations of the proportions of fever associated with malaria parasites, the differences across regions were not statistically significant (p = 0.108).Mean haemoglobin concentration showed insignificant differences between regions. In general, there was some degree of variations on mean haemoglobin concentration across regions. The northern and central-northern regions recorded mean haemoglobin of 9.4 g/dl  and 9.4 g/dl , respectively. A slightly higher mean haemoglobin concentration of 10.2 g/dl  and 10.4 g/dl  were recorded in the central and in the southern regions, respectively.According to age groups, mean haemoglobin concentration, increased significantly with age only within the central (p < 0.05) and southern (p < 0.005) regions. In the northern regions also increased, but without significant differences among age groups.Overall, the highest levels of anaemia prevalence were recorded in the northern and central-northern regions 77.9% and 79.4% respectively Figure . AlthougThe levels of severe anaemia were higher among children in the northern and central northern regions 2.2% and 1.6%. The lowest prevalence of severe anaemia was recorded in the southern region (0.3%), and the differences observed between regions were statistically significant (p = 0.031). Overall, the peak was recorded among children in the 12 – 23 months age group. However, the northern region recorded the highest peak (3.9%) of severe anaemia prevalence among children aged below 12 months. Unlike prevalence of anaemia, severe anaemia decreased sharply with age. In the southern region there were no records of severe anaemia among children aged 5 years and above.P. falciparum parasites, while among non-anaemic 37.6% had malaria infection associated (p = 0.011).Comparison among anaemic and non-anaemic subjects in all regions, revealed a significant association between prevalence of anaemia and malaria parasite infections. Among anaemic children 54.0% had Plasmodium infections and anaemia in children less than 10 years of age across different ecological settings in Mozambique.Planning and monitoring of malaria control programmes requires good quality data that can define disease burden across geographical and ecological areas in a country. A nationwide survey was performed to assess the malaria disease burden, by estimating the prevalence and intensity of Overall, the prevalence of malaria infection and parasite density among children was markedly age-dependent, peaking during the first year of life, and thereafter declining with increasing age, as described previously in other malaria endemic areas across tropical Africa ,17, whicet al during the 1950s, which showed highest prevalence of malaria infections in the centre-northern region and in the coastal areas of the country [This study highlighted variations on the intensity of malaria infection across the country, decreasing from northern to southern regions and from low lands in the coastal and plateau strata to the highlands stratum. In general the heterogeneity in malaria transmission and its intensity within regions could be explained by the regional variations on the amount of rainfall, average air temperatures, humidity and also on the human population distribution. These findings corroborate with the results from the first and only attempt to evaluating the malaria situation nationwide conducted by Soeiro  country . DespiteData from a longitudinal field research with a strong focus on epidemiology and entomology conducted during the 1970's, in Garki have demonstrated that malaria in tropical Africa is characterized by high levels of transmission and the pattern of malaria endemicity is maintained by highly effective vectors. Regardless of high coverage rates of residual spraying with an effective insecticide against the mosquito vectors, combined with mass drug administration at high frequency and coverage, reduction on malaria transmission to low levels was observed, but interruption could not be achieved . SimilarP. falciparum, continues to be a major scourge throughout the country and therefore represents a large public health problem. Young children bear the brunt of the infection and this implies exposure to intense malaria transmission. Parasite rates in children may well guide to a classification of the malaria endemicity, as described by Metselaar and van Thiel [This study confirmed that malaria especially that caused by an Thiel . The metan Thiel ,22. In gP. falciparum malaria plays a key role in the burden of anaemia [Moreover, results of this study strongly support the finding that  anaemia ,17,26. T anaemia ,25. Simi anaemia . Neverth anaemia ,23. AnaeP. falciparum malaria parasites and approximately 3.8 million are expected to be anaemic. The majority of these children inhabit the coastal and plateau strata in the northern regions.The burden of malaria disease and anaemia-related malaria during childhood constitutes a major public health problem. According to 1997 population census projection, if we apply the age regional and stratum specific prevalence rates for malaria infection and anaemia, at a position in time in the country we can estimate absolute numbers