Plasmodium falciparum. Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children  monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM  after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM . Assays of these two functional mechanisms were poorly correlated . Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.Severe malaria (SM) is a life-threatening complication of infection with  Plasmodium falciparum, it is estimated that at least 2 will develop SM and that 1 of them will die  afflicts young children, commonly under the age of 5 years, in areas with stable and high malaria transmission intensity –3. Child–will die . Furtherwill die .P. falciparum infections are asymptomatic while others result in a severe and life-threatening illness that may cause lifelong disability has long been debated and, remarkably, still remains unclear  are targets of antibodies that have been shown to inhibit or reverse sequestration of iRBCs, inhibit formation of rosettes , and pro36–Here, we designed a case-control study of SM nested within a longitudinally monitored birth cohort of children at the Kenyan coast. We analyzed samples collected prospectively, before hospital admission, with well-characterized clinical phenotypes of SM for antibodies against five merozoite antigens, parasite schizont lysate, and the intact iRBCs. We investigated the mechanisms of action of antibodies directed against merozoites using assays of growth inhibition activity (GIA)  and anti2 around Kilifi County Hospital (KCH), which is the first-level referral facility for the county and tracks a population of approximately 260,000 individuals  . It coveividuals . Quarterividuals . A markeividuals , 46, whiividuals  and the A detailed description of the study cohort has been provided elsewhere . BrieflyParents of children who were unwell outside the follow-up visits were advised to seek medical attention at KCH, and the children were treated according to the Kenya National and WHO recommended guidelines. In the event of admission to the hospital, KBC participants were identified using a unique personal identification number that links their demographic data to the clinical and laboratory information collected.Written informed consent was obtained from the parents/guardians of all the children. Ethical approval was granted by the KEMRI National Ethics Committee.We conducted a matched case-control study nested within the longitudinally monitored birth cohort described above. Index cases were children admitted between 2002 and 2010 with parasites detectable by microscopy and one of the following syndromes of severe malaria: (i) severe anemia, defined as Hb of <5 g/dl; (ii) impaired consciousness, defined as a Blantyre coma score (BCS) of less than 5; and (iii) chest indrawing or deep breathing, i.e., respiratory distress as a marker of metabolic acidosis . ChildreCases were individually matched to a maximum of three controls by age, date of sample collection, and area of residence. Controls were selected from KBC participants who did not present to KCH with severe malaria during the 8-year period.P. falciparum in blood samples collected during the quarterly follow-up visits and during admission has been described previously  fusion proteins in E. coli (19) was expressed as a GST fusion protein and was based on the Wellcome parasite strain  that was similar in PfRh2a and PfRh2b was based on the 3D7 parasite strain and was expressed in E. coli as a GST-fused protein  or 100 μl of schizont extract diluted 1:1,000, as previously described . After 15 min, the reaction was stopped with 25 μl H2SO4, and the absorbance was read at 492 nm. Eleven serial dilutions of a purified immunoglobulin reagent  prepared from a pool of Malawian adults presumed to be malaria immune . For wells where the subtraction resulted in an OD value that was less than zero, the OD value was converted to zero. A pool of sera obtained from Kilifi adults was included on a single well in each plate as a positive control to allow standardization of day-to-day and plate-to-plate variation. The coefficient of variation (CV) recorded for the serum pool across plates and across days was less than 5%. Twenty sera obtained from non-malaria-exposed United Kingdom adults were also included as negative controls for each antigen tested. For quality control, all samples were assayed in duplicate, and those that had a CV greater than 20% were repeated.A well-established standard enzyme-linked immunosorbent assay (ELISA) protocol was used to measure antibody titers against the recombinant antigens –62 and P– extract . Wells oescribed . After oa immune  were inca immune . For eacP. falciparum 3D7 trophozoite parasites (24 to 30 h postinvasion) at 0.3 to 0.5% parasitemia and 1% hematocrit was added to individual wells of sterile 96-well U-bottom plates . Thereafter, 5 μl of dialyzed test sera was added in duplicate, and the plates were placed in a sealed humidified gas chamber and incubated at 37°C in 5% O2, 5% CO2, and 90% N2. Duplicate wells with a pool of sera from nonimmune United Kingdom adults were included as a negative control and 10 mg/ml of purified MIG as a positive control. Ten microliters of fresh complete medium was added to the wells after the first growth cycle, and the plates were incubated for a further 40 h. At the end of the second growth cycle, 10 μg/ml of ethidium bromide (molecular grade) diluted in 1× PBS was added to each well, and the plates were incubated for 30 min at room temperature in the dark. The plates were centrifuged at 1,200 rpm for 1 min, and the pellet was resuspended in 200 μl of 1× PBS. Parasitemia was determined by analyzing 70,000 events on an FC500 (Beckman Coulter) flow cytometer.GIA was measured after two cycles of parasite replication according to previously established protocols , 65. Brig for 40 min at room temperature. The PMNs were collected and washed in PMN buffer [0.1% bovine serum albumin (BSA) and 1% d-(+)-Glucose Hybri Max (Sigma) in Hanks balanced salt solution (HBSS)]. Lysis of residual RBCs was performed by addition of ice-cold 0.2% sodium chloride for 30 s, followed by 1.6% sodium chloride. The cells were washed, resuspended in PMN buffer, and enumerated using a fast-read disposable chamber.Polymorphonuclear cells (PMNs) were isolated from whole blood collected in EDTA tubes, layered onto Polymorphprep (Axis-Shield Diagnostics) at a ratio of 1:1, and separated at 500 × P. falciparum. Parasitophorous-vacuole-enclosed membrane structures (PEMS) were isolated as previously described (trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane  was added for 8 h to allow growth of late-stage pigmented trophozoites into schizonts without rupture. The treated cultures were pelleted by low-speed centrifugation, resuspended in PBS at 1.8 × 106 schizonts/ml, and immediately frozen at −20°C to lyse the RBCs.Experiments were performed with the 3D7 laboratory strain of escribed . Briefly5 schizonts/ml. The wells were washed three times with 1× PBS and blocked for 1 h at 37°C with casein (Thermo Scientific). One hundred microliters of test serum diluted 1:50 in 1× PBS was added to individual wells in duplicate and incubated for 1 h at 37°C. Three washes were performed, followed by addition of 50 μl of 4-aminophthalhydrazide (Sigma) at 0.04 mg/ml and 50 μl PMNs at 1 × 107 cells/ml. Each serum sample was assayed in duplicate using PMNs from a single donor. The plates were immediately read on a Varioskan flash luminometer (Thermo Scientific). Chemiluminescence readings from each well were obtained for 1 s every minute for an hour .Levels of IgG (measured as median fluorescence intensity [MFI]) against the surface of intact RBCs infected by the 3D7 laboratory strain were determined using flow cytometry, as previously described . BrieflyData analysis was performed using STATA 11.2 . Antibody seropositivity was defined as a cutoff above the mean plus 3 standard deviations of 20 non-malaria-exposed United Kingdom adult sera. An individual was classified as a high or low GIA responder if their GIA was above or below the median GIA level, respectively. Seropositivity for ADRB was defined as a cutoff above the mean plus 3 standard deviations of 10 healthy malaria-naive United Kingdom sera obtained from phase I/IIa malaria vaccine trials at the Jenner Institute, University of Oxford , 70. SerWe have previously defined threshold antibody concentrations that are associated with protection against uncomplicated malaria episodes in two independent cohorts , 71. HerConditional logistic regression (CLR) models were used to derive odds ratios (OR) and 95% confidence intervals (CI) using data from risk sets that had at least one matched control per case . A risk sr = 0.52; P < 0.0001)  . During  0.0001) . Interes 0.0001) .Samples were collected every 3 months from birth up to 2 years of age. The analysis presented here is drawn from antibody responses measured in serum samples collected at the time point immediately prior to the severe malaria episode for cases and their individually matched controls, as illustrated in The median time between date of sampling and date of index case admission was 2.7  months and 2.5  months for cases and corresponding controls, respectively . Of the The female/male ratio, median age at the time of index case admission, and proportion of children who had fever and asymptomatic parasitemia during the quarterly visits were similar between the cases and controls  Table 1Table 1. We also compared the dynamics of the antibody titers between the cases and controls using samples collected at all the 3-month time points before the admission of the cases . A large proportion of the children had low antibody titers, often fluctuating below the seropositivity cutoff at all the time points. Some children had detectable antibody levels that often fluctuated either above the cutoff  or around the cutoff . Importantly, the individual patterns of antibody responses were similar between the cases and controls for the antigens tested .sr = 0.94 for FVO versus 3D7, sr = 0.85 for HB3 versus FVO, and sr = 0.82 for HB3 versus 3D7; P < 0.001), MSP-2, and MSP-3 were strongly correlated , responses to only one allelic form of these antigens are shown in 19, and PfRh2 between the index cases and controls  and those who were parasite negative . IgG levels were higher in the children who were parasite positive than in those who were parasite negative across the age categories for all the antigens tested. However, this difference was significant only in children over 13 months of age for anti-AMA1, anti-MSP-2, anti-MSP-3 and anti-parasite schizont lysate antibodies . In