Plasmodium falciparum malaria that acts by inhibiting the phosphorylation of erythrocyte membrane band 3 by an erythrocyte tyrosine kinase. Because tyrosine phosphorylation of band 3 causes a destabilization of the erythrocyte membrane required for parasite egress, inhibition of the erythrocyte tyrosine kinase leads to parasite entrapment and termination of the infection. Moreover, because one of the kinase inhibitors to demonstrate antimalarial activity is imatinib, i.e. an FDA-approved drug authorized for use in children, translation of the therapy into the clinic will be facilitated. At a time when drug resistant strains of P. falciparum are emerging, a strategy that targets a host enzyme that cannot be mutated by the parasite should constitute a therapeutic mechanism that will retard evolution of resistance.With half of the world’s population at risk for malaria infection and with drug resistance on the rise, the search for mutation-resistant therapies has intensified. We report here a therapy for  The inaugural meeting of the World Health Organization in 1948 designated malaria as one of six consensus global health priorities . The absPlasmodium cause malaria, but the most lethal of the species is Plasmodium falciparum. During the intra-erythrocytic phase of its life-cycle, the parasite matures through ring, trophozoite, and schizont stages that are characterized by increasing tyrosine phosphorylation of the erythrocyte membrane protein, band 3 [P. falciparum promotes tyrosine phosphorylation of band 3 in order to facilitate its egress from the erythrocyte, thereby enabling its propagation. In exploring this hypothesis, we learned that others have discovered that the P. falciparum genome surprisingly encodes no classical tyrosine kinase [P. falciparum kinase with the ability to phosphorylate tyrosines [P. falciparum life cycle by preventing its egress from the red cell. To test this hypothesis, we screened inhibitors of the five known erythrocyte tyrosine kinases  for theP. falciparum infection of human erythrocytes (RBCs) promotes a gradual but significant increase in band 3 tyrosine phosphorylation [The major erythrocyte membrane protein, band 3  forms the predominant bridge connecting the red cell membrane to its spectrin/actin cytoskeleton via an association with ankyrin –24. In prylation  despite rylation , 14. Becrylation , this obP. falciparum, we examined the abilities of inhibitors of the five known erythrocyte tyrosine kinases to prevent parasite propagation in vitro. As shown in P. falciparum reinvasion/proliferation in fresh human RBCs. In contrast, imatinib, PRT062607, gefitinib, R406, bafetinib, nilotinib, and PP-121 all showed measurable antimalarial activity, with the latter three displaying higher potencies than the former four . As shown in To obtain an accurate assessment of the anti-malaria potency of imatinib in vitro, synchronized We next determined whether imatinib might cause elimination of the parasitemia by inhibiting the parasite-induced tyrosine phosphorylation of band 3, as hypothesized in the proposed mechanism. As shown in P. falciparum cultures. As shown in the light micrographs of The proposed mechanism of imatinib action further posits that inhibition of band 3 tyrosine phosphorylation should prevent the membrane destabilization required for merozoite egress at the end of the parasite’s life cycle. To test this step in the mechanism, we next monitored both the development and eventual escape of parasites from untreated and imatinib-treated synchronized When the concentration of imatinib was increased to 10 μM, the trend of parasite entrapment followed by disintegration was still observed, however, additional indications of growth arrest and interruption of parasite maturation preceded parasite entrapment, suggesting that this higher dose was more generally toxic. Indeed, all of the parasitized cells appeared pyknotic by the 96 h time point, and few if any infected cells displayed signs of normal maturation at any time point. These data suggest that lower doses of imatinib may terminate parasitemia by prevention of parasite egress, whereas higher doses likely induce parasite death by additional unrelated mechanisms. A quantitative analysis of the fraction of infected RBCs at each stage of parasite development following treatment with either 0 or 5 μM imatinib is shown in Patients with symptomatic malaria experience severe fever roughly every 48 h, demonstrating that the parasite’s life cycle is moderately synchronized in vivo . Not surP. falciparum malaria infected blood samples to imatinib were performed in Mbale, Uganda. As seen in 50 of 1.37 μM . Surprisingly, these data suggest that imatinib has greater anti-malarial activity against random field isolates of P. falciparum than laboratory-adapted strains of the same parasite  in cancer patients was required. Le Coutre et al.  report t μM Figs  and 5, osee also ), suggesnd century BCE, all malaria therapies have targeted malaria-encoded processes that are critical to parasite survival. Artemisinin, originally obtained by chewing the plant, Artemisia annua, is thought to function by promoting oxidative stress and inhibiting hemozoin formation within the infected red cell [Plasmodium food vacuole and inhibits the formation of hemozoin among other mechanisms [P. falciparum cation ATPase (PfATP4), P. falciparum protein kinase G (PfPKG), and P. falciparum phosphatidylinositol-3 kinase (PfPI3K) in the parasite [Since the 2red cell . Quininechanisms , 44. Synchanisms , and sevparasite , 46–49. parasite . These oparasite .In this article, we describe a novel antimalarial agent that blocks parasitemia by inhibiting a process encoded in the host cell’s genome. As we have shown elsewhere, phosphorylation of band 3 on tyrosines 8 and 21 is catalyzed by syk tyrosine kinase , leadingP. falciparum encoded dual-specificity kinase might be capable of catalyzing the tyrosine phosphorylation of band 3, or that imatinib might inhibit this kinase or some other parasite protein and thereby block parasitemia, the likelihood that such an unknown target constitutes the site of action of imatinib seems remote, especially since the crystal structure of imatinib bound to syk shows a very high degree of structural complementarity and the remarkable specificity of imatinib for a very limited number of tyrosine kinases constitutes its source of notoriety [To inhibit this process, we treated the malaria infected cultures with imatinib, a tyrosine kinase inhibitor that has known activity against syk . The resotoriety , 16.It is important to emphasize that the malaria parasite engages actively in erythrocyte membrane remodeling from the moment that it enters the red cell. Thus, novel malaria-encoded proteins are repeatedly inserted into the host cytoskeleton and plasma membrane , 55, whiP. falciparum, both in red cells and in hepatocytes in vitro. Millholland and coworkers [P. falciparum co-opts an erythrocyte Gs protein to activate signaling pathways required for intra-erythrocytic parasite growth and invasion. Finally, Brizuela et al. [It is important to note that previous work from prominent labs has provided additional motivation for seeking a malaria therapy that targets an erythrocyte-mediated process. Thus, Doerig and colleagues  have shooworkers  have simoworkers , 62. In oworkers  have dema et al.  have repIn conclusion, we suggest that imatinib constitutes an excellent candidate for clinical evaluation as an anti-malaria therapy for several reasons: i) it is FDA approved for use in both adults and children, ii) it is generally non-toxic and can be taken daily in perpetuity, iii) well tolerated concentrations demonstrating complete antimalarial activity can be achieved in patient’s plasma, iv) imatinib has been well studied in nearly all human populations, and iv) imatinib’s mechanism of action renders development of resistance mutations unlikely. With the focus of the malaria field on the design of mutation-resistant drug cocktails that might collectively enable eradication of parasitemia with a single pill, the contribution of a drug like imatinib could prove valuable.Plasmodium falciparum strains used in these studies, Dd2 (MRA-156) and FUP Palo Alto (MRA -915), were obtained from Malaria Research and Reference Reagent Resource Center (MR4). The protein tyrosine kinase inhibitors bafetinib, PP121, R406, PRT062607, dasatinib, and