accordingly. Thus, approximately 2.6 million of children less than 10 years of age are expected to harbour Integrated and collaborative malaria control interventions are warranted. Intermittent preventive treatment, insecticide treated nets, mass de-worming, iron, and vitamin A supplementation programmes have already proven to be cost-effective interventions, particularly in areas lacking adequate health care services. In future malaria vaccines may also contribute to improving control and prevention efforts. Moreover, estimations of the disease burden caused by malaria are essential for planning cost-effectively malaria control interventions, monitoring and advocacy.The authors declare that they have no competing interests.SJAM, made a substantial contribution on conception and design of the study, co-ordinated and supervised data collection in all regions, executed all statistical analysis and wrote the manuscript. SLRC, co-ordinated and supervised data collection in the central-northern region, revised the manuscript. LQ made a major contribution on study design and helped with statistical analysis. PA gave a major contribution on conception and study design and helped to draft and critically revised the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria, children in endemic areas develop antibodies specific to antigens on the parasite-infected red cell surface of the infecting isolate, antibodies associated with protection against subsequent infection with that isolate. In some circumstances induction of antibodies to heterologous parasite isolates also occurs and this has been suggested as evidence for cross-reactivity of responses against the erythrocyte surface. The role of these relatively cross-reactive antibodies in protection from clinical malaria is currently unknown. We studied the incidence of clinical malaria amongst children living on the coast of Kenya through one high transmission season. By categorising individuals according to their pre-season parasite status and antibody response to the surface of erythrocytes infected with four parasite isolates we were able to identify a group of children, those who failed to make a concomitant antibody response in the presence of an asymptomatic parasitaemia, at increased susceptibility to clinical malaria in the subsequent 6 months. The fact that this susceptible group was identified regardless of the parasite isolate tested infers a cross-reactive or conserved target is present on the surface of infected erythrocytes. Identification of this target will significantly aid understanding of naturally acquired immunity to clinical malaria amongst children in endemic areas.Following infection with  Evidence from the passive transfer of antibodies from immune to non-immune individuals suggests this immunity is, at least in part, antibody mediated (Non-sterile immunity to mediated . Humans mediated . Among cmediated .The induction of antibodies during an acute episode to the infected red cell surface of parasite isolates apparently not involved in that episode (heterologous parasites) has been suggested as evidence for some cross-reactivity in these responses . HoweverP. falciparum infection and enhanced anti-erythrocyte surface antibody responses to a range of isolates  Centre for Geographic Medicine Research Coast (CGMRC) situated at Kilifi District Hospital, 50 km north of Mombasa on the coast of Kenya. The hospital serves approximately 240,000 people. Individuals investigated during these immuno-epidemiological studies were resident in Kilifi District, in an area called Chonyi. This study site has been described in detail elsewhere . Inhabit2.2Sera collection and active surveillance were conducted as part of a study examining the clinical epidemiology of malaria under differing transmission conditions . In brieP. falciparum malaria. Children were followed up at 1, 2, 3 and 6 weeks after treatment and further serum samples obtained. The precise details of recruitment and sampling of this cohort have been published elsewhere , 3D7 and one clinical isolate, P1, obtained from a 5-year-old child admitted to Kilifi District Hospital with moderately severe malaria  of uninfected erythrocytes following incubation in negative control plasma; b = MFI of parasitised erythrocytes following incubation in negative control plasma; c = MFI of uninfected cells incubated in immune plasma or test antibody and d = MFI of parasitised erythrocytes incubated in immune plasma or test antibody.Reactivity against the infected erythrocyte surface was scored as mean fluorescent intensity using the method of 2.5Plasmodium falciparum (A4 strain) schizont extract was coated onto wells in PBS according to the methods described by 2O2 and O-phenylenediamine . The reaction was stopped with 25 μl of 2 M H2SO4 per well and absorbance read at 492 nm.2.6All statistical analysis was performed using Stata8™ . The relationship between the presence of parasites and reactivity to the surface of erythrocytes infected with each isolate was assessed using logistic regression. The outcome in every case was positivity for recognition of each isolate as classified using the cut-off of the