contrast, GIA levels were consistently higher in the parasite-negative children than in the parasite-positive children , and an opposite trend was observed for ADRB activity . However, neither of these differences was statistically significant.sr > 0.50; P < 0.05) for both cases     and the = 0.002) .P = 0.14) (P < 0.001) (Our previous study demonstrated that the breadth of anti-merozoite antibody responses was associated with better antibody function (as measured by opsonic phagocytosis of merozoites) . Here, w = 0.14) . In cont< 0.001) .P = 0.04 and OR = 0.40, 95% CI = 0.17 to 0.98, P = 0.04, respectively) in multivariate analysis (P = 0.029). Antibodies to the iRBCs were not associated with protection .We examined the association between antibody titers (measured at the time point closest to admission) to different merozoite antigens, IgG to the intact iRBCs, GIA, and ADRB activity with the odds of developing SM during the first 2 years of life. Antibody seropositivity to all the antigens tested was not associated with protection against developing SM, with the exception of responses to AMA1 (FVO and 3D7), which were associated with a significant reduction in the odds of developing SM  versus noncerebral malaria (n = 28)  and SMA (n = 18) versus non-SMA (n = 41) . There was no association between antibodies to all the antigens tested with regard to protection against either impaired consciousness  or SMA .To determine whether the protective effects of antibodies were comparable across individual syndromes of severe malaria, separate analyses were conducted for the outcome of impaired consciousness (P = 0.04), which possibly reflects the protection accorded by anti-AMA1 antibodies in this study . In contrast, antibodies to one antigen were significantly associated with protection . Remarkably, children classified as being positive responders to a combination of both functional assays had very low and significantly reduced odds of developing severe malaria  during the follow-up period compared to children who had responses to either of the two functional assays  .P < 0.001).Following from our observation that the highest antibody levels were observed in children less than 6 months of age, we tested whether our significant results described above were driven by the presence of maternally transferred antibodies. First, we investigated the proportion of children who were seropositive for AMA1 and were below 6 months of age. Among those who did not develop SM, 91.8% were seropositive for AMA1 and were below 6 months of age compared to 50.0% among the index cases. The difference in proportions was statistically significant (P = 0.33).Second, we tested whether the protective effect of achieving positive responses in both GIA and ADRB was driven by the presence of maternal antibodies. We compared the proportions of cases and controls who were positive responders in both assays and were less than 6 months of age. Sixty-six percent of the children who did not subsequently develop SM and were below 6 months of age were classified as positive responders in both functional assays compared to 50.0% among the cases. This difference was not statistically significant (Although the vast majority of deaths due to SM occur in young children, few studies have examined the host immunological factors that might contribute to susceptibility to severe illness or resistance in the age group –19. Two –in vitro assay for measuring the functional activity of naturally acquired and vaccine-induced antibodies (78––P. falciparum. These mechanisms may potentially act to limit the exponential growth of parasites, which is often linked to the development of SM (The GIA assay is currently the most established and widely used tibodies . Howevertibodies  or develtibodies , 78–81. odies 78–. Other modies 78––38. Our nt of SM .Levels of antibodies to all the antigens tested here were low compared to those in semi-immune adults resident in the same area and even lower than the threshold concentrations we have previously reported to be important for protection against clinical malaria in this area . A carefPlasmodium yoelii challenge (19, and PfRh2 were not independently associated with reduced odds of developing SM, also reported independently for antibodies against MSP-119 and MSP-3 (Despite these low antibody levels, we show for the first time that anti-AMA1 antibodies were independently associated with significant protection against SM in this age group. This result remained significant and strong even after adjustment for responses to all the other merozoite antigens analyzed here, as well as prior exposure defined by schizont ELISA reactivity. Our findings lend further support to a renewed interest in improving AMA1-based vaccines by demonstrating that such a vaccine could potentially protect against the most severe forms of malaria in the youngest children at the highest risk of death. AMA1 antibodies may protect by inhibiting the essential process of erythrocyte invasion . Howeverhallenge . In theihallenge . These fnd MSP-3 .We have previously argued that high levels of antibodies against merozoite antigens are required for protection against clinical episodes of both UM and a less rigorously defined SM , 64. TheNotably, GIA and ADRB were poorly correlated, as has been reported for the relationship between GIA and opsonic phagocytosis of merozoites . Our datMaternal antibodies are widely believed to play a key role in protection among infants in multiple infectious diseases –97; howe–SM often presents in three major overlapping clinical syndromes in areas with high and sustained levels of malaria transmission intensity: impaired consciousness, respiratory distress, and severe malaria anemia . The preFinally, it has been argued that a decline in malaria transmission intensity, such as was observed in our setting and has been reported from many parts of Africa, could have detrimental effects on naturally acquired immunity . This isin vitro correlates of immunity to objectively prioritize known and predicted antigens being evaluated as vaccine targets in preclinical and clinical studies.In summary, we provide the first evidence that antibodies against AMA1 and the combined ability of total IgG to inhibit parasite growth and to mediate the release of reactive oxygen species from PMNs were remarkably strongly associated with reduced odds of developing SM in African children less than 2 years of age. Assessment of a combination of assays that measure multiple mechanisms of antibody function could be useful


Asymptomatic malaria infections represent a major challenge in malaria control and elimination in Africa. They are reservoirs of malaria parasite that can contribute to disease transmission. Therefore, identification and control of asymptomatic infections are important to make malaria elimination feasible. In this study, we investigated the extent and distribution of asymptomatic malaria in Western Kenya and examined how varying parasitemia affects performance of diagnostic methods including microscopy, conventional PCR, and quantitative PCR. In addition, we compared parasite prevalence rates and parasitemia levels with respect to topography and age in order to explore factors that influence malaria infection. Over 11,000 asymptomatic blood samples from children and adolescents up to 18 years old representing broad areas of Western Kenya were included. Quantitative PCR revealed the highest parasite positive rate among all methods and malaria prevalence in western Kenya varied widely from less than 1% to over 50%. A significantly lower parasitemia was detected in highland than in lowland samples and this contrast was also observed primarily among submicroscopic samples. Although we found no correlation between parasitemia level and age, individuals of younger age group (aged <14) showed significantly higher parasite prevalence. In the lowlands, individuals of aged 5–14 showed significantly higher prevalence than those under age 5. Our findings highlight the need for a more sensitive and time-efficient assay for asymptomatic malaria detection particularly in areas of low-transmission. Combining QPCR with microscopy can enhance the capacity of detecting submicroscopic asymptomatic malaria infections. Asymptomatic malaria infection is a major obstacle to controlling and eliminating malaria in many African countries. Asymptomatic infections represent reservoirs of malaria parasite that can contribute to resurgence of disease transmission at the right conditions , 2. It iIn areas where malaria endemicity is low control programmes need increasingly sensitive tools for identifying submicroscopic hidden malaria infections and monitoring malaria transmission intensity . DiagnosP. falciparum infections in broad areas of western Kenya, (2) whether there is a difference in parasitemia level in asymptomatic infections especially the submicroscopic infections between high-transmission and low-transmission areas as well as among age groups, and (3) whether microscopy and PCR-based approaches give different level of sensitivity and specificity when diagnosing asymptomatic infections at different transmission settings. Characterizing and quantifying parasitemia in asymptomatic infections will provide more in-depth information for malaria control and elimination planning.The present study sought to determine (1) the prevalence of asymptomatic Scientific and ethical clearance was given by the institutional scientific and ethical review boards of the Kenya Medical Research Institute, Kenya and the University of California, Irvine, USA. Written informed consent/assent for study participation was obtained from all consenting heads of households, parents/guardians (for minors under age of 18), and each individual who was willing to participate in the study.2 area of Western Kenya  and HB3 (MRA-155) were used as positive controls in all amplifications, and water and uninfected samples were used as negative control to ensure lack of contamination.Parasite DNA was extracted from dried blood spots by the Saponin/Chelex method . The finPFPK2) were tested on a subset of samples for optimal amplification conditions in conventional PCR and quantitative PCR. We selected gene regions and amplification conditions that produced reproducible results and detected the most positive cases, respectively, in conventional PCR and QPCR to minimize potential bias inherent in the PCR methods. A semi-nested amplification of the P. falciparum-specific microsatellite PFPK2 [In our pilot study, three different gene regions  that targeted on a falciparum-specific rRNA region. Amplification was conducted