nilotinib were purchased from Selleck Chemicals. Imatinib mesylate and N-desmethyl imatinib were from AlsaChim.The P. falciparum strains Palo Alto and Dd2 were then cultured at 1–5% hematocrit, as previously described but with minor modifications [2, 5% CO2, and 94% N2 atmosphere in complete media (wash media supplemented with 0.5% Albumax II (Gibco)). Parasites were synchronized by isolating late stage parasites on a Percoll density gradient (Sigma), washing 3x to remove the Percoll, and adding the schizont stage parasitized cells to fresh RBC cultures. After 4 h incubation to allow for re-invasion of fresh RBCs, cultures were treated with aqueous 5% sorbitol for 5 min at room temperature to lyse any residual late stage parasitized cells, preserving primarily ring stage parasites and uninfected RBCs [Fresh blood was collected from healthy adult volunteers into acid-citrate-dextrose (ACD) glass vacutainer tubes (BD Biosciences) and prepared as previously described . Writtenications . Parasitted RBCs , 67. AftP. falciparum Palo Alto or Dd2 strain infected erythrocytes were treated with the above tyrosine kinase inhibitors at 2% hematocrit and 0.5–2% parasitemia. All inhibitors were solubilized in anhydrous DMSO at a 10mM stock concentration and serially diluted in anhydrous DMSO prior to addition to malaria cultures. Untreated cultures were run in parallel with the same final concentration of DMSO as the drug treated cultures. Parasitemia was measured by flow cytometry at the desired time points and thin smears were prepared for evaluation of parasite integrity and morphology by light microscopy (see below).Synchronous ring stage Malaria infected erythrocyte cultures were stained with SYBR Green I DNA stain ; excitation wavelength, 488nm) to quantitate residual percent parasitemia using a Becton Dickinson FACSCalibur flow cytometer according to well defined procedures , 69. BriP. falciparum-infected cells at 20% parasitemia and the desired stage of parasite maturation were incubated for 24h in the presence of different concentrations of imatinib  and then lysed at 0°C in hemolysis buffer  containing protease and phosphatase inhibitor cocktails . Erythrocyte membranes were prepared according to standard procedures [ocedures  and storP. falciparum-infected patients at the Mbale Regional Referral Hospital, Mbale, Uganda, following approval from the hospital’s IRB and from the Uganda National Council for Science and Technology. Research was conducted according to the principles expressed in the Declaration of Helsinki. Infected erythrocytes were isolated by three washings in wash medium and resuspended at 2% hct in wash media supplemented with 10% heat-inactivated human serum. Infected cultures were added to culture plates pre-dosed with DMSO, dihydroartemisinin, or imatinib concentrations in triplicate and maintained under a reduced oxygen atmosphere at 37°C. After a 50 h incubation, 10 μl of the infected blood cell suspension were spotted onto filter paper in triplicate and dried at room temperature for 20 minutes or stored at -20°C until use. Malaria parasite DNA was extracted according to standard procedures [P. falciparum as previously described with modifications [™ Real-Time PCR Detection System (Biorad) using the following program: 95°C for 3 min, followed by 45 cycles at 95°C for 15 min and 60°C for 45 min. Samples were run in duplicate. Percent parasitemia was estimated using a standard curve obtained from serial dilutions of P. falciparum DNA in cultures with known parasitemia using CFX Manager™ Software 3.1 (Biorad). The 50% inhibitory concentrations (IC50) were calculated by nonlinear regression analysis using ICEstimator software 1.2 [Blood samples were acquired with written informed consent from ocedures  and DNA ications . Brieflyware 1.2  and by cS1 FigSynchronous P. falciparum strain Palo Alto cultures were treated during ring stage (0.5% parasitemia) with increasing concentrations of bafetinib, PP-121, imatinib, PRT062607. After 60 h incubation, % parasitemia of infected cultures was determined by flow cytometry. Results were obtained with each concentration examined in triplicate. Error bars are within the size of the point marker.(TIF)Click here for additional data file.S2 FigSynchronous P. falciparum strain Palo Alto cultures were treated during ring stage (0.5% parasitemia) with increasing concentrations of bafetinib, dasatinib, imatinib, nilotinib, PP-121, PRT062607. Infected cultures were analyzed every 24 hours to determine % parasitemia by flow cytometry. Results were obtained with each concentration examined in triplicate.(TIF)Click here for additional data file.S3 FigSynchronous P. falciparum strain Dd2 cultures were treated during the late ring stage (2% parasitemia) with increasing concentrations of bafetinib, dasatinib, R406, gefitinib, imatinib, nilotinib, PP-121, PRT062607. Infected cultures were analyzed every 11–24 hours to determine % parasitemia by flow cytometry. Results were obtained with each concentration examined in triplicate.(TIF)Click here for additional data file.S4 FigP. falciparum (Dd2 strain) cultures at different stages of their life cycle, ring stage ~7hpi, late ring stage ~14hpi, trophozoite stage ~21 hpi, late trophozoite stage ~28hpi, and schizont stage ~36hpi. Parasitemia was measured by flow cytometry every 11 hours after treatment until the untreated parasites had progressed 24 h into their second infective cycle. Results were obtained with each concentration examined in triplicate.Imatinib (8 μM) was added to synchronized (TIF)Click here for additional data file.S5 FigSynchronized ring stage (12hpi) P. falciparum  cultures at 0.75% parasitemia were treated with the indicated concentrations N-desmethyl imatinib. After 60 h incubation, % parasitemia of infected cultures was determined by flow cytometry. Results were obtained with each concentration examined in triplicate.(TIF)Click here for additional data file.S1 TableConditions are as shown in (TIF)Click here for additional data file.


P. falciparum, P. malaria, P.vivax, P. ovale. Southeastern corner of Iran, including Sistan and Baluchestan, Hormozgan and the tropical part of Kerman Province, are endemic region of malaria. This study aimed to find out clinical findings in malaria caused by various plasmodium species in moderate transmission area of southern Kerman Province.Malaria is a big problem of public health in many tropical countries where socioeconomic development is deficient. Four species of plasmodium are capable of infecting human: This study was conducted in health centers of Kahnooj, Manujan, Ghale-Ganj, Roudbar and Fariab in south of Kerman Province, Southeastern Iran during 2005–2009. Three hundred and thirty patients with positive malaria parasite slides entered the study. Frequencies of several malaria clinical presentations were investigated in four plasmodium species.P. falciparum, 314 with plasmodium vivax and 6 with mixed infection. The symptoms of fever, chills and sweating were present in 74.6% of subjects. Other complaints were joint pain, headache, fatigue, vomiting, and diarrhea. Splenomegaly was detected in 17.57% of the individuals.54.2% of considered patients were male. Mean age of patients was 22.8±17.8 yr. Younger and older patient were 6 months and 80 yr, respectively. Ten patients were infected with Malaria should be considered in differential diagnosis of all acutely febrile patients in endemic area. Classic symptoms of fever, chills and sweating may not present in all of patients. P. falciparum, P. vivax, P. malariae, P. ovale).Malaria is the most important parasitic infection in the world that poses major health challenges. Despite years of continual efforts, this infection is still a threat to approximately 40% of the world’s population in about 100 countries  and 151(45%) were male and female, respectively. Mean age of patients was 22.8±17.8 yr old. Younger and older patient were 6 months and 80 yr, respectively. Ten patients were infected with lciparum . ClassicP. falciparum symptoms  had uncomplicated malaria presented by fever, chills, headache, sweating, myalgia and joint pain such one other study that designed in Colombia  Pakistansymptoms , 17, 18.h others . Vomitinfalciparum and vivax but not in mixed infection in this study similar to those results found others  have been completely observed by the authors.