mean response from 20 non-exposed donors plus 3 SD Initially the data were explored for additional factors affecting the likelihood of recognising each isolate. In every case these were identified as age (in categories of 6 months duration) and previous exposure to malaria infection . The likelihood of recognition of each isolate was then compared between those individuals who were parasite-positive and those who were parasite-negative at the time of the cross-sectional survey when the plasma samples were drawn.To explore the relationship between antibody responses, the presence of microscopically detectable parasites at the time of serum collection and subsequent clinical malaria, a multiple logistic model was performed after identification of significant confounding variables. These were identified as age (categorised as a factor of 6 months duration), and exposure (estimated from responses to whole schizont extract). The outcome variable was at least one episode of malaria as defined previously after censoring the first 30 days of follow-up . Individuals were categorised according to whether or not they had detectable parasitaemia at cross-sectional survey and whether they were positive or negative for recognition of each isolate. This variable was then factorised and analysed as the main explanatory variable in a multiple logistic regression.When investigating the likelihood of having a greater number of episodes of clinical malaria, an ordered logistic regression was used. Individuals were scored 0, 1, 2, 3 or 4 according to the number of episodes of malaria they suffered during the 6 month follow-up; the resultant odds ratio (OR) gives the likelihood of being in a higher numbered group  depending on which structured antibody/parasite group the individual fell into. Again confounding variables of age and exposure were included and the first 30 days of follow-up were censored.Where differences between two continuous variables were assessed, no assumptions of distribution were made and the Wilcoxon rank sum test was used to calculate significance. Where more than two variables were compared, the Kruskal Wallis test was used. All correlations were performed using Spearman’s rank correlation coefficient.P values less than 0.05 were considered significant.Kaplan–Meier survival curves were plotted using time in days to first episode as previously defined. Throughout the analysis, 33.1Antibody responses to A4U increased significantly with age in individuals who were parasite-positive or parasite-negative at cross-sectional survey . ResponsP = 0.002; A4 40-cycle OR 5.05 (95% CI 2.00–12.75), P = 0.001; 3D7 OR 4.62 (95% CI 1.92–11.12) P = 0.001; P1 OR 2.62 (95% CI 1.01–6.83) P = 0.049).However, in those individuals who were parasite-positive at the time of sampling, there was a significantly increased likelihood of recognition of all three laboratory parasite lines and the clinical isolate P1 (A4U OR of recognition if parasite-positive compared with parasite-negative 2.53 (95% CI 1.41–4.54) 3.2P < 0.0001). Limiting the analysis to risks of a clinical attack 30 days after the initial cross-section continued to demonstrate a significant increased likelihood of becoming a case if parasite-positive ).Parasite prevalence at cross-section among children aged 1–10 years in Chonyi was 43.2%. Overall the OR for an individual developing clinical malaria in the 6 months following the cross-sectional survey if they were parasite-positive compared with parasite-negative was 3.42  and whether or not they had a microscopically detectable parasitaemia. Kaplan–Meier survival curves were drawn with each of the four groups using responses against each isolate in turn. It is clear that for responses against three of the parasite isolates tested, those individuals with asymptomatic parasite carriage and no concomitant antibody response appeared to be highly susceptible to mild clinical malaria .P = 0.022). The same group was observed to be similarly susceptible when responses to the other three parasite isolates were considered.The likelihood of acquiring at least one episode of clinical malaria in the subsequent 6 months was investigated according to group in a multiple logistic model with age and exposure as confounding variables and excluding the first 30 days of follow-up. When the outcome examined was the total number of malaria episodes experienced by each individual over the follow-up period, an ordered logistic regression model was used with the same confounding variables included and the first 30 days of follow-up excluded. Again the group of children who were antibody-negative and parasite-positive were significantly more likely to experience a greater number of malaria episodes in the subsequent 6 months. This was true for all parasite lines tested .t-test P < 0.0005 for all comparisons between the community samples and the samples from children presenting to hospital, both acute presentation and all follow-up samples).In order to examine antibody responses in a known susceptible group of children, sera from children presenting to hospital with acute malaria were tested for recognition of A4U. Sera were collected at each