in a 20ul reaction mixture containing 2ul of genomic DNA, 10ul 2×SYBR Green qPCR Master Mix (Thermo Scientific), and 0.5uM primer. Reaction was performed in CFX96 Touch Real-Time PCR Detection System (BIORAD), with an initial denaturation at 95°C for 3 min, followed by 45 cycles at 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min with a final 95°C for 10 sec. This was then followed by a melting curve step of temperature ranged from 65°C to 95°C with 0.5°C increment to determine the melting temperature of each amplified product. Each assay included both positive controls (7G8 and HB3 isolates) and negative controls (uninfected samples and water). A standard curve was produced from 10-fold dilution series of P. falciparum control plasmid ranging from 105 to 101 copies/μl to evaluate qPCR amplification efficiency (E). The 10-fold serial dilutions were included in each plate run as internal standard and the amplification efficiency was calculated from the corresponding threshold cycle (Ct) values of the dilutions for each plate samples. The amplification efficiency ranges from 90.5–92.8% among all runs. The negative controls showed Ct values of above 40. Samples yielding Ct values higher than 40 were considered negative for Plasmodium species. Melting curve analyses were performed for each amplified sample to confirm specific amplifications of the target sequence. The parasite gene copy number (GCN) in a sample was quantified based on the threshold cycle using the follow equation: GCNsample = 2Ctsample)E×, results of conventional PCR were used as the standard. Sensitivity of microscopy was calculated as true positives/, and specificity was calculated as true negatives/. The χ2-test was used to test for the significance of differences in parasite prevalence rates by microscopy versus PCR, as well among lowland versus highland sites. One-tailed T-test was used to test for the significance of differences in parasite gene copy number and parasite density among samples of different transmission settings (high versus low transmission sites), and across age groups . In addition, we calculated Pearson's correlation coefficient (r) for continuous data including (1) parasite density based on microscopy and parasite gene copy number based on QPCR; (2) parasite gene copy number and age in R (R Core Team 2013).Microsoft Excel software (Microsoft Office 2010) and MedCalc software  were used for sensitivity and specificity calculations. For all samples . Sensitivity of microscopy in the highland samples (34.8%) was shown to be 20% lower than in the lowland samples (53.3%), though specificity values of microscopy in both samples were similar  whereas conventional PCR detected 2,355 cases (21.1%), which was about 8% more of the total samples than microscopy (χ similar . Such di similar .1/μl (range 0.2×101 to 6.7×101/μl), which was apparently lower than those microscopic-positive and conventional PCR-positive samples (geometric mean gene copy number of 6.5×103/μl and range 6.3×101 to 2.6×106/μl). Likewise, the parasite density in the microscopic-positive and conventional PCR-negative samples (geometric mean 3.8×102 parasite/μl and range 4×101 to 3.5×103 parasites/μl) was also found to be lower than the microscopic-positive and conventional PCR-positive samples (mean 8.3×103 parasite/μl and range 4×101 to 1×105 parasites/μl).For the 1,470 samples that were detected positive by microscopy, 80%  but not all of them were detected positive by conventional PCR assay . Among t3/μl and range 1.1×101 to 2.7×106/μl in the lowlands versus a mean of 1.8×102/μl and range 0.08×101 to 2×105/μl in the highlands  was found to be significantly lower than in microscopic-positive samples  and highlands  counted on a stained thin blood film  in both highland and lowland sites . HoweverThe present study is by far the most comprehensive survey that examines asymptomatic infections in broad areas of Western Kenya and includes a deep sampling at community level based on both microscopy and PCR-based methods. We explore and compare parasite density and gene copy number among sites of different transmission settings as well as across age groups. It is noted that our samples represent only children and adolescents up to 18 years old but not the entire demographic population. This community-based study uncovers the extent of asymptomatic malaria in areas such as the eastern and southern shore of the Lake Victoria where malaria prevalence is previously unknown. The information obtained from this study is important because it provides a basis for identifying priority areas for disease control concerning asymptomatic malaria. Our data indicate that lowland areas in the northern shore of the Lake Victoria have the highest asymptomatic malaria infection rate; and as expected, highland fringe areas show relatively low transmissions. It is, however, noteworthy that not all lowland sites surrounding the lakeshore area have high transmissions, for example, both microscopic and PCR-based malaria prevalence rates were moderate in eastern and southern shore areas. Thus, apart from topography , there cOur comparisons of parasite gene copy number among sites using QPCR confirm that submicroscopic asymptomatic infections in the low transmission setting of the highlands have lower level of parasite gene copies than those in the high transmission setting of the lowlands and that such difference impacts diagnostic outcomes. Although the comparison was made on an imbalance sample size given that PCR-positive cases in the lowland are more than six times higher than those in the highland, the samples included in the QPCR assay well represent the majority of the study sites in Western Kenya. The contrast in parasite gene copy number supports the hypothesis that frequent infection in high-transmission areas increase the average parasite density in infected asymptomatic individuals, whereas in lower transmission areas asymptomatic infections are more likely to have reached a submicroscopic phase . These sOur study showed that microscopy detected a significantly smaller number of asymptomatic infections compared to PCR-based method, which is consistent with several previous findings that indicated molecular assay a more sensitive and time-efficient approach for asymptomatic malaria , 32–35. Previous studies found that both uncomplicated clinical malaria and asymptomatic infections peak in young children –9 and thIn summary, this study highlights the need for a more sensitive and time-efficient assay for asymptomatic malaria in areas of low-transmission settings because of the prevalence of submicroscopic infections with low parasite density and gene copy number. Combining PCR with microscopy can enhance the capacity of detecting low-density asymptomatic malaria infections and allow for an improved characterization of the current reservoir of infections that is largely hidden and heterogeneous. The combined diagnostic approaches are of key importance in providing precise malaria prevalence information for novel surveillance control strategies targeting the most infectious reservoirs.S1 TableAsterisks indicate sites where prevalence rate detected by microscopy is higher than that by nested PCR.(DOCX)Click here for additional data file.S2 TablePCR was used as gold standard.(DOCX)Click here for additional data file.S3 Table‘-’ denote data not available. Locality information of sites is presented in (DOCX)Click here for additional data file.S4 TableCt values were calculated from three independent runs and standard error values are provided. ‘-’ denote samples not detected by quantitative PCR.The mean (DOCX)Click here for additional data file.S1 FigPearson’s product moment (r) correlation coefficients were indicated.(TIF)Click here for additional data file.


In African highland areas where endemicity of malaria varies greatly according to altitude and topography, parasitaemia accompanied by fever may not be sufficient to define an episode of clinical malaria in endemic areas. To evaluate the effectiveness of malaria interventions, age-specific case definitions of clinical malaria needs to be determined. Cases of clinical malaria through active case surveillance were quantified in a highland area in Kenya and defined clinical malaria for different age groups.Plasmodium falciparum positive blood smear. Individuals in the same cohort were screened for asymptomatic malaria infection during the low and high malaria transmission seasons. Parasite densities and temperature were used to define clinical malaria by age in the population. The proportion of fevers attributable to malaria was calculated using logistic regression models.A cohort of over 1,800 participants from all age groups was selected randomly from over 350 houses in 10 villages stratified by topography and followed for two-and-a-half years. Participants were visited every two weeks and screened for clinical malaria, defined as an individual with malaria-related symptoms  at the time of examination or 1–2 days prior to the examination in the presence of a Incidence of clinical malaria was highest in valley bottom population  compared to mid-hill  and up-hill  populations. The optimum cut-off parasite densities through the determination of the sensitivity and specificity showed that in children less than five years of age, 500 parasites per μl of blood could be used to define the malaria attributable fever cases for this age group. In children between the ages of 5–14, a parasite density of 1,000 parasites per μl of blood could be used to define the malaria attributable fever cases. For individuals older than 14 years, the cut-off parasite density was 3,000 parasites per μl of blood.Clinical malaria case definitions are affected by age and endemicity, which needs to be taken into consideration during evaluation of interventions. Evaluation of malaria interventions, such as long-lasting impregnated nets (LLINs), indoor residual spraying (IRS), drugs and vaccines trials depends on clinical definition of the disease, which is still a challenge due to lack of distinct malaria specific clinical features. Malaria case definitions for interventions differ from those used in clinical care in that high specificity is needed and this is complicated by the relatively high prevalence of asymptomatic parasitaemia in endemic areas . ParasitThe clinical signs of malaria are nonspecific and parasitaemia accompanied by clinical symptoms consistent with malaria does not necessarily imply clinical malaria especially in endemic areas . To measClear case definitions of malaria are an essential means of evaluating the effectiveness of present and proposed interventions in malaria. Several studies have estimated parasite cut-off densities in different settings, using the point at which the sensitivity and specificities are both highest as the optimum case definition. This definition is most appropriate for clinical trials for which higher specificity is more important than that for clinical diagnosis and treatment, where high sensitivity is paramount. Definitions range from any to 7,000 parasites per microlitre (μl) of blood depending on age , 7, seaset al.