At the time of malaria infection, 12/24 SN children and 15/23 SP children (P = 0.29) stimulated with malaria antigens demonstrated memory recall as defined by CD4-derived cytokine production. This compares to 7/23 SN children and 16/23 SP children (P = 0.009) at the convalescent timepoint. 46.2% of cytokine-producing CD4+ T cells expressed a single cytokine after stimulation with malaria antigen during the malaria episode. This fell to 40.9% at follow-up with a compensatory rise of multifunctional cytokine secretion over time, a phenomenon consistent with memory maturation. The majority (53.2–59.5%) of responses derived from CD45RA−CD62L− effector memory T cells with little variation in the phenotype depending upon the time point or the study cohort. We conclude that detectable T cell memory responses can be measured against both malaria and schistosoma antigens and that the presence of Schistosoma haematobium may be associated with long-term maintenance of T memory to malaria.Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4–8 years, are protected from malaria compared to matched schistosomiasis-negative (SN) children. The effect of concomitant schistosomiasis upon acquisition of T cell memory is unknown. We examined antigen-specific T cell frequencies in 48 Malian children aged 4–14 to a pool of malaria blood stage antigens, and a pool of schistosomal antigens, at a time point during a malaria episode and at a convalescent time point ~6 months later, following cessation of malaria transmission. CD4 Plasmodium falciparum and Schistosoma spp. are co-endemic parasitic diseases with worldwide distribution. Revised estimates suggest that falciparum malaria causes 212 million infections annually, while schistosomiasis affects an estimated 207 million with 92% residing in Africa  and merozoite surface protein 1 (MSP1)] ] , alteredlevation , and suplevation , comparein vivo (H2 cytokine production pattern) may alter responses to a secondary stimuli that depend upon TH1 cytokine production, such as malaria antigens , were age, gender, and residence-matched to a child without schistosomiasis [schistosomiasis-negative (SN)] prior to malaria transmission. Children were followed weekly over the malaria transmission season (25 weeks) and at a dry season follow-up appointment (~6–9 months after enrollment at a time when standing water pools had dried and schistosoma transmission had ceased). The primary endpoint of the clinical trial was time to first clinical malaria infection. Clinic personnel were available 24 h-a-day throughout study duration to detect, examine, and treat symptomatic malaria episodes. A clinical episode of malaria was defined as P. falciparum parasitemia and axillary temperature ≥37.5°C on active surveillance, or parasitemia and symptoms leading to treatment-seeking behavior in the absence of other clear cause on passive surveillance. All children were pre-treated with albendazole to eliminate concomitant helminth infections and study samples were drawn at the time of their first clinical malaria episode  and again at the final dry season appointment. Children were optimally treated for schistosomiasis with praziquantel at the final appointment.Bandiagara pop. ~13,600) is located in Mali, West Africa, and has intense seasonal transmission (July–December) of r season . S. haemthe area , 27, wit,600 is ln adults . BrieflyThis trial was carried out in accordance with the recommendations of the University of Bamako and the University of Maryland Institutional Review Boards (IRBs) with written consent obtained from all legal guardians. Village permission to conduct research was obtained from village chiefs, government officials and traditional healers prior to study initiation. Individual written informed consent was obtained from the parent or legal guardian of each child prior to screening and enrollment in accordance with the Declaration of Helsinki. All children displaying gross hematuria or symptoms of genitourinary pathology were treated with praziquantel (40 mg/kg) therapy and discharged from the study.and the presence of at least 10 × 6 cells10 for each child at each time point. A secondary analysis from the same study, which was part of a larger, previously unpublished set of experiments, examined samples with only a single transmission season time point utilizing a second flow cytometry panel of antibodies.Patient whole blood (7–10 mL) was collected at the study clinic into sterile eppendorf and EDTA tubes on admission, prior to institution of anti-malarial therapy, and immediately refrigerated. Sera was processed as previously described . Blood w2 and washed. A portion of cells (2.0 × 106) was removed to serve as negative (media) and positive  controls. The remaining PBMC were split into two aliquots consisting of ~1.0 × 106 cells each and stimulated with a malaria antigen pool [consisting of AMA1 (42)] (S. haematobium antigen pools [soluble egg antigen and soluble worm antigen protein (SWAP)]. Antigen stimulation for the assay was optimized at 5 μg/mL/antigen. The malaria antigens chosen represent two vaccine candidates being tested at the same field site in Mali, while the S. haematobium antigens are commonly used in schistosoma research. The 3D7 stain of malaria is well documented at the Malian site and strain-specific protection against an AMA1 vaccine has been established  from an SP child and an age-matched SN child and was performed by an investigator blinded to the age and schistosoma status of the samples. Thawed PBMC were rested overnight at 37°C, 5% CO of AMA1  and MeroA1 (42)] , and S. ablished . AMA1 haablished . Using aablished , all celPeripheral blood mononuclear cells were stained with fluorochrome-labeled monoclonal mouse anti-human antibodies (mAb) against surface antigens , CD4-Pacific Orange , CD8-FITC , CD19 /CD14 Invitrogen, clone TüK4)/Vivid-Pacific Blue, CD45RA Quantum Dots (Qdot) 655 (clone 5H3), and CD62L APC-EF780 , followed by fixation/permeabilization by using cytofix/cytoperm solution (eBiosciences) and intracellular staining with mAb to IFN-γ-APC , IL-2-phycoerythrin (PE)-Cy7 , TNF Alexa Flour 700 , IL-17A PerCP-Cy5.5 , and CD69-PE .A subset of unpaired PBMC (from the transmission season) were stained with CD3-ECD, CD4-APC-Cy7 , CD8-PE-Cy7, CD19/CD14-PacBlue, biotinylated CD45RA  PacOrange, CD62L PE-Cy5 , IFN-γ-PE , IL-4 Alexa488 , and IL-10 APC  mAb as described above.+) and macrophages/monocytes (CD14+) were excluded from analysis. Doublets/aggregates were subtracted from analysis and gate placement determined with the aid of Fluorescence Minus One controls. Specimens were included in the analysis if (1) the cell viability was >80% after thawing and (2) cells were shown to be functionally active as determined by the production of IFN-γ by at least 0.2% CD3+ cells after stimulation with SEB. A response was considered specific if (1) the differential in the number of positive events in the stimulant pool compared to the media control was significantly increased by χ2 analyses; and (2) the net percentage of cytokine-producing cells was ≥0.1% in stimulant pool as compared to the media control. A response was considered positive if the production of one or more cytokines, meeting the pre-defined criteria, was measured in response to antigen stimulation of PBMC.Cells were resuspended in 1% formaldehyde fixation buffer and analyzed using a BD LSR II SORP 4-laser flow cytometer. PBMC from healthy subjects were used as internal controls in the experiments. A total of 244,000–1,000,000 events  in the forward and side scatter (FS/SS) lymphocyte gate were collected per sample. List-mode data files were analyzed using WinList 7.1 3D . An amine reactive dye  was used as a dead cell discriminator and B lymphocytes (CD19t-test (two-tailed) or Mann–Whitney U test were used to compare continuous and/or nonparametric data and χ2 analysis, using Mantel–Haenszel or Fisher Exact (two-tailed) as appropriate, was performed for categorical data. Cellular multifunctionality was assessed using the WinList 3D 7.1 FCOM to enumerate phenotypes. Spearman rank correlation coefficient was calculated utilizing GraphPad Prism 5. A significance level of P < 0.05 was considered statistically significant.Statistical analysis was performed on GraphPad Prism 5 , and demographic and immunologic data were stratified and evaluated by age group (age 4–8 and 9–14 years). Student 10 × 6 PBMC/time point10  were thawed and examined. Evaluative data was available from 24 SP children (mean egg count 58 eggs/10 mL urine) and 24 SN children, all but two of who developed 1–4 malaria episodes during the transmission season (Table n = 3 time points). SP children had a statistically longer time to first clinical malaria infection and a trend toward a reduced geometric mean parasite density at the time of malaria infection but experienced similar numbers of malaria episodes over the course of a single transmission season  in these age-matched populations . The majority of cytokine-producing cells were found to derive from CD3+CD19−CD4+CD8− T cells with a minority (<0.03%) observed from CD8+ T cells. The amount of cytokine measured after malaria antigen stimulation was low, but significant, with a geometric mean value of less than 0.2%. More SP children [15/23 (65%)] had detectable cytokine expression (one or more cytokines) than SN children , during the malaria transmission season at the time of their malaria episode . When paired dry season samples were analyzed, fewer SN children [7/23 (30.4%)] had a recall response to malaria antigen (defined as one or more cytokine response) as compared to SP children, who not only retained the low-level memory response to malaria antigen but had a higher number of responders  Individual cytokines results demonstrate statistically significant results for IFN-γ, TNF-α, and IL-2  and dry season) were examined e Figure , 15, we P = < 0.0001, 95% CI (4.69–271), χ2 analysis]. As noted with the malaria antigen, the net percentage of cytokine expression, in those that had a demonstrable cytokine response, was not significantly different between groups , χ2 analysis]. Of the SN children with detectable response to Sh antigen, none had evidence of urinary egg secretion at follow-up. One SN child demonstrated a particularly robust response to antigen at both the wet and dry season time point, without detectable egg secretion, suggesting that this individual may have been previously exposed or developed inherent or putative resistance to urinary schistosomiasis (Paired PBMC were stimulated with Sh antigen as described above. Significant production of at least one cytokine was measured after Sh antigen stimulation in the majority of SP children [17/23 (74%)] compared to 2/24 (8.3%) of SN children  were IFN-γ+TNFα+IL2+-expressing at the time of an acute malaria episode. Over time an incremental but persistent increase in multifunctional cell cytokine production was noted across all cytokine combinations , 42.1% double-positive (P = 0.27), 15.5% triple-positive (P = 0.38) and 0.8% (P = 0.58) quadruple positive. Raw data can be found in Figure Among the multifunctional CD4/11.9 88.