follow-up visit and again tested for recognition of the surface of unfixed A4U. As can be seen in 3.4In order to assess the specificity of individual responses scatter diagrams of paired responses, normalised on a log scale, were plotted . A disti4While it is accepted that children develop ‘protective’ antibodies specific to the infecting isolate following infection , it is nPreviously, associations between antibody responses to isolates not immediately involved in an episode of clinical malaria and subsequent protection have been conflicting . In the We have shown that failure to mount an antibody response against the surface of erythrocytes infected with three laboratory parasite lines, including two phenotypes of a genetically identical isolate, A4 and the reference line 3D7 as well as a locally obtained clinical isolate, was associated with susceptibility to clinical malaria in children with an asymptomatic parasitaemia. The lack of antibody response in children of the same age, from the same area, presenting as a case of acute clinical malaria to hospital, and thus by definition susceptible, supports this claim. It is interesting that up to 6 weeks following successful treatment this group of children did not make a response comparable with those children from the community who were well at the time of serum sampling, thus supporting the possibility that the inability to mount an antibody response in the presence of parasites may be associated with susceptibility.This increased susceptibility amongst those harbouring parasites but without a concomitant antibody response might reflect a poor inherent ability to respond appropriately in the presence of challenge, as has been suggested in a number of studies . These pP. falciparum infection, present in many studies (It has been hypothesised that development of immunity against clinical malaria is associated with the ability to maintain chronic asymptomatic infections . Chronic studies , and truOf course, the importance of chronic infection in the development of anti-malaria immunity is at odds with the observation in this study that being parasitaemic per se was associated overall with an increased susceptibility to mild clinical malaria. This should be interpreted with caution as it is not an invariable finding and previous surveys in this area have shown no association between asymptomatic parasite carriage at the end of a dry season and protection from, or susceptibility to, severe malarial disease . It is nThe lack of any association between the presence of anti-erythrocyte surface antibodies and protection from clinical malaria among children without the presence of an asymptomatic microscopically detectable parasitaemia is interesting. It is likely that these antibody responses are short-lived  and thusvar genes into discrete antigenic groups (The apparent lack of isolate specificity of this important antibody response, unlike that acquired following an episode of disease  suggestsc groups , it was The biological function of such an antibody response remains highly speculative. There are a number of mechanisms through which antibody directed at the infected erythrocyte surface could mediate protection. While it is known that sera from individuals resident in malaria endemic areas are able to disrupt cytoadherence to various cell-lines in vitro , the relIdentifying both the target for this response and its biological effector mechanism are of considerable importance both for vaccine development and improved understanding of the acquisition of immunity in children in malaria endemic areas.


Clinical association studies have yielded varied results regarding the impact of glucose-6-phosphate dehydrogenase (G6PD) deficiency upon susceptibility to malaria. Analyses have been complicated by varied methods used to diagnose G6PD deficiency.We compared the association between uncomplicated malaria incidence and G6PD deficiency in a cohort of 601 Ugandan children using two different diagnostic methods, enzyme activity and G6PD genotype . Although roughly the same percentage of males were identified as deficient using enzyme activity (12%) and genotype (14%), nearly 30% of males who were enzymatically deficient were wild-type at G202A. The number of deficient females was three-fold higher with assessment by genotype (21%) compared to enzyme activity (7%). Heterozygous females accounted for the majority (46/54) of children with a mutant genotype but normal enzyme activity. G6PD deficiency, as determined by G6PD enzyme activity, conferred a 52%  reduced risk of uncomplicated malaria in females. In contrast, when G6PD deficiency was defined based on genotype, the protective association for females was no longer seen . Notably, restricting the analysis to those females who were both genotypically and enzymatically deficient, the association of deficiency and protection from uncomplicated malaria was again demonstrated in females, but not in males .This study underscores the impact that the method of identifying G6PD deficient individuals has upon association studies of G6PD deficiency and uncomplicated malaria. We found that G6PD-deficient females were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency is described using enzyme activity. These observations may help to explain the discrepancy in some published association studies involving G6PD deficiency and uncomplicated malaria. Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first step of the pentose phosphate pathway, which converts glucose into pentose sugars for glycolysis and other biological reactions. The pentose phosphate pathway is the only source of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in erythrocytes, and therefore the principal means by which erythrocytes counteract oxidative stress P. falciparum malaria, leading to approximately 1 million deaths each year Malaria has exerted a tremendous selective pressure on the human genome, with an estimated 300–660 million cases of Most clinical screening tests measure the enzymatic formation of NADPH from NADP, such as the semiquantitative fluorescent spot test or the quantitative spectrophotometric test Participants for a randomized comparison of three combination antimalarial regimens were recruited from the Mulago III parish of Kampala, Uganda, as previously described This study was conducted according to the principles expressed in the Declaration of Helsinki. The clinical trial and this study were approved by the Uganda National Council of Science and Technology and Institutional Review Boards of Makerere University and the University of California, San Francisco. All patients' parents or guardians provided written informed consent for the collection of samples and subsequent analysis.msp1, msp2, and four microsatellites All participants were asked to come to a study clinic for all their healthcare needs; the clinic was open seven days a week. Episodes of malaria were diagnosed by passive surveillance, and, in addition, subjects were assessed every 30 days. Subjects presenting to the clinic with fever (tympanic temperature ≥38.0°C) or history of fever in the previous 24 h had a thick blood smear assessed for parasites. If the smear was positive, the participant was treated for malaria, regardless of parasite density. Molecular genotyping was performed to distinguish new infections (incident events) from recrudescences (which were not considered incident events), following a step-wise algorithm including assessment of polymorphisms in the P. falciparum genes 9 erythrocytes, and recommends a cutoff of 110 mU/109 erythrocytes to differentiate normal from deficient G6PD activity.Fresh whole blood samples were collected at enrollment and sent to a certified lab in Kampala for G6PD activity assessment. A single assessment was performed by estimating the rate of NADPH production from NADP using a commercially available quantitative spectrophotometric test . Strict adherence to manufacturer's protocol was followed including appropriate use of control samples. The manufacturer reports G6PD values as mU/10aq Polymerase (recombinant), 4 mM MgCl2, and 0.4 mM of each dNTP), 0.2 µM of each primer . A specimen was unavailable for one individual, leaving a sample population of 600 individuals. Genotypes for the G6PD A- variant  were determined by PCR followed by restriction endonuclease digestion. PCR was performed using 2× PCR Master Mix  were determined by PCR as above with primers as previously described Sixteen individuals who were found to be enzymatically deficient but were genotyped as wild-type for the 202 variant were sequenced to assess other alleles that define the G6PD A- variant (G680T and T968C). The amplification reactions were performed as described above, using primers described elsewhere Statistical analysis was performed using Stata version 10 . Predictor variables included age, sex, sickle cell trait, G6PD enzyme deficiency, G6PD genotype, bed net usage, materials used in household construction, household water source, and index of household wealth, household crowding, antimalarial treatment arm, and distance from potential mosquito breeding sites. Time-dependent covariates  were evaluated at the precision of 1 day. Our outcome measure was malaria incidence, as measured by incident episodes of malaria per person-year at risk. Subjects who were in the cohort as of April 1, 2005, but were terminated from the study prior to completion of follow-up contributed person-time equivalent to the duration of time they spent in the study.Univariate and multivariate analyses using generalized estimating equations with control for repeated measures in the same subject were used to estimate associations between predictor variables of interest and malaria. P<0.05 was considered statistically significant. Variables that were significant in univariate analysis were included in the multivariate model.9 erythrocytes, 20 females (6.9%) and 42 males (13.5%) had deficient G6PD enzyme activity.A total of 600 individuals, consisting of 289 females and 311 males, were tested for G6PD enzyme activity . The shaThirty-six males (11.6%) were hemizygous, 61 females (21.1%) were heterozygous and five females (1.7%) were homozygous for the G202A mutation. The allele frequency of G202A was 0.12 for both males and females. All genotypes were in Hardy-Weinberg