[The distribution pattern of febrile malaria may be used to infer environmental risk factors and thus providing crucial information for the development of new prevention or intervention methods. The topography in the highlands of western Kenya is made up of hills and valleys and this affects the epidemiology of malaria , 10, 12.et al. showed tIn this study, Clinical malaria cases were quantified through active case surveillance and asymptomatic malaria infection screening and used the data to define age-specific clinical cases in highland areas in western Kenya. This was part of a study to test different combinations of malaria interventions in the area.Plasmodium falciparum is the predominant malaria species in the study sites and it is transmitted by Anopheles gambiae, Anopheles arabiensis and Anopheles funestus[The study was conducted in a highland site in Iguhu, Kakamega south district, western Kenya. Ten clusters of villages were selected which were stratified by topography. Individual households are spread almost everywhere from the bottom of the valleys , through the middle of the hills (mid-hill) up to the top of the hills (hill top). The Yala River transects the site and most of the mosquito breeding takes place in cultivated swamps in the valley and also at the edges of the several streams in the site, therefore malaria transmission intensity varied from valley bottom  to hill tops  . Malariafunestus, 16, 17.P. falciparum positive blood smear [A cohort of over 1,800 participants of all ages was selected randomly from over 350 houses in the 10 villages in Iguhu for the longitudinal study. All houses in the study sites had been mapped and assigned a unique identification number which has been previously described . Particiod smear . During During the dry (February and March) and rainy (June and July) seasons, which represent the low and high malaria transmission seasons in the area respectively, the cohort population was screened for asymptomatic malaria infection. Thick and thin blood smears were made from each individual. Temperature, symptoms of malaria, if any, were recorded. Four surveys were done, two each in 2008 and 2009. Blood smears were not analysed immediately and, therefore, participants who had positive parasitaemia had no treatment. Nevertheless, each participant already had an ID card to access free medical care in the health facilities.The thin and thick blood smears were air-dried. The thin smears were fixed in methanol and stained in 4% Giemsa for 30 minutes. Two experienced laboratory technicians who were blinded to each other’s results examined the slides under × 1,000 oil immersion to obtain a species specific parasite count. Results were later compared and if there were any discrepancies in slide readings, a third and more experienced technician was brought in to confirm the diagnoses. Parasite density was scored against 200 leukocytes when the slide was positive; otherwise, the whole slide was carefully scanned before being declared negative. Parasite densities were converted to number of parasites per microliter of blood, assuming a leukocyte count of 8,000 cells/μL . For quaEthical clearance was given by the Ethical Review Committee (ERC) of the Kenya Medical Research Institute, Kenya and Institutional Review Board (IRB) of University of California, Irvine, USA. Written consent was obtained from all participants. Written consent for children (<18 years of age) was provided by the participants and their parents or guardians. Inclusion criteria were: provision of informed consent, age >6 months at recruitment, and no reported chronic or acute illness except malaria. Exclusion criteria were: those who were unwilling to participate in the study, those who planned to move out of the study area and those with no reported chronic or acute illness except malaria.t-test with Bonferroni correction. Asymptomatic malaria infection rate was calculated as ratio of infected individuals over total samples. The differences in incidence rate of clinical malaria and asymptomatic infection rates between the topographical sites were determined by chi-square test, between season and year differences were tested the same way.Data were entered into Microsoft Excel spreadsheet and processed and analyzed using SPSS software package  and JMP Statistical software . SampledThe case definitions were derived using logistic regression methods. The logistic regression model used was P is the probability that a participant with parasites at a density x had a fever, and where τ is the power function of the parasite density. The power function was derived using maximum likelihood estimation for the different age groups and was used to model the relationship between fever and parasite density as a continuous function. The cutoff parasite density for different study area and age groups were determined by the intersection of sensitivity and specificity curves. The malaria attributable fraction (MAF) was calculated as the reduction in incidence that would be observed if the population were entirely unexposed, compared with its current  exposure pattern.where Incidence rate of clinical malaria among the study participants detected through active case surveillance was found to be associated with topography of the area. It was clustered around the valley bottoms. The incidence rate of clinical malaria was highest in the population living in the valley bottoms  compared to the population in the mid-hill   and in the up-hill population  . The population in the mid-hill sites had a higher incidence of clinical malaria than the uphill residents  followed by those living in the mid-hill (4.49%) and up-hill (1.85%) sites . Infections at all sites were also higher during the rainy season . 0.78% folThe optimum cut-off parasite densities through the determination of the sensitivity and specificity showed that in children less than five years of age, 500 parasites per μl of blood could be used to define the malaria attributable fever cases for this age group. In children between the ages of 5–14, a parasite density of 1,000 parasites per μl of blood could be used to define the malaria attributable fever cases. For individuals older than 14 years, the cut-off parasite density was higher. The cut-off value to determine the malaria attributable fever was found to be 3,000 parasites per μl of blood. Figure From the model, the mean malaria-attributable fraction or the probability that any individual fever case was attributable to malaria was 0.65 in children less than five years, 0.58 in the age group 5–14 years and was 0.32 in the age group of ≥ 15 years. As expected, the adult group had significantly lower malaria-attributed fraction among the three age groups (Table This study quantified clinical malaria cases through active case surveillance and asymptomatic screening for malaria infections. Fever and parasite densities were used to define age-specific clinical malaria cases in a highland area in western Kenya. There was clustering of clinical malaria cases and asymptomatic malaria infections around the bottom of the valley both through active case surveillance and the screening of the cohort for asymptomatic infections respectively and with very clear seasonality. Individuals 15 years and older had higher cutoff points of parasite density used in defining clinical malaria than those younger than that age. From the malaria attributable fraction estimations, many of the fevers in the population could be attributed to clinical malaria.Malaria transmission in most parts of the highlands of East Africa and indeed in western Kenya is spatially and temporally driven by climate variability and topography , 23, 24.et al.[The results reveal an increasing trend in the incidence of clinical malaria in the area. Malaria infections increased each year during the study period. These results supports what other studies have reported in western Kenya , 26 on tet al., supportThe differences in cut off values for parasite density with increase in age could be attributed to the fact that older people could tolerate higher densities of parasites than younger ones, due in part to immunity build up. The cut-off value for parasites densities to define clinical malaria for individuals older than 15 years was 3,000 parasite per μl of blood showing how much they could tolerate malaria parasite in their bodies without getting sick. In children under five years of age, they only require 500 parasites per μl of blood and children between 5–14 years require 1,000 parasites per μl of blood. These underlie how immunity build up affects asymptomatic parasite carriage. Immunity to malaria infection depends on endemicity of the disease in the area. Thus, people living in the valley bottoms of our study area may have more immunity to malaria than the mid-hill and uphill population. Endemicity of malaria could also be a factor in determining cut-off values for parasite density in defining clinical malaria , 28. In In this area, over 60% of participants with fever or a history of fever were not parasitaemic, underlying the fact that fever is not a specific marker for clinical malaria. It was found that for people above the cut-off value for the definition of clinical malaria, over 90% of the fevers could be attributed to malaria. These results show the high specificity of our results and the fact that fever alone with parasitaemia cannot be used to define clinical malaria. One limitation to this study was that fever cases might have been missed in between home visits that might not have been added to the overall fever cases.It can be concluded that in highland areas where malaria transmission varies by topography, clinical malaria should be defined by age and also by endemicity when evaluations of interventions are being conducted. However, the situation where intervention groups and control groups are being evaluated will bring challenges because whereas the control groups might have higher parasitaemia, the intervention groups might have lower parasitaemia which might bring distortions in the definition for clinical malaria even in the same age group or transmission settings.


Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis.Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during  During a malaria infection, the vascular endothelium becomes more adhesive, permeable, and prone to trigger blood clotting. These changes help the parasite adhere to blood vessels, but endanger the host by obstructing blood flow through small vessels. Endothelial nitric oxide (NO) would normally counteract these pathological changes, but NO signalling is diminished malaria. NO synthesis is inhibited by asymmetric dimethylarginine (ADMA), a methylated derivative of arginine that is released during normal protein turnover. We found the ratio of ADMA to arginine to be elevated in Gambian children with severe malaria, a metabolic disturbance known to inhibit NO synthesis. ADMA was associated with markers of endothelial activation and impaired tissue perfusion. In parallel experiments using mice, the enzyme responsible for metabolizing ADMA, dimethylarginine dimethylaminohydrolase (DDAH), was inactivated after infection with a rodent malaria. Based on these studies, we propose that decreased metabolism of ADMA by DDAH might contribute to the elevated ADMA/arginine ratio observed during an acute episode of malaria. Strategies to preserve or increase DDAH activity might improve NO synthesis and help to prevent the vascular manifestations of severe malaria. Current estimates of world-wide mortality due to malaria range from 367,000 to 755,000 deaths per year, mostly in African children ,2. PrompPatients with severe malaria exhibit impaired endothelium-dependent vasodilation  and reduDDAH1 polymorphism is associated with susceptibility to severe malaria : 0.40 [0.30–0.51] μmol/L) or uncomplicated malaria (0.40 [0.33–0.47] μmol/L) compared to healthy children  compared to children with uncomplicated malaria  or healthy children ; children with lower arginine tended to have lower ADMA . In chilLactate is a biomarker of impaired tissue perfusion that is associated with mortality from severe malaria in children –43. In oWe measured soluble vascular cell adhesion molecule (sVCAM) as a biomarker of endothelial activation. Plasma sVCAM was elevated in children with severe malaria at the time of admission compared to children with uncomplicated malaria or healthy children , a circulating marker of parasite biomass, was higher in children with severe malaria compared to uncomplicated malaria  compared with uninfected controls  though the tissue levels of ADMA were higher, and the DDAH activities were lower than in uninfected mice . We also calculated the correlation between hepatic DDAH activity and plasma ADMA/Arginine ratio in mice, and found a negative trend, i.e., mice with lower DDAH activity tended to have higher ADMA/Arginine ratios in plasma , i.e., hepatic DDAH activities were greater in mice that had higher tissue levels of ADMA. This may reflect induction of DDAH activity in response to tissue levels of ADMA. Among ted mice . The parPlasmodium berghei ANKA, a model of severe malaria. P berghei infection caused inactivation of hepatic DDAH1, accumulation of intracellular ADMA in liver tissue, elevation of the ADMA to arginine ratio in plasma, and decreased levels of nitrite in blood. Although these findings in the mouse model cannot be directly extrapolated to human malaria, it raises the possibility that the elevated ADMA/arginine ratio observed in children with severe malaria could be due in part to inactivation of DDAH1.We have analyzed ADMA and arginine concentrations in plasma from children with severe malaria to determine whether malaria is associated with disruption of ADMA/arginine homeostasis. We found that children with acute severe malaria have uncompensated hypoargininemia, i.e., low arginine with an elevated ADMA/arginine ratio. The hypoargininemia persisted over the 28 days of follow-up, while the ratio of ADMA to arginine returned to normal. We interpret this as a transient inability to metabolize ADMA at a sufficient rate to compensate for low arginine during acute infection. Although plasma ADMA levels were below normal in patients with severe malaria, ADMA was positively correlated with lactate, a biomarker of severity, and sVCAM, a biomarker of endothelial activation, suggesting that higher ADMA levels are associated with adverse pathophysiologic changes. DDAH1 metabolizes ADMA, so we examined changes in DDAH1 activity in mice infected with a −3 in cerebral malaria, 12.3 [10.0–15.1] ×10−3 in non-cerebral severe malaria, 12.6 [10.7–15.1] ×10−3 in moderately severe malaria and 7.1 [5.8–9.0] ×10−3 in healthy children [Our results extend upon a previous report of ADMA and arginine levels in Tanzanian children. Weinberg et al. observed ADMA/arginine ratios of 13.2 [11.1–16.4] ×10children . These vchildren . As a reIn both Gambian and Tanzanian children with severe malaria, ADMA and the ADMA/Arg ratio were each correlated with lactate. This could be mediated through the vasoconstrictive or pro-adhesive effects of ADMA on vascular endothelium especially in the setting of hypoarginemia, with subsequent impairment of tissue perfusion leading to anaerobic glycolysis and lactate generation. Inter-individual differences in hepatic blood flow could also be responsible for the strong correlation between ADMA and lactate, since the clearance of each is dependent on hepatic perfusion. Impaired perfusion of liver tissue has been observed in adults with severe malaria  and coulIn both Gambian and Tanzanian children with severe malaria, ADMA was correlated with biomarkers of endothelial activation . This could be through direct effects of ADMA on endothelial cells  or via tP. falciparum histidine-rich protein 2 (PfHRP2) has been previously assessed as a quantitative marker of parasite biomass [ biomass ,52. PfHR biomass  and togeArginine depletion and disruption of ADMA/arginine homeostasis have also been observed in adults with moderately severe and severe malaria . In contDisruption of ADMA/arginine homeostasis in children with severe malaria could be due to increased protein methylation, accelerated proteolysis of methylated proteins or impaired clearance of free ADMA. One might expect plasma ADMA to be directly elevated during a severe malaria infection, due to the combination of increased release of ADMA from erythrocytes undergoing hemolysis  and the DDAH1, suppression of DDAH2 expression did not result in altered plasma ADMA concentrations [DDAH1 [DDAH1 expression in liver significantly lowered plasma ADMA [DDAH1 is known to regulate ADMA/arginine homeostasis: heterozygous knock-out of DDAH1 in mice increased plasma ADMA, decreased NO-dependent vasodilation, and elevated blood pressure . Converstrations . The livs [DDAH1  and metas [DDAH1 . Inductisma ADMA . Endothesma ADMA ,66, whicsma ADMA ,67,68 thsma ADMA , in addiPlasmodium infection appears to accelerate the degradation of DDAH1 protein in hepatic tissue. Oxidative stress is a potential trigger of DDAH degradation that is present during malaria infection [phox subunit of NADPH oxidase in smooth muscle cells increased oxidative stress, decreased DDAH protein levels, decreased DDAH activity and caused accumulation of both intracellular and extracellular ADMA [phox-transfected smooth muscle cells with the proteasome inhibitor epoxomicin raised DDAH protein concentrations and reduced intracellular ADMA, demonstrating that oxidative modification of DDAH protein may target it for degradation by the proteasome. Plasmodium infection causes oxidative stress in liver tissue of mice [nfection ,70. Overlar ADMA . Treatme of mice , which cThe liver may be exposed to reactive oxygen species generated by the increased populations of neutrophils and pigment-laden monocytes found in the hepatic vasculature during malaria infection ,73. Incri of eNOS [Elevation of ADMA relative to arginine favors NOS inhibition because ADMA is a competitive inhibitor of NOS . ADMA al of eNOS ,77. Even of eNOS ,79. Impa of eNOS ,7. NO sy of eNOS , which m of eNOS  and L-NA of eNOS . Taken tPlasmodium infection are needed to establish a causal relationship between DDAH degradation and disruption of ADMA/arginine homeostasis. While restoring ADMA/Arginine homeostasis might be necessary to improve endothelial NO synthesis, it might not be sufficient: impaired endothelial NO synthesis is likely to be limited by arginine deficiency and oxidation of tetrahydrobiopterin. NO that is produced will have a limited half-life due to reactions with cell free hemoglobin, superoxide, and other radicals that have increased abundance during malaria infection.Therapeutic strategies that preserve or enhance DDAH activity during In summary, through clinical observational studies of Gambian children and controlled experiments mice, we have identified hepatic DDAH dysfunction as a potential mechanism disturbing ADMA/Arginine homeostasis and limiting nitric oxide synthesis in severe malaria.Patient enrollment and sample collection were conducted following ethical review and approval by the Gambian Government/MRC Joint Ethics Committee and the Ethics Committee of the London School of Hygiene & Tropical Medicine . Analysis of plasma samples and de-identified clinical data at the National Institutes of Health was exempted from further ethical review by the NIH Office of Subjects Research (Exemption #5161). Written informed consent was provided by a parent or guardian on behalf of children enrolled in the study. Families who declined to participate were provided standard medical care.Animal studies  within the previous 48 hrs and the absence of severe criteria. Acute severe malaria was defined as parasitemia of >5000 parasites/μl, a history of fever and one or more of the following: severe anemia (Hb < 6g/dl), severe acidosis (serum lactate >7 mmol/L), cerebral malaria (Blantyre coma score 2 or less in the absence of hypoglycemia or hypovolemia with the coma lasting for at least 2 hrs), and severe prostration (inability to sit unsupported in children >6 months or inability to suck in children <6 months). Patients with severe malaria were admitted and treated with quinine, and patients with uncomplicated malaria were treated with chloroquine plus sulfadoxine-pyrimethamine according to Gambian Government Treatment Guidelines [Acute uncomplicated malaria was defined as asexual idelines .