%] were IEM. TCM accounted for 14–22% of cytokine secretion with slightly more TCM noted in the small subset of SN compared to SP population . A minority of cytokine was detected from cells of TEMRA (5–11%) population and naïve T cells (2–4%).There was little variation in memory subpopulation in which cytokine production was observed, between seasons or study cohorts. SP and SN results were combined for analysis and reported as geometric mean values Figure . The majn = 63, mean age 8.3 years, range 4–13 years), which were thawed and examined as part of a previously unpublished data set. Evaluative data were available from 30 SP children and 30 SN children who developed between 1 and 5 malaria episodes during a single transmission season . Among PBMC stimulated with malaria antigen AMA1, 4/30 (13.3%) SN vs. 10/30 (33%) SP expressed IFN-γ ; 1/30 (3.3%) SN vs. 12/30 (40%) SP expressed IL-4 ; and 1/30 (3.3%) SN vs. 7/30 (23.3%) SP expressed IL-10   and CD62L+ T central memory (TCM), relative to the cytokine expression profile. Of those children that had an inflammatory expression profile characterized by IFN-γ expression, the percentage of TEM predominated as compared to those children with a dominant IL-4 expression profile . Additionally, those children with an IL-4 expression profile (predominantly SP children) had a higher percentage of TCM as compared to those children with a dominant IFN-γ expression   populations. However, when SP children were stratified between those expressing an IL-4 profile (n = 9) and those with an IFN-γ profile (n = 4), children expressing IFN-γ were significantly older .A secondary experiment utilized unmatched transmission season samples from Malian children from this same study . SP children also had similar memory responses to Sh antigens at both time points. The quality of these cellular responses, as measured by cellular multifunctionality, increased over the 6- to 9-month time interval with elevated dual, triple, and quadruple cytokine-secreting cells to malaria and Sh antigens at the convalescent time point.Very little is known about the immunologic perturbations that one parasitic infection inflicts upon the immune response to another infection when they co-occur in the human host. We have demonstrated an age-specific reduction in n season . In this+ T cells is well established in malaria. However, the presence of CD4+ T cell memory response in chronically infected individuals with schistosomiasis and the kinetics of these cells is poorly understood. While CD4+ T cell memory cells are thought to provide protection against re-infection by schistosomes, the prevailing belief is that chronic helminth infection leads to suppression of the CD4 T cell memory compartment. Moreover, as we have demonstrated, helminth infections can modulate the host adaptive response against itself and other “bystander” antigens  had significantly elevated CD4+ TCM populations. Helminth-infected children comprised the majority of those with a dominant IL-4 expression profile, with only one uninfected exception; however, a subset of SP children demonstrated an inflammatory-dominant profile. The subset of SP children with an inflammatory-dominant profile were older than those with an IL-4 dominant profile . This fact would argue against the inflammatory-dominant profile in SP children being due to an acute Sh infection as Malian children would have been exposed at a much younger age. This is also consistent with the finding that IFN-γ production in schistomiasis-infected individuals is associated with acquisition of anti-helminth immunity  with written consent obtained from all legal guardians. Village permission to conduct research was obtained from village chiefs, government officials, and traditional healers prior to study initiation. Individual written informed consent was obtained from the parent or legal guardian of each child prior to screening and enrollment in accordance with the Declaration of Helsinki. All children displaying gross hematuria or symptoms of genitourinary pathology were treated with praziquantel (40 mg/kg) therapy and discharged from the study.KL obtained funding; KL, OD, CP, and MS participated in study design; KL, AD, CA, ID, CP, and OD participated in study conduct and regulatory review. KL and MS participated in immunologic assays and interpretation; KL, CP, and MS participated in writing of manuscript; all authors participated in editing of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium vivax now accounts for 84% of cases countrywide. Targeting residual malaria transmission entrenched in the Amazon is the next major challenge for ongoing elimination efforts. Better strategies are urgently needed to address the vast reservoir of asymptomatic P. vivax carriers in this and other areas approaching malaria elimination.Malaria burden in Brazil has reached its lowest levels in 35 years and P. vivax transmission in farming settlements in the Amazon Basin of Brazil. Over six months, 41 cases detected by passive surveillance triggered four rounds of RCD , using microscopy- and quantitative real-time polymerase chain reaction (qPCR)-based diagnosis, comprising subjects sharing the household (HH) with the index case (n = 163), those living in the 5 nearest HHs within 3 km (n = 878), and individuals from 5 randomly chosen control HHs located > 5 km away from index cases (n = 841). Correlates of infection were identified with mixed-effects logistic regression models. Molecular genotyping was used to infer local parasite transmission networks.We evaluated a reactive case detection (RCD) strategy tailored for P. vivax biomass in study subjects. Clustering patterns were temporally stable. Four rounds of microscopy-based RCD would identify only 49.5% of the infections diagnosed by qPCR, but 76.8% of the total parasite biomass circulating in the proximity of index HHs. However, control HHs accounted for 27.6% of qPCR-positive samples, 92.6% of them from asymptomatic carriers beyond the reach of RCD. Molecular genotyping revealed high P. vivax diversity, consistent with complex transmission networks and multiple sources of infection within clusters, potentially complicating malaria elimination efforts.Subjects in index and neighbor HHs were significantly more likely to be parasitemic than control HH members, after adjusting for potential confounders, and together harbored > 90% of the  Plasmodium vivax carriers clustered in hard-to-reach rural communities is a major challenge faced by countries approaching malaria elimination across Latin America and Asia. Routine surveillance targets subjects presenting with fever or reporting recent fever, but overlooks asymptomatic infections that might be otherwise detected by periodic mass blood surveys of the entire population at risk. Here we show that subjects living in close proximity to malaria cases detected by routine passive surveillance are much more likely to carry both symptomatic and asymptomatic infections than randomly selected inhabitants in the same farming settlements in the Amazon Basin of Brazil. Four rounds of microscopy-based screening for malaria parasites targeted at these high-risk subjects would identify 49.5% of the parasite carriers  detected by a more sensitive molecular method. Whether subpatent and asymptomatic carriers outside the identified clusters of symptomatic infections, beyond the reach of our screening, represent a significant parasite reservoir remains undetermined. The extensive genetic diversity found in local P. vivax populations suggests that multiple sources of infection fuel ongoing residual transmission within malaria clusters, further complicating current elimination efforts.Addressing the vast reservoir of asymptomatic  Plasmodium vivax, the human malaria parasite with the widest global distribution [P. vivax typically causes less severe cases and deaths than P. falciparum, but its distinctive biological features pose major challenges for malaria elimination strategies focused on early diagnosis and prompt treatment of blood-stage infections. First, low-density P. vivax infections, which are often subpatent  and asymptomatic, predominate in areas approaching malaria elimination, rendering accurate clinical and laboratory diagnosis more difficult [P. vivax infections detected by polymerase chain reaction (PCR) in a rural Amazonian cohort were missed by microscopy; 57% of them caused no clinical signs or symptoms suggestive of malaria and 33% were both subpatent and asymptomatic [P. vivax remains silent within human hosts for several months as hypnozoites, the dormant liver stages that may eventually cause relapses [P. vivax transmission is favored by the earlier production within human hosts of blood stages that are infective to mosquito