equilibrium. To confirm that the G202A mutation occurred only in the background of the G376A mutation, we genotyped 110 randomly selected samples for the G6PD A variant. Of these 110 samples, 23 carried the G202 mutation, all of which also carried the G376A mutation. No individuals carried the G376A mutation in isolation.9 erythrocytes (Interquartile Range (IQR) = 80) for wild-type males and 61 mU/109 erythrocytes (IQR = 38.5) for hemizygous males with the G202A allele. For females, the median value of enzyme activity was 225 mU/109 erythrocytes (IQR = 101) for wild-type females, 135 mU/109 erythrocytes (IQR = 77) for heterozygous females, and 63 mU/109erythrocytes (IQR = 148) for homozygous G6PD A- females. Enzyme activity was stratified by gender and genotype . The medOnly 6 of 269 (2%) males with normal G6PD enzyme activity carried the G6PD A- allele . HoweverWe assessed the sensitivity and specificity of the phenotypic test as compared to genetic analysis. For this analysis heterozygous females were classified as deficient. Using a cutoff of 110 mU/10∧9 erythrocytes to define G6PD deficiency in males, the test was 83.3% sensitive and 95.6% specific. In males, the test had a positive predictive value of 71.4% and a negative predictive value of 97.7%. In females, the test was 24.2% sensitive and 98.2% specific, with a positive predictive value of 80.0% and a negative predictive value of 81.4%.We previously showed in this cohort that G6PD deficiency, as assessed solely by enzymatic assay, was associated with a significantly lower risk of uncomplicated malaria in females (relative risk (RR) = 0.48, 95% CI 0.31–0.75, p-value = 0.001) but not males  As seen in both 9 erythrocytes, enzymatic G6PD deficiency was significantly associated with a decreased risk of uncomplicated malaria in females , and differences in the malaria phenotype being assessed. A potential major source of discrepancy between studies was the method of screening for G6PD deficiency. We compared the results of the association between G6PD deficiency and malaria using both enzymatic and genetic assessments. We found that G6PD deficiency had no significant association with the incidence of uncomplicated malaria in males regardless of which assay was used. However, in females, when G6PD deficiency was defined by enzyme activity, deficiency was associated with a 52% reduced risk of uncomplicated malaria. This level of protection was comparable to that seen in individuals who use insecticide-treated bed nets (RR = 0.51) and somewhat greater than that conferred by sickle cell trait (RR = 0.68) As described earlier, the mosaic distribution of G6PD deficiency is due to variable X chromosome inactivation 9 erythrocytes, G6PD deficiency was consistently protective in females, with risk reductions between 42–54%. However, at higher cutoff values (<130 mU/109 erythrocytes), protection from malaria was not seen. These results suggest that lower levels of enzyme activity are protective against malaria in females. Sample sizes were too small to meaningfully assess the association of levels of G6PD enzyme activity levels <90 mU/109 erythrocytes in females. Notably, however, there was no evidence of a protective effect against uncomplicated malaria in the 23 males with the lowest G6PD activity levels (<70 mU/109 erythrocytes). Prior studies have seen protection from malaria primarily in heterozygous females To further explore the relationship between G6PD enzyme activity and the risk of malaria, we determined whether the degree of deficiency affected an individual's risk of malaria. With a cutoff of 90–120 mU/10Of 62 people with enzymatically determined G6PD deficiency, we were unable to identify a deficiency-causing variant in ∼25% (16/62) after assessing the three common G6PD A- variants found in African populations. Most notably, nearly 1 in 3 males who were enzymatically deficient, were wild-type at the G202A, G680T and T968C loci. It is likely that other mutations exist and that they may mediate altered enzyme activity in these individuals This study underscores the impact that the method of identifying G6PD deficient individuals has upon studies of G6PD deficiency and malaria. We found that G6PD-deficient females, but not males, were significantly protected against uncomplicated malaria, but this protection was only seen when G6PD deficiency was described using enzyme activity. The lack of association seen in heterozygous females was likely due to the mosaic population of erythrocytes, as protection was again seen when the analysis was restricted to females who were both heterozygous and enzymatically deficient. These observations may help to explain discrepancies in some published association studies involving G6PD deficiency and uncomplicated malaria. Ultimately, it may be that both methods have a role in the detection and characterization of G6PD deficiency. In the setting of association studies, whilst enzymatic assays seem to more closely approximate biologic function and correlate with protection, the addition of genetic testing may uncover novel disease causing variants and thereby aid in our understanding of this widespread enzymopathy.