°C. Three patients with severe malaria died and 65 completed follow-up visits; plasma sample were available from 47 of them. Eighty-five patients with uncomplicated malaria completed follow-up visits; plasma samples were available from 65 of them. Thirty-one healthy, afebrile, aparasitemic Gambian children of similar ages were also studied at a single visit.Ninety-six children with severe malaria and 102 children with uncomplicated malaria were enrolled during the 2005–2008 malaria seasons. Four milliliters of blood were collected in heparinized vacutainers (BD) at the time of initial presentation and at a follow-up visit 28 days later. Blood samples were immediately refrigerated, placed on ice for transport, and processed within 2 hours of collection. Plasma was frozen at -80P. falciparum parasitemia was determined by bright-field microscopy of giemsa-stained blood smears. 50 fields were counted at high power. Full blood counts were obtained with an automated instrument . Lactate was measured with a handheld Lactate Pro device . Soluble VCAM (sVCAM) and plasma haptoglobin concentrations were determined by ELISA  according to the manufacturer’s instructions.P. falciparum HRP2 was measured in duplicate in plasma by ELISA (Cellabs) according to the manufacturer’s instructions. A standard curve was constructed using serial dilutions of the PfHRP2 standard and run with every plate. Laboratory staff were unaware of clinical status of the subjects.G-monoethyl-L-arginine (MEA) as an internal standard before undergoing solid-phase extraction . The eluted cationic amino acids were dried, resuspended in water, and derivatized with ortho-phthalaldehyde (OPA) in 3-mercaptopropionic acid. Derivatized samples were separated by reverse-phase liquid chromatography over a 1×100 mm C18(2) column  and fluorescence detected at excitation and emission wavelengths of 340nm and 455nm, respectively. Concentrations were determined by integrating peak area with reference to the internal standard (MEA) and daily external standards .Each plasma sample was diluted in PBS containing NData are expressed as median and inter-quartile range (IQR). Statistical analyses were performed with GraphPad Prism 6.02 software and the R computing environment. P-values of less than 0.05 were considered significant. Mann-Whitney test was used to compare median values between healthy children and children with uncomplicated or severe malaria. Wilcoxon matched-pairs signed rank test was used to compare acute and recovery values in children with uncomplicated or severe malaria. Correlations were calculated on transformed data using Pearson’s correlation test, except in the case of correlation with haptoglobin in which Spearman’s method was used. When two separate groups were combined, partial correlations were calculated. Multiple linear regression analysis was used to assess the independent contributions of ADMA or arginine to correlations with hemoglobin, HRP2, sVCAM, or lactate. In these linear models, arginine and ADMA were the explanatory variables, and the direction, strength and significance of the association was assessed by the beta value and p-value. Mann-Whitney test was used to compare values between infected mice and uninfected controls. Data are available in supplemental files S1 TableData are presented as Pearson’s correlation coefficients, partial correlation coefficients and p-values. df, degrees of freedom. The results in rows (A) and (B) correspond to (DOCX)Click here for additional data file.S2 TableData are presented as Pearson’s correlation coefficients, partial correlation coefficients and p-values. df, degrees of freedom. The results in rows (C) and (D) correspond to (DOCX)Click here for additional data file.S1 Figr, and p-value are provided in the upper left corner of each graph of ADMA and arginine in healthy children , children with uncomplicated malaria , and children with severe malaria . Day 28 follow up values are presented in the lower panels. The slopes of the linear regression models are presented in the lower right corner of each graph, along with the p-value for F-tests comparing the slopes of each group as indicated.Pearson’s correlation coefficient, (PDF)Click here for additional data file.S2 FigIn the No Compensation model, ADMA remains constant as arginine concentration falls, causing the ADMA/Arg ratio to rise according to a reciprocal function. In the Full Compensation model, ADMA is regulated to maintain a constant ADMA/Arg ratio in the setting of falling arginine concentration. The models are based on ADMA and arginine measurements from healthy Gambian children . During acute severe malaria, children exhibit incomplete compensation of ADMA in the setting of hypoargininemia (red circle), but they return to a compensated state by day 28 despite persistent hypoargininemia (dark gray circle). Children with uncomplicated malaria also exhibit incomplete compensation of ADMA in the setting of hypoargininemia and return to a compensated state by day 28 (orange and light gray circles), though the disturbance is less than observed in severe malaria. Arrows indicate the shift from admission to day 28 values.(PDF)Click here for additional data file.S3 FigX-y plots are presented to support the correlation analyses detailed in (PDF)Click here for additional data file.S4 FigP berghei-infected mice; open circles represent measurements obtained from uninfected control mice. Dotted line represents the linear regression of data from infected mice and dashed line is from uninfected mice. Pearson’s r and p-values for the correlations of each group and combined groups are provided in Filled circles represent measurements obtained from (PDF)Click here for additional data file.S5 Fig2 = 0.9972). This confirmed that ADMA metabolism is constant and approximates Vmax kinetics when 2.5 mmol/L ADMA is present. Citrulline production was negligible in the absence of exogenous ADMA . (B) Citrulline (25 μmol/L) was stable during a 2 hr incubation at 37°C , demonstrating that citrulline is not degraded or metabolized by enzymes in liver homogenate and is thus a reliable indicator of DDAH activity. (C and D) After addition of 2.5 mmol/L ADMA to liver homogenate, the rate of citrulline production was linear with respect to the protein concentration of liver homogenate in the assay .DDAH activity in liver homogenates was assessed by quantifying citrulline production in the presence of saturating concentrations of ADMA substrate (2.5 mmol/L). (A) After addition of 2.5 mmol/L of ADMA to liver homogenate, citrulline increased linearly over time (R(PDF)Click here for additional data file.S1 MethodsDetailed description of animal experiments and laboratory methods are provided in the supporting information.(PDF)Click here for additional data file.S1 DataData are provided in.csv format for inspection and analysis.(CSV)Click here for additional data file.S2 DataData are provided in.csv format for inspection and analysis.(CSV)Click here for additional data file.


Identifying the prevalence and distribution of P. falciparum parasites with deleted pfhrp2 is important for malaria control.Malaria rapid diagnostic tests (RDTs) play a key role in malaria management and control. The PfHRP-2 based RDT is the most widely used RDT for malaria diagnosis in Ghana. Deletion of pfhrp2 deletant P. falciparum parasites circulating within different regions of Ghana.The purpose of this study was to identify and confirm the prevalence of pfhrp2 and pfhrp3 genes were amplified by polymerase chain reaction (PCR), resolved by agarose gel electrophoresis and visualized under UV light.DNA was extracted from the membrane of spent CareStart™ PfHRP-2 RDT kits and dried filter paper blood blots using Chelex-100. Exon 2 of pfhrp2 gene was deleted in 18/54 (33.3 %) of the microscopy confirmed and 36.2 % (34/94) of the PCR confirmed RDT positive samples collected in Accra. No RDT sample, confirmed to contain parasites by either PCR or microscopy was negative by pfhrp2 exon 2 PCR in Cape Coast. A survey of an additional 558 DBS revealed that 22.4 % (46/205) and 40 % (44/110) of PCR positive samples in Accra and Cape Coast, respectively, lacked the exon 2 region of pfhrp2 and possibly the entire pfhrp2 gene.Microscopic analysis of blood smears from samples that were PfHRP-2 RDT positive revealed a parasite prevalence of 54/114 (47.4 %) and 2/26 (7.7 %) in Accra and Cape Coast, respectively. PCR analysis increased parasite prevalence in the RDT positive samples to 94/114 (82.5 %) and 6/26 (23.1 %) in Accra and Cape Coast respectively. The exon 2 of the P. falciparum parasites, which lack pfhrp2 exon 2 gene have been identified in two communities in Ghana. Continuous nationwide monitoring of the prevalence of pfhrp2 deletant parasites would be essential to malaria control. The use of RDT kits that are effective at malaria diagnosis despite deletion of pfhrp2, such as the PfHRP-2/PfLDH combo RDT kit could enhance the diagnosis of clinical malaria in Ghana.A high number of  Anopheles mosquito. Early diagnosis is very important for disease management and the effective treatment of malaria. Before the advent of malaria rapid diagnostic tests (RDTs), diagnosis was based on microscopy of thick blood smears, which is still the gold standard for malaria diagnosis. However, in a number of rural and semi urban settings where lack of equipment, trained personnel and electricity prevents this essential diagnosis, health practitioners diagnose malaria based solely on clinical evaluation of symptoms [Malaria is one of the deadliest infectious diseases of humanity, which causes significant mortality and morbidity in the tropics, particularly in Africa . Malariasymptoms . RDTs ofsymptoms .Plasmodium falciparum specifically or discriminately detect both P. falciparum in addition to another human malaria parasite or indiscriminately detect all human malaria parasites [P. falciparum specific antigen with the advantage of being highly abundant and heat stable however, the PfHRP-2 antigen remains in circulation for up to 4 weeks after the malaria parasites have cleared [P. falciparum, ten detect P. falciparum as a part of a mixed infection with other human malaria parasites, one is Plasmodium vivax specific and 115 detect and distinguishing P. falciparum from either P. vivax mixed infections or mixed infections containing all the other human malaria parasites, P. vivax, P. ovale and P.malariae [Presently there is a very large demand for malaria RDT kits, as the World Health Organization (WHO) has recommended its use and majority of National Malaria Control Programmes have accepted it as the first step in the diagnosis of malaria. Due to the importance of the results of this initial screen, the WHO has established two programmes, the Foundation for Innovative New Diagnostics (FIND) malaria RDT quality assurance programme and WHO-FIND malaria RDT lot testing programme whose main mandate are to ensure accurate diagnosis of malaria . Malariaarasites , 5. The  cleared , 7. Some cleared , 9. Thusmalariae .pfhrp2 and or pfhrp3 genotype of the parasite [The accuracy of malaria RDT results can be affected by test antibody stability, product design and quality as