vectors (mature gametocytes) as well as by the faster development within vectors of stages that are infective to humans (sporozoites) [Over one-third of the world's population is currently at risk of infection with ribution . Where bifficult . Accordirelapses . Finallyhttp://portalsaude.saude.gov.br/index.php/o-ministerio/principal/leia-mais-o-ministerio/662-secretaria-svs/vigilancia-de-a-a-z/malaria/11346-situacao-epidemiologica-dados). Most transmission hotspots are located in remote sites across the Amazon Basin. This vast region, with 60% of the country's territory but only 13% of its total population, accounts for 99.5% of all malaria cases diagnosed in Brazil; 84% of them are due to P. vivax [Brazil is one of the 14 (out of 21) malaria-endemic countries in the region of the Americas that achieved a ≥ 75% reduction in their case incidence rates between 2000 and 2015, as called for by the United Nations' Millennium Development Goals . This coEliminating residual foci when malaria is steadily declining and reaching pre-elimination stages is the next major challenge in Brazil . RoutineP. vivax endemicity and substantial spatial clustering of malaria episodes [P. vivax; and (c) molecular genotyping of parasites may help to determine the epidemiological connections among cases found by RCD.Here, we evaluate a reactive case detection (RCD) strategy  that hasepisodes ,13. We s2 between the Abunã and Iquiri Rivers, in the Acre River Valley, with a population of 14,120 inhabitants. Nearly half of them live in the town of Acrelândia , located 35 km northeast of Plácido de Castro  and 112 km east of Rio Branco . With its equatorial humid climate, this area receives the most rainfall  between December and March. The mean annual temperature is 24.5°C. Subsistence agriculture and cattle raising are the main economic activities, with coffee, bananas, and rice being the predominant cash crops.Acrelândia is a municipality situated in the easternmost corner of Acre State, Amazon Basin of Brazil, bordering with the States of Amazonas and Rondônia . The munhttp://portalsaude.saude.gov.br/index.php/o-ministerio/principal/leia-mais-o-ministerio/662-secretaria-svs/vigilancia-de-a-a-z/malaria/11346-situacao-epidemiologica-dados). Although both P. falciparum and P. vivax were transmitted in the early 2000s, with the latter accounting for over 80% of all infections [P. vivax has been observed in slide-confirmed indigenous cases since 2010. Selective indoor spraying with residual insecticides, implemented in early 2008, and the widespread distribution of long-lasting insecticide-treated bed nets, since 2010, are likely to have contributed to the dramatic reduction of malaria transmission  in Acrelândia in recent years. The main local vector is the highly anthropophilic and exophilic Anopheles darlingi [An. benarrochi s.l., An. oswaldoi s.l., and An. konderi s.l. are also often found in larval habitats [Acrelândia has experienced a major decline in malaria incidence in recent years , but yeafections , only P.darlingi , but An.habitats .The study design is summarized in P. vivax infection diagnosed by routine surveillance during the enrollment period and were recruited in one of the two only government-run malaria clinics currently operating in Acrelândia ; (b) 163 index HH members—subjects who shared the HH with the index case; (c) 878 neighbor HH members—subjects living in the five nearest houses, within a radius of up to 3 km  away from the index HH (neighbor HH members); (d) 841 control HH members—subjects living in five randomly chosen houses located in the same or nearby locality but > 5 km  away from the index HH. The 5-km radius was chosen based on the maximal flight range reported for An. darlingi in the Amazon [The vast majority of the 1,923 study participants were enrolled between January and July 2013, except for those who were later recruited to replace individuals leaving the site before study completion (see below). Inclusion criteria were: (a) all subjects aged ≥ 3 months living in the selected HHs who provided written informed consent, or with parental written informed consent and assent when age-appropriate; and (b) subjects with plans to remain in the study site for the next six months. Exclusion criteria included mental disorders and any chronic or acute condition that, in the opinion of the field nurse, might affect the results of the study or the subjects' ability of understanding the study objectives and of providing informed consent. Participants were classified into the following categories: (a) 41 index cases—had a e Amazon  to ensurP. vivax up to 180 days after the primary infection [Home visits were made to eligible subjects on day 0  and 30 (±5), 60 (±5), and 180 (±10) days after the first visit, with a total of 6,172 observations. Visits were scheduled to maximize the probability of finding infections that had been acquired at the same time as the index case (day 0), those that were secondary to the index case or other infections diagnosed at baseline (day 30), new infections originating from these secondary infections (day 60), as well as most relapses, which are usually recorded in local strains of P. vivax parasitemia on 5,969 clinical samples. To this end, we isolated DNA templates using Qiagen  kits. We used QIAamp DNA blood kits for 200-μL venous blood samples and QIAmp DNA micro kits for 5-μL capillary blood samples eluted from three 3-mm bloodspots excised from FTA Micro Cards . Final DNA elution volumes were 20 μL for bloodspots and 200 μL for whole blood. Each 20 μL qPCR mixture contained 2 μL of template DNA (corresponding to approximately 2 μL of venous blood or 0.5 μL of finger-prick blood), 10 μL of 2× Maxima SYBR Green qPCR master mixture  and 0.5 μM of each of the primers P1 (ACG ATC AGA TAC CGT CGT AAT CTT) and V1 (CAA TCT AAG AAT AAA CTC CGA AGA GAA A) [P. vivax 18S rRNA gene. Standard curves were prepared with serial tenfold dilutions of the target sequence, cloned into pGEM-T Easy vectors , to allow for parasite load quantitation (number of parasites/μL of blood). We used a Step One Plus Real-Time PCR System  for PCR amplification with a template denaturation step at 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 1 minute at 60°C, with fluorescence acquisition at the end of each extension step. Amplification was followed by a melting program consisting of 15 sec at 95°C, 15 sec at 60°C, and a stepwise temperature increase of 0.2°C/sec until 95°C, with fluorescence acquisition at each temperature transition. The detection threshold of this diagnostic qPCR is approximately 3.3 parasites/μL of blood. No-template controls  were run for every qPCR microplate. To estimate the relative contribution of particular subgroups of infected individuals to the total P. vivax biomass found in the study population, we summed up all individual qPCR-derived P. vivax densities and calculated the fraction corresponding to each subgroup. Because parasite densities are expressed as parasites per blood volume, we assumed that average whole-body blood volumes (volemias) do not differ across subgroups.Laboratory diagnosis of malaria was based on microscopic examination of thick smears and qPCR. A total of 6,028 thick blood smears were stained with Giemsa and examined for malaria parasites under 1000 × magnification in our laboratory in the town of Acrelândia. At least 100 fields (corresponding to approximately 0.3 μL of blood) were examined before a slide was declared negative. We additionally used qPCR to detect and quantify A GAA A) , which amerozoite surface protein-1 gene (msp1F3 [Pv3.27 [MS16 [P. vivax infections diagnosed during the study (66 from index cases and 295 from other study participants) were tested; 343 (95.0%) of them were successfully genotyped at all 3 loci. The minimal detectable peak height was set to 200 arbitrary fluorescence units. Because stutter bands may be occasionally observed in microsatellite genotyping, we scored two alleles at a locus only when the minor peak was > 33% the height of the predominant peak. Only two samples, out of 343 that were fully genotyped, had minor alleles with peaks of > 200 fluorescence units that were discarded (one in each sample) because they failed to reach one-third of the height of the major peak in the same sample. Samples were considered to contain multiple clones if at least one locus showed more than one allele. Multilocus genotypes were defined as unique combinations of alleles at each locus analyzed. Although our descriptive analysis considered the most abundant allele (as judged by peak heights) at each locus for assigning predominant genotypes to multiple-clone infections, we also considered all possible genotypes in pairwise comparisons of samples from the same subjects (recurring infections) or within the same cluster (comprising the index and neighbor HHs). For this purpose, we listed all combinations of alleles, considering major and minor peaks, in each pair. Samples were considered to harbor identical parasites when they had exactly the same predominant (or only) genotype; they were considered to harbor similar parasites when the same alleles (considering major and minor peaks) were found at all loci, but their predominant genotypes were different.Parasite genotyping was carried out on microscopy- and qPCR-positive samples to classify infections as either (a) recrudescences, homologous relapses or persisting untreated infections , (b) related infections in different subjects  or (c) unrelated, sporadic infections or heterologous relapses (different genotypes), as described in  (msp1F3 ) and two [Pv3.27  and MS1627 [MS16 . PCR proP. vivax infection was defined as any episode of parasitemia detected by thick-smear microscopy, qPCR, or both. Subpatent infections were defined as infections confirmed by qPCR but missed by microscopy. We defined symptomatic malaria as a laboratory-confirmed infection, irrespective of the parasite density, diagnosed in a