well as the transport and storage conditions of the kits and sample parasite density . Accuratparasite , 10, 11,parasite , 13 as wparasite , 15.pfhrp2 deletant (pfhrp2−) parasites as well determines the prevalence of P. falciparum parasites with deletions in pfhrp2 and pfhrp3 (pfhrp3−) in two communities in Ghana.In some facilities in Ghana, where microscopy is unavailable, malaria is treated based on RDT results. It is thus very important to monitor the accuracy of RDT results as well as identify factors that affect the diagnostic ability of malaria RDTs. So far the main studies conducted in Ghana have determined the sensitivity and specificity of different brands of malaria RDT kits, including the CareStart™ and Paracheck RDT Kit , 16–18, This study was approved by the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research, University of Ghana. Prior to enrollment, the study was explained to all participants after which written informed consent was obtained. Parental consent was obtained from parents and guardians of all children in addition to child assent obtained from children between 12 and 17 years.Abura Dunkwa, also known as Abura, is the district capital for Abura-Asebu-Kwamankese district and the Cape Coast Metropolis of the Central Region with a rural population of 31,768 for children under 14 years of age . The CenObom is in the Ga south municipality of the Greater Accra Region with a rural population of 22,368 for children under 14 years of age . MalariaThe study utilized a total of 226 spent PfHRP-2 based RDT kits as well as 558 filter paper blood blots from consenting healthy children within the two study sites in 2015. Thick and thin blood smears as well as DBS samples were obtained from healthy school children as part of a monthly malaria-screening programme from February through May. In April, RDT was performed according to manufacturers instructions in addition to the DBS and blood smears. The spent RDT cassettes were stored at room temperature for a maximum of 1 week, after which their membranes were processed for DNA. Approximately 50 μl of finger-pricked blood was spotted on to filter paper to make the DBS and thick blood smears. DBS were kept in sealed plastic bags with a desiccant and stored at −20 °C for no longer than 2 weeks after which they were processed for genomic DNA (gDNA). The slides containing the thick and thin blood smears were air dried and stored in slides boxes.P. falciparum parasites on a Giemsa-stained thick blood smear. A sample was considered PCR positive when P. falciparum parasite genotyping using standard WHO genotyping procedures yielded a product. A positive RDT result was referred to as RDT positivity results, while a sample was considered positive for P. falciparum by RDT when the positive test strip was confirmed by microscopy or PCR. RDT positivity is used frequently as an indication of malaria in some facilities in Ghana where microscopy is unavailable.A sample was defined as negative by microscopy when two independent microscopists confirmed the absence of Thick and thin blood smears as well as dried filter paper blood spots (DBS) were each made from a drop (~50 μl) of finger-prick blood. The blood smears were processed and then stained with 10 % Giemsa for 15 min. The stained slides were subsequently air-dried and viewed under 100X oil immersion microscope. Two independent microscopists read the slides and parasitaemia was determined as the  % of malaria parasite infected RBCs observed per 200 white blood cells (WBCs).2 disks from each of the dried blood spot (DBS). Each sample pieces was put into a 1.5 ml microcentrifuge tube containing 200 µl TE. The sample tubes were heated at 97 °C for 15 min on a dry heating block, centrifuged at 10,000g for 30 s after which the supernatant transferred into a 500 μl tube for storage at −20 °C. For the chelex extraction, 150 μl of 6 % chelex in PBS was added to the tube with the punched DBS disks. The tubes were then incubated at 95 °C for 30 min with intermittent mixing by vortexing followed by a quick centrifugation step. The samples were centrifuged at 6000g for 6 min, after which 120 μl of the supernatant was transferred into a 500 µl tube for storage at −20 °C.Genomic DNA was isolated from the membranes of the previously used PfHRP-2 RDT kits and dried filter paper blood spots (DBS) using either Tris–EDTA (TE)  or cheleThe WHO malaria parasite genotyping protocol  was foll® Fast PCR Master Mix UP. The DNA was initially denatured at 96 °C for 10 min followed by 41 cycles of denaturation at 95 °C for 50 s, annealing at 55 °C for 50 s (pfhrp2 gene) or 51 °C (pfhrp3 gene) and extension at 68 °C for 1 min. The final extension was performed at 72 °C for 5 min then to 4 °C. Genomic DNA from Dd2.The PCR amplification was adapted from Baker et al.  with verpfhrp2−), HB3 (pfhrp3−) and 3D7 (wild type) were used as controls for the PCR amplifications. The primers used in the amplification of the exon 2 regions of pfhrp2 and pfhrp3 were pfhrp2-F1, pfhrp2-F2 and pfhrp2-R1 for pfhrp2; pfhrp3-F1, pfhrp3-F2 and pfhrp3-R1 for pfhrp3, as previously listed [ loaded on the same gel.Crosstab descriptive analysis was performed using IBM SPSS Statistics (version 22). Microsoft Excel was used to draw the table and graphs.Parasite carriage observed by microscopy in Obom (Accra) and Abura (Cape Coast) for February through May of 2015 was 41.2 % (120/291) and 0.7 % (2/267) respectively  and 41.2 % (110/267) was identified by PCR genotyping in Accra and Cape Coast respectively during the period of February through May 2015  of the samples did not yield a product after PCR amplification and were classified as pfhrp3−  of the PCR positive samples contained rcent 35/79 of theP. falciparum parasites that lacked exon 2 of pfhrp2. Forty-five percent (49/109) of the samples lacked exon 2 of pfhrp3. One sample was omitted in the pfhrp3 PCR analysis. Overall, 27.5 % of the samples contained parasites that lacked exon 2 of both pfhrp2 and pfhrp3  of the PCR positive samples contained 10 of theTwo hundred and twenty six (226) spent PfHRP-2 RDT kits were obtained from school children in Accra and Cape Coast in April, 2015. Four of the test kits collected in Accra gave invalid results. An RDT positivity rate of 75 % (114/152) was observed in Accra and 35.1 % (26/74) in Cape Coast  and 1/74 (1.4 %) of the samples from Accra and Cape Coast, respectively  of the samples lacked both pfhrp2 and pfhrp3  to 7.4 % (4/54) in microscopy-confirmed RDT positive samples. The prevalence of pfhrp2−/pfhrp3+ samples however decreased from 31.9 % (30/94) to 25.9 % (14/54)  of the PCR-confirmed RDT positive samples lacked pfhrp2− parasites that had intact pfhrp3 (pfhrp2−/pfhrp3+) was to be 14 % (25/205) in Accra and 13 % (14/110) in Cape Coast and the prevalence of parasites with intact pfhrp2 that were pfhrp3− (pfhrp2+/pfhrp3−) was 16 % (29/201) in Accra and 17 % (19/109) in Cape Coast   parasites; each gDNA sample was analyzed by PCR genotyping prior to pfhrp2 and pfhrp3 exon 2 PCR.To estimate the true prevalence of P. falciparum parasite prevalence was high, the PCR estimate of parasite prevalence was comparable to the RDT positive rate . Microscopic evaluation of the RDT samples increased the false positive rate to 60/114 (52.6 %) in Accra. PCR genotyping identified 26/38 negative branded RDT kits to be positive for P. falciparum, out of these 26 samples, 12 were positive by microscopy.False positive PfHRP-2 RDT results are not uncommon in malaria endemic settings as the PfHRP2 antigen persists for weeks after parasite clearance; however, in Accra where ate Fig. b. DespitP. falciparum parasites causes the persistence of PfHRP2 to become more evident.In Cape Coast, the RDT positive rate was higher than parasite estimation by both microscopy and PCR, confirming PfHRP2 antigen persistence. There were only two positive microscopy slides over the entire 4 months (Table pfhrp2 gene (Table pfhrp2.False negative RDT results are obtained when parasite carriage is confirmed by either microscopy or PCR, however the RDT kit produces a negative test results. This can have severe consequences in malaria endemic settings where negative RDT kit results are not confirmed by any other diagnostic tests such as microscopy. The prevalence of false negative RDT results increased from 18/38 when the samples collected in Accra were confirmed by microscopy to 26/38 when confirmed by PCR (Table pfhrp2 by exon 2 PCR, confirming our observation that factors other than pfhrp2 deletion, including the high prevalence of submicroscopic parasites accounted for the false negative RDT results (Table In Cape Coast, PCR genotyping confirmed the presence of 12 false negative RDT tests. All 12 samples were positive for ts Table .pfhrp3 for the lack of pfhrp2 could also contribute to the positive RDT results obtained in samples that lacked pfhrp2. Eighty-eight percent of the samples from Accra that were pfhrp2− but were positive by PfHRP2 RDT and PCR genotyping were pfhrp3+ (Fig. Persistence of PfHRP-2 antigen from a recent past infection could explain the false positive RDT test, however possible compensation of pfhrp2−/pfhrp3− parasites have been found to be as high as 25.7 % in some countries within the Amazon basin [pfhrp2−/pfhrp3− was 28 % in samples obtained from Cape Coast over February through May (Fig. pfhrp2−/pfhrp3− parasite. The prevalence of double pfhrp2−/pfhrp3− parasites obtained in Accra over the months of February to May was 4.3 %, which was similar to 3 % that obtained in the samples collected in April.Double on basin . The preP. falciparum prevalence and density is relatively low in most parts of Ghana. Although this is a major limitation, PfHRP-2 RDT kits are used for malaria diagnosis over this period, making this study highly important.This study was carried out in the off-peak malaria season, where Plasmodium falciparum parasites that lack pfhrp2 alone or in addition to pfhrp3 have been identified in two regions of Ghana based on the assumption that deletions in exon 2 of these genes represents deletions of the entire gene. Malaria RDT testing is highly suitable for diagnosis in communities where parasite densities are high but become less accurate when parasite densities are low and also where pfhrp2 deletant parasites are prevalent. More accurate diagnosis of malaria would be obtained in countries such as Ghana where pfhrp2 deletant parasites exits when a more sensitive RDT kit such as the pLDH/PfHRP-2 combo RDT kit that is able to detect pfhrp2 deletant parasites is used. More studies are needed to evaluate RDT use in the peak malaria season as well as identify the possible influence pfhrp2 and pfhrp3 sequence diversity has on the diagnostic read out of PfHRP-2 based RDT kits across Ghana.


Plasmodium vivax and Plasmodium falciparum whose respective prevalence influences the frequency of mixed plasmodial infections. The accuracy of their diagnosis is influenced by the sensitivity of the method used, whereas neither microscopy nor rapid diagnostic tests allow a satisfactory evaluation of mixed plasmodial infections.The two main plasmodial species in French Guiana are In the present study, the frequency of mixed infections in different part of French Guiana was determined using real time PCR, a sensitive and specific technique.From 400 cases of malaria initially diagnosed by microscopy, real time PCR showed that 10.75 % of the cases were mixed infections. Their prevalence varied considerably between geographical areas. The presence, in equivalent proportions, of the two plasmodial species in eastern French Guiana was associated with a much higher prevalence of mixed plasmodial infections than in western French Guiana, where the majority of the population was Duffy negative and thus resistant to vivax malaria.P. falciparum/P. vivax areas, in order to deliver optimal care for patients suffering from malaria. This may involve the use of rapid diagnostic tests capable of detecting mixed infections or low density single infections. This is important as French Guiana moves towards malaria elimination.Clinicians must be more vigilant regarding mixed infections in co-endemic  Plasmodium vivax and Plasmodium falciparum.Plasmodium malariae is much rarer, only representing 1 % of cases [In the early 2000s, French Guiana was one of the most malaria-affected territories among the South American regions . Betweenof cases , 4. For of cases , but it of cases . In thesof cases .P. falciparum was more frequent in western French Guiana, where the Maroon populations live. This population is resistant to P. vivax because, as most Africans, they do not express the Duffy antigen. Eastern French Guiana was mostly populated by Amerindians and the incidence of P. vivax is similar to that of P. falciparum [P. vivax malaria in eastern French Guiana and a decrease of P. falciparum malaria in western French Guiana, notably along the Maroni River [P. falciparum in French Guiana has thus decreased from 46 % of all malaria cases in 2005 to 30 % in 2014 [The presence of lciparum . During ni River . Overall in 2014 .P. falciparum, artemether and lumefantrine (Riamet®) is employed since 2007. Thus, the identification of the infecting plasmodial species is essential to select the appropriate treatment.The specific treatment used in French Guiana depends on the infecting species, the severity of disease and the patient’s condition. Chloroquine is administered in uncomplicated vivax malaria, in association with primaquine. For ® Pf/Pan), cannot distinguish between single P. falciparum infections and a mixed P. falciparum/P. vivax infections. This may lead to inadequate treatment, since misdiagnosis of a P.falciparum/P. vivax mixed infection as a P. falciparum infection may lead to failure to administer primaquine and hence will lead to vivax relapses, while misdiagnosis of P. falciparum/P. vivax as P. vivax infection may lead to the use of chloroquine for resistant, potentially severe, P. falciparum infections. Therefore, it is important for clinicians to have local data on the frequency of mixed infections. This problem concerns areas with resistant P. falciparum and/or P. vivax with frequent relapses, which was the case in French Guiana [Although errors in species identification are rare, it is common to miss mixed species infections by the microscopic examination of blood smears, particularly when one of the species is predominant in the patient’s blood, which is a frequent situation , 11. In h Guiana , 13. It h Guiana –19.P.falciparum/P. vivax infections in eastern and western French Guiana were thus determined using real-time PCR and compared to microscopic results.In the present study, the frequency of mixed Between 2000 and 2008, malaria diagnoses in the health centres and at the Hospital were performed using thin and thick blood smears, and were then confirmed by an experienced microscopist at the Department of Parasitology and Mycology at the Hospital. Given the low prevalence of mixed infections using microscopy, PCR diagnosis was implemented in order to estimate the prevalence of mixed infections.®) or in EDTA vacutainers, through the Cayenne Hospital or the National Reference Centre for Malaria, respectively. When filter paper was available and when the physician on site was informed  the filter paper was sent to the Hospital where PCR was performed. Thus, whether a patient eventually got PCR, it was not linked to the particulars of the patient or the malaria episode leading to the consultation, but to the health care professional rotation. Thus, this was not likely to be a recruitment bias.The study included samples from patients with clinical malaria having consulted remote health centres or Cayenne Hospital between 2000 and 2008. Samples were collected for diagnostic purposes on filter paper (WhatmanAmong the 400 samples collected (representing 10 % of French Guiana cases), 331 came from health centres located in the eastern part of French Guiana: Cacao, Saint-Georges-de-l’Oyapock, Régina, Camopi, Trois-Sauts, and Cayenne Hospital , as recommended by the manufacturer.The DNA from the samples conserved on filter paper or at −20 °C were extracted using the DNeasyP. falciparum primer sense sequence was: Pf1 5′-ATTGCTTTTGAGAGGTTTTGTTACTTT-3′, that of the antisense primer was: Pf-2 5′-GCTGTAGTATTCAAACACAATGAACTCAA-3′ and that of the probe was: Pf-probe FAM-CATAACAGACGGGTAGTCAT-MGB. The size of the amplicon was 95 pb. The sequence of the P. vivax sense primer was: Pv-1 5′-CGCTTCTAGCTTAATCCACA TAACTG-3′, that of the antisense primer was: Pv-2 5′-AATTTACTCAAAGTAA CAAGGACTTCCAAG-3′ and that of the probe was: Pv-probe VIC-CGCATTTT GCTATTATGT-MGB. The amplicon size was 142 pb. The amplification and the detection of DNA were performed in duplex, using the PCR Applied Biosystem 7300 analytic system. Real-time PCR was performed in a final volume of 25 µl, in presence of an internal positive control (IPC) . Samples were analysed in two tubes, the first one contained P. falciparum and P. vivax primers and probes and the second one the internal control of the absence of inhibitor, the IPC. Each reaction mix contained 12.5 µl of Master Mix Gene Expression (Applied Biosystem), 300 nM of each primer , 150 nM of each probe  and 5 µl of matrix DNA. The IPC was used as recommended by the manufacturer. PCR starts with a 10-min phase at 95 °C, followed by 50 cycles of 15 s at 95 °C and 1 min at 60 °C.DNA was amplified using real time PCR as described by Veron et al. . The amp2 test.Proportions of mixed infections were compared between regions using the χThe retrospective use of anonymous patient files on the site of patient care is authorized by the French National Commission on Informatics and Liberties (CNIL). All the human blood samples and the data collected retrospectively were anonymized in a standardized case report form and in database.P. falciparum and 140 P. vivax infections in the eastern French Guiana and 62 P. falciparum and 6 P. vivax infections in western French Guiana. Forty-three samples, within the 400 collected samples, corresponded to mixed P. falciparum/P. vivax infections, which represented a frequency of 10.75 %, versus 2 % in microscopy. Forty-two originated from Eastern French Guiana and only one came from the western part. The latter was an isolate from Maripasoula, a municipality which marks the end of the Maroon territory and the beginning of Amerindian territory. Thus, in eastern French Guiana the proportion of mixed infections was 12.7 % whereas in western French Guiana the prevalence was only 1.4 %. This difference was statistically significant (P < 0.0001). The municipalities of Saint-Georges-de-l’Oyapock (n = 11), Camopi (n = 9), Trois-Sauts (n = 10), and Cayenne (n = 10) were the sites with the highest number of mixed infections observed  will kill P. vivax, treatment of latent hypnozoites with primaquine will be omitted thus leading to the risk of P. vivax relapses, notably as P. falciparum malaria re-activates latent hypnozoites [Mixed infections have been associated with less severe malaria by some , 30 and e region . Conversnozoites , 34, 35.nozoites . HoweverPlasmodium species at low parasite densities.PCR is much more sensitive than microscopy and rapid diagnostic tests, notably to detect mixed infections –19. HoweP.vivax/P. falciparum infections is of clinical importance because interactions between the different species simultaneously infecting the same patient could result in significant changes in the course of the infection and disease, and thus affect therapeutic strategies. The development of rapid diagnostic tests for the detection of mixed infections or the systematic and simultaneous use of two rapid diagnostic tests allowing: (1) the detection of low densities of P. vivax, and, (2) the detection of low densities of P. falciparum, to improve mixed infections diagnosis and treatment, and further decrease malaria as French Guiana moves towards malaria elimination.Microscopy often fails to reveal mixed infections or low density single infections. In French Guiana, there is a singular situation with inhabitants of different ethnic origins and therefore malaria susceptibilities, and with differences in local epidemiology, which should be known by clinicians who in routine care do not have access to real-time PCR and thus have a non-negligible risk of overlooking mixed infections. The detection or the anticipation of mixed