subject reporting one or more signs and symptoms investigated , up to seven days before the interview. Subjects with laboratory-confirmed infections, irrespective of the parasite density, but no malaria-related signs or symptoms up to seven days prior to the interview, were classified as asymptomatic parasite carriers. All study participants with vivax malaria infection confirmed by onsite microscopy, either symptomatic or not, received free-of-charge treatment with chloroquine  and primaquine (0.5 mg of base/kg/day for 7 days) following the latest antimalarial therapy guidelines of the Ministry of Health of Brazil [A laboratory-confirmed f Brazil . Accordi2 of Fisher exact tests tests to contingency tables. Statistical significance was defined at the 5% level (two-tailed tests) and 95% confidence intervals (CI) were estimated whenever appropriate.Data were entered and cleaned using EpiInfo version 3.5.2 software  and analyzed with STATA version 14.1 software . Proportions were compared by applying standard χP. vivax infection in one or more RCD rounds, regardless of any symptoms, stratified by diagnostic method: microscopy  and qPCR  in one or more RCD rounds, regardless of any symptoms. Index cases were excluded from this analysis. Individual-level variables included in the model were: age, gender, occupation, past migratory history, travel history during the last month, overnight stays in the forest in the last month (for hunting or fishing), use of vector control measures such as bed nets, history of slide-confirmed P. vivax malaria within the past six months, history of malaria-related symptoms and signs within the previous week, and use of antimalarials within the past month. HH-level variables were: HH location, determined with the use of global positioning system (GPS) receivers, and HH size. Regression models were run to identify correlates of Due to the nested structure of the data, which introduces dependency among observations that can affect model parameter estimates, we used the “melogit” STATA command to build mixed-effects logistic regression models that included the grouping variables as random factors. We had repeated observations  nested within subjects  who were clustered within HHs . Finally, HHs were clustered within index cases , since the inclusion of each index case triggered the recruitment of matched study participants  checklist to document its compliance with STROBE guidelines . Written informed consent was obtained from all study participants or their parents/guardians.P. vivax infection within the past six months. Eighteen percent reported overnight stays in the forest in the last month. Study participants were distributed into seven localities of the municipality of Acrelândia: Gleba Porto Luiz (PTL), Projeto Santo Antônio Peixoto (SAP), Projeto São João do Balanceio (SJB), Projeto Redenção (RED), Projeto Orion (ORI), Reserva Porto Dias (PTD), and Projeto Pedro Peixoto (PEP). Individual HH locations are shown in P. vivax infections diagnosed in Acrelândia between January and July 2013; between 19 (March) and 98 (July) cases were diagnosed monthly during the study period. Index cases were tentatively classified as indigenous, relapsing, or imported according to classical eradication-era criteria [P. vivax infection within the past six months), and 24 were considered indigenous . Index cases, other index HH members and neighbor HH members were recruited in all localities listed above, except for PEP; 22 (53.6%) index cases lived in PTL.The study population comprised 1,923 subjects , who were aged between 3 months and 89 years  and had been living in the study site for 1 month to 64 years . Most  were males and 1,613 (83.9%) were born in the Amazon. Nearly half of them reported previous slide-confirmed malaria episodes, but <5% had a criteria ; 6 were Plasmodium vivax positivity rates differed significantly across HH types ; interestingly, the highest prevalence of infection was observed in index HHs not only at the first visit but also over the following RCD rounds.A total of 108 infections were diagnosed by microscopy by our field team over the entire study period. HH types . They weP = 0.220), suggesting that targeting neighbor HH members on day 0 would not be more effective for diagnosing additional patent infections than randomly screening subjects living in the same locality. In contrast, positivity rates in all other RCD rounds were similar between index HHs and neighbor HHs  and significantly higher than those in control HHs , indicating that screening index and neighbor HH members would be similarly effective for diagnosing new infections between days 30 and 180  to 4.4% (day 60) . In the  and 180 . OverallP. vivax prevalence diagnosed by qPCR in index HHs (excluding the index case), neighbor HHs and control HHs during the four RCD rounds (P. vivax in the community that would be entirely missed by RCD.We next compared D rounds . Not surP < 0.001 in both comparisons) but they were similar when comparing neighbor to control HHs . Therefore, targeting neighbor HHs in search of new infections would not be more effective than randomly sampling community members on days 0 and 30. However, similar infection rates were found in index and neighbor HHs on day 60  and day 180 . Furthermore, significantly higher infection rates were detected by qPCR in neighbor than control HHs in the last two RCD rounds .Significantly different qPCR positivity rates were found across HH types, ranging from 6.8% to 15.0% in index HHs, 5.0% to 7.0% in neighbor HHs, and 0.3 to 5.6% in control HHs . On daysMicroscopy diagnosed 1.9-fold and 1.2-fold more symptomatic than asymptomatic infections in index and neighbor HHs, respectively , suggestP. vivax biomass circulating in the study population. To this end, we used data from 276 qPCR-positive samples with available qPCR-based parasite-density measurements (We thus sought to estimate the relative contribution of index and neighbor HH members (who would be screened during RCD) and control HH members (who would be missed by RCD) to the total urements . Overallurements , who hadP. vivax biomass , while those that were missed by microscopy but detected by qPCR had much lower parasite densities . If conventional microscopy were the only diagnostic method available to identify infections and guide malaria treatment, a large proportion of the parasite biomass carried by index HH members (92.4%) and neighbor HH members (79.2%), but only 47.9% of that harbored by control HH members, would be detected and potentially removed by an effective antimalarial chemotherapy.Individuals with patent infections harbored 80.7% of the P. vivax biomass measured in study subjects.Parasite densities had a unimodal right-skewed frequency distribution in asymptomatic carriers  in the fully adjusted logistic model, thus regardless of the HH type. Interestingly, a history of slide-confirmed P. vivax infection within the past six months, in the fully adjusted model, was the only other variable significantly associated with increased risk of current infection detected by either microscopy  or qPCR . These findings suggest that selectively targeting individuals reporting recent P. vivax infections, regardless of their proximity to an index case, might be a practical way of finding current infections.We used mixed-effects logistic regression analysis to test whether the association between residence in the proximity to an index case and prevalence of  members . Similar members . Each adP. vivax populations, with 146 unique (predominant or only) genotypes found in 343 completely typed samples (267 from index and neighbor HHs and 76 from control HHs). Most (272 or 79.3%) subjects harbored mixtures of genotypes. Overall, six common genotypes accounted for 30.1% of all infections and were widely distributed across study localities . Overall, 96 (35.9%) samples had genotypes that were either identical or similar to those found in other parasites within the same cluster, while 171 (64.0%) genotypes were unique . Therefore, nearly two thirds of all infections were genetically unrelated to each other within a putative malaria cluster. However, most (23 or 56.1%) clusters had at least one identical or similar genotype shared by two or more parasite samples.We next examined parasite genotypes within the 41 clusters surrounding index cases, each comprising one index HH and five neighbor HHs see . BetweenP. vivax infections that resulted in no further onward transmission within the cluster.Examples of identical parasite strains within cluster #4 , suggesting a common source of infection, are shown in We next examined all instances of recurrent infections within clusters. A total of 130 genotypes were recovered from 54 individuals who had parasite recurrences  during the study. Genotypes recovered from most (41 or 75.9%) recurrent infections were different from those found in the primary infection, even when considering minor alleles in pairwise comparisons involving multiple-clone infections. These results are consistent with unrelated, sporadic infections or heterologous relapses. However, 11 (20.4%) subjects had one or more episodes of recurrent parasitemia with identical or similar genotypes and two (3.7%) had consecutive infections involving both identical and different genotypes . OverallOnly six control HH members presented qPCR-detected parasite recurrences during the study. In all cases, different genotypes were found in the primary and subsequent infection. Although all infections were subpatent and left untreated, we found no example of chronic infection with the same strain reappearing in consecutive visits to control HH members.P. vivax endemicity and spatial clustering of malaria episodes.Malaria burden in Brazil has reached its lowest levels in more than three decades. This success led the Ministry of Health to launch, in November 2015, a Plan for Elimination of Malaria in Brazil, in alignment with the new Sustainable Development Agenda of the United Nations aimed to reduce the global number of malaria cases by 90% until 2030, and to eventually eliminate malaria in 35 countries . To thisP. vivax infections are strongly clustered at the HH level in rural Amazonia [P. vivax positivity in all RCD rounds suggests a simple way of identifying high-risk subjects that can be further explored in the design of narrowly focused and intensive active surveillance strategies. In addition, although qPCR detected nearly twice more infections than conventional microscopy in index HHs, subpatent infections contributed only 7.6% of the total P. vivax biomass circulating in index HH members. Combined, these findings suggest that a highly sensitive molecular method is not an essential component of RCD programs targeting index HH members. Interestingly, the number of slide-confirmed P. vivax infections diagnosed in Acrelândia declined substantially after our study was completed; 117 and 54 P. vivax infections (99 and 40 of them classified as autochthonous) were recorded in this municipality in 2014 and 2015, respectively .Our results provided further evidence that Amazonia ,13. In fLarge-scale RCD programs have been either limited to index HHs or extended to other HHs in the vicinity of the index HHs ,29. It hP. vivax biomass harbored by study participants was found in neighbor HH members, highlighting their potential contribution to ongoing malaria transmission in the community. One possible explanation for the increased prevalence of infections in neighbor HH members diagnosed on days 60 and 180 is that at least some of them may be secondary to those diagnosed in index HH members on days 0 and 30. We thus conclude that neighbor HHs must be screened in RCD programs in endemic settings similar to our study site. Defining the appropriate timing for microscopy- or qPCR-based screening is crucial for finding these additional cases in a cost-effective way.Here we show that malaria positivity rates among neighbor HH members on day 0, when the index case diagnosis triggered the initial investigation, were not significantly different from those found in randomly selected inhabitants in the same communities. Therefore, targeting neighbor HH members at the time of index case diagnosis did not appear to be more cost-effective than screening randomly chosen individuals for identifying new infections in our rural Amazonian setting. Nevertheless, much higher microscopy positivity rates were found in neighbor HHs, compared to control HHs, over the next months, with a similar trend observed for qPCR-based diagnosis in the last two RCD rounds. Furthermore, nearly two-thirds of the An. darlingi with blood from asymptomatic P. vivax carriers vs. 22% for the mosquitoes fed with blood from symptomatic carriers [P. vivax asexual blood-stages and mature gametocytes are positively correlated to each other [A major limitation of RCD-based strategies is that asymptomatic parasite carriers will be missed if no single symptomatic infection is found in their vicinity. A recent survey in coastal Kenya, for example, has revealed different features of clusters of asymptomatic vs. symptomatic infections, such as the greater temporal stability of the former over > 10 years; these clusters with purely asymptomatic or some symptomatic infections did not necessarily overlap spatially in all localities . qPCR-decarriers . Under tch other , asymptoch other ,35. ReasP. falciparum outbreaks in Peru [P. vivax population in our study site as well as in endemic settings nearby [Malaria clusters in our study localities were not synonymous with isolated, common-source outbreaks involving one or a few closely related parasite genotypes, as in recently described  in Peru  and Pana in Peru . To the s nearby –40, withP. vivax largely predominates, such as most Latin American countries [Results shown here may help decision-makers to design malaria elimination strategies in low-endemicity settings where ountries . We concS1 ChecklistPLoS Neglected Tropical Diseases, this paper includes the STROBE checklist to document its compliance with STROBE guidelines.As required for all observational studies published by (PDF)Click here for additional data file.S1 FigThe map also indicates the location of the nearest towns, Acrelândia, Plácido de Castro, Senador Guiomard, and Rio Branco , and the BR 364 interstate highway, which connects the States of Acre, Rondônia, and southern Amazonas to the rest of the country.(PDF)Click here for additional data file.S2 FigPhotographs by Alessandra Fratus.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Database(XLSX)Click here for additional data file.S2 Database(XLSX)Click here for additional data file.


Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia.Genetic analyses of P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor.Genotyping was undertaken on 713 sympatric HE = 0.847 vs 0.625; p = 0.017) and polyclonality  were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections.Higher diversity (P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods.The results demonstrate the greater refractoriness of  Anopheles vector distributions, and ethnic and socio-economic features [Indonesia has one of the highest burdens of malaria in Southeast Asia. Despite recent progress in reducing case incidence, more than a quarter of a million clinical cases were reported in 2013 with 230 million people at risk of infection [Plasmodium that commonly infect humans are present in Indonesia [P. falciparum has historically been the predominant species, in recent years the proportion of P. vivax cases has risen [P. vivax species, particularly in Papua Province [Further complicating the picture, all five species of ndonesia . Differeas risen . This trProvince , 5.P. vivax and P. falciparum diversity and structure, and inform associated patterns of transmission intensity, foci and stability, thus complementing traditional malariometric surveillance [P. vivax and P. falciparum transmission between four sites in Indonesia with varying endemicity, highlighting regional heterogeneity within and between species [Population genetic approaches can define eillance –9. A rec species . However species , and litP. falciparum and P. vivax circulating in Timika, the capital of Mimika district, Papua Province. The region is an epicentre for antimalarial resistance [The aim of this study was to describe the population diversity and structure of co-endemic sistance , 5 and tP. vivax and 5.2% due to P. falciparum) [The study was conducted in Timika, Papua Province in eastern Indonesia, which has the greatest burden of malaria in the country. Timika is located within Mimika Baru, one of the largest sub-districts in Mimika district. The population of 202,350 people is increasing in size by an estimated 16% per year, mainly due to economic migration, and is composed of different ethnic groups including Highland Papuans, Lowland Papuans, and Non-Papuans. Unstable malaria transmission occurs mainly in the lowland areas, where approximately 90% of the population lives. In 2013, malaria prevalence by microscopy was estimated to be 12.2% (5.7% due to Plasmodium species are treated with dihydroartemisinin-piperaquine (DHP).The Rumah Sakit Mitra Masyarakat (RSMM) is the main hospital in the district, serving a population of approximately 150,000 people. Located in the lowland area, RSMM provides medical healthcare to approximately 300 patients per day. Since 2006, local antimalarial guidelines have recommended that all patients with uncomplicated malaria due to any Plasmodium cases was performed using ArcGIS software (version 10.2.1).Parasite DNA was collected within the framework of two studies. The first was an ongoing ex-vivo drug susceptibility surveillance study conducted between March 2011 and August 2014. The second was a cross-sectional study conducted between April and July 2013. Patient and parasitological details were recorded for each case. For the cross-sectional study, GPS coordinates were recorded of the residence of all individuals assessed. The majority of houses were located in one of 16 villages, within the 3 subdistricts Mimika Baru, Mimika Timur, and Kuala Kencana. Geospatial mapping of residences with Plasmodium species was undertaken using a nested PCR protocol with 2 μL gDNA template in duplicate [P. falciparum, P. vivax, P. malariae, and P. ovale small-subunit rRNA DNA clones  were used as positive controls.For parasitaemia detected by microscopy, species and parasite density were determined by examination of Giemsa-stained thick blood films. Genomic DNA (gDNA) was extracted from 50 μL packed red blood cell (RBC) pellets using the QIAamp 96 DNA Blood Kit (Qiagen) for the cross-sectional study and from 2 mL of packed RBCs using the QIAamp DNA Mini Kit (Qiagen) for the ex-vivo surveillance study cases. Confirmation of uplicate . P. falcP. falciparum and P. vivax genotyping was performed using short tandem repeat (STR) markers. Nine STR markers  were amplified in P. falciparum infections using previously described methods [P. vivax, nine STR markers that have been defined as a consensus panel within the Asia Pacific Malaria Elimination Network (APMEN)  were amplified using the APMEN protocol [ methods . For P. protocol –16. CapiHE), population differentiation was measured using the pairwise FST metric. Both HE and FST were calculated using Arlequin software (version 3.5) [P. vivax, measures of the HE, genetic differentiation and LD (described below) were undertaken on the full marker set and on a sub-set of five markers  defined as exhibiting balanced diversity in a recent study [Samples with more than one allele at any locus were defined as polyclonal infections. The multiplicity of infection (MOI) for each sample was defined as the maximum number of alleles at any locus. Population diversity was measured using the expected heterozygosity (ion 3.5) , 18. Fornt study .K (number of sub-populations), ranging from 1–10. The model parameters included admixture with correlated allele frequencies. The most probable K was derived by applying the delta K method [IAS) using the web-based LIAN 3.5 software [STRUCTURE software version 2.3.3 was used to assess population structure . The simK method . STRUCTUK method . Multi-lsoftware . The sigsoftware . Unrootesoftware . Mantel’software .HE was undertaken using the Mann-Whitney U test and Kruskal-Wallis test.Statistical analysis was conducted using SPSS v.20.0 for windows software (IBM SPSS Statistics), with a significance threshold of 0.05. Data on patient gender, ethnicity and proportion of polyclonal infections was compared between subgroups using Pearson’s Chi-square test. The significance of difference between patient age, parasite density, MOI and Ethical approval for this study was obtained from the Eijkman Institute Research Ethics Commission, Eijkman Institute for Molecular Biology, Jakarta, Indonesia (EIREC no. 47/2010 and EIREC no. 67/2013), Faculty of Medicine, University of Gadjah Mada (Ref: KE/FK/763/EC), and the Human Research Ethics Committee of the Northern Territory Department of Health & Families and Menzies School of Health Research, Darwin, Australia (Ref: HREC-2010-1434).P. vivax and 94 P. falciparum cases were collected from the surveillance study, with genotyping successfully completed in 96% (104) of P. vivax and all P. falciparum cases. The cross-sectional household survey, undertaken between April and July 2013, assessed 2,476 participants by blood film examination and PCR analysis, of whom 877 (35.4%) had peripheral parasitaemia detected by PCR. P. vivax and P. falciparum accounted for 749 (85.4%) malaria cases. Genotyping was successful in 60% (240/397) of P. vivax and 80% (280/352) of P. falciparum cases. A total of 73 (30.4%) P. vivax and 21 (7.5%) P. falciparum samples failed to amplify any of the markers; all of these had submicroscopic parasitaemia.Between March 2011 and August 2014, 108 P. falciparum or P. vivax parasitaemia are presented in P. vivax and P. falciparum cases did not reveal any local high prevalence clusters for either species in the cross-sectional study Demographic and parasitological details on the individuals with P. falciparum and P. vivax markers are summarised in P. falciparum markers (TA42 HE = 0.315 and TA109 HE = 0.176), all markers exhibited a minimum 5% minor allele frequency. With the exception of TA42 (20.6%) in P. falciparum populations, all other markers exhibited >80% genotyping success in both species. The MS8 assay presented artefacts that affected the reliability of the allele calling and therefore, this locus was excluded from further analysis.The diversity and genotyping success rate for each of the P. vivax exhibited higher genetic diversity than P. falciparum  and a higher proportion of polyclonal infections . The mean MOI was higher for P. vivax (1.6) than P. falciparum (1.2) (p<0.001) , even after excluding complex infections . There was no evidence of LD after restricting to the 5 balanced markers , although LD became significant after removing complex samples . LD analysis using the unique haplotypes found no evidence of epidemic transmission in either species (p<0.001) . In contP. vivax . Analysi species .P. falciparum MLGs (106 infections) were observed in more than one individual (range 2–13). Only 22 MLGs (68 cases) had GPS metadata available. Amongst these, 3 pairs of infections (8.8% (6/68)), were shared between members of the same household. Seven pairs of infections ) were less than 1 Km apart. The median distance between infections was 5.3 Km (range 2 meters to 19.5 Km) (Thirty three 19.5 Km) .P. falciparum, no substructure was found in the P. vivax population. The neighbour-joining analysis  to K1 and the remaining 89.4% of isolates demonstrating predominant ancestry to K2 .In contrast to analysis  illustrary to K2 . Neighbory to K2 . AssessmP. falciparum subpopulations  (p<0.001) (IAS of 0.041 (p<0.01) for K1 and 0.033 (p<0.01) for K2  . Howeverp<0.001) . LD anal) for K2 ; the marp = 0.084), sex (p = 0.734), ethnicity (p = 0.972), or occupation (p = 0.096) (p <0.001) (p = 0.762). The geographical distribution of both subpopulations is shown in p = 0.851).Investigation of the demographic profiles of the K1 and K2 clusters did not find any significant difference in terms of age (= 0.096) . Interes <0.001) . No signP. falciparum population, the Timika genotypes (Papua) were compared with published data on 168 P. falciparum isolates collected from 4 surveys in other Indonesian islands  underlying the sub-structure in the st Timor . Principst Timor . Furtherst Timor ; Table 5 islands .P. falciparum and P. vivax populations circulating in Timika, Papua Indonesia. At this high spatial resolution, we demonstrate contrasting transmission patterns of both co-existing species, and reveal admixture of two divergent P. falciparum subpopulations, one comprised of submicroscopic infections with high genetic relatedness to isolates from other Indonesian islands.This study utilised a comprehensive collection of clinical and community samples to describe the genetic microepidemiology of P. vivax compared to P. falciparum populations [P. falciparum, there was no evidence of substructure in P. vivax. LD was also markedly lower in P. vivax than P. falciparum, even after accounting for admixture in the latter. These patterns are consistent with more intense and stable transmission in the local P. vivax population relative to P. falciparum. The patterns of diversity in the P. falciparum population were similar to those found in low to meso-endemic regions in Southeast Asia, but markedly less diverse than in hyper-holoendemic regions in Africa [P. vivax, the patterns of P. vivax diversity in Timika were most comparable to those reported in other hypo-mesoendemic settings in South-East Asia and the Pacific [P. vivax compared to P. falciparum transmission in this location, including recurrences from relapsing infections, the earlier appearance of the transmissible sexual stages in P. vivax, and the larger reservoir of submicroscopic infections in this species [Similarly to previous reports, we found higher population and within-host diversity in co-endemic ulations , 27–30. n Africa , 31. Alt Pacific . Several species .P. falciparum population exhibited several clusters of identical infections, neither species demonstrated evidence of epidemic transmission dynamics, indicating moderately stable transmission at the time of the study. The identical infections enabled assessment of shared origins of infection, revealing a small proportion (8.8% (6/68)) of isolates that were shared amongst household members, likely reflecting transmission events taking place in the vicinity of the household. However, the large majority of shared infections (70.5%) were over 1 Km apart and more likely to reflect a reservoir outside of the household. Together, the patterns of parasite relatedness, diversity and LD suggest that broad ranging interventions are still needed to further interrupt transmission of P. falciparum and P. vivax before more targeted strategies can be implemented in the area. Furthermore, although the widespread deployment of ACT has been effective in reducing the P. falciparum population, radical treatment targeting the dormant liver stages will be required to make a similar impact on P. vivax transmission.Although the P. falciparum population was the distinct substructure at this small spatial scale. The marked differentiation, elevated IAS on pooling the K1 and K2 subpopulations, lack of evidence of epidemic transmission, together with the moderate genetic diversity within each subpopulation was indicative of admixture. However, it is unclear how sympatric P. falciparum subpopulations are genetically isolated with no sign of geographical, temporal or demographic boundaries. We contemplated three possibilities: 1) the importation of these cases, 2) the rise of a new, fitter population, and 3) an unidentified biological barrier.The most notable revelation in the Timika P. falciparum genotypes with data from Bangka, Kalimantan, Sumba and West Timor revealed that the K1 isolates were more closely related to non-Papuan than Papuan isolates, and K2 was strikingly different from the rest of Indonesia. In accordance with geographic distance, the K1 sub-population demonstrated the greatest genetic similarity to the two most proximal islands, Sumba and West Timor, where P. falciparum remains hypo-mesoendemic. Owing to the mining industry and the National Transmigration Programme, there has been a great deal of human movement in Timika. Thus, it is feasible that the K1 subpopulation is the product of infections acquired outside Timika. Imported infections undermine local intervention efforts and present the threat of drug resistance introduction. Our results emphasise the need to develop geographical markers to help to differentiate between imported and local malaria cases. A recent study presented a barcode of organellar genome markers to determine the geographic origin of P. falciparum isolates [Comparison of the Papuan isolates , althougP. falciparum structure in Timika is the rise and spread of a fitter subpopulation in response to recent selective pressure such as the change in policy from chloroquine to ACT treatment in 2006. Parasites carrying mutations conferring chloroquine resistance may lose their survival advantage after drug pressure is removed, as has been demonstrated by the decrease in K76T pfcrt frequency in several African countries [P. falciparum and P. vivax, this treatment regimen may have had a greater impact on P. falciparum, since ACT alone has no activity against the dormant liver stages of P. vivax. However, more studies are needed to confirm this.The second possibility for the unusual ountries . AssuminAnopheline populations and their vectorial capacity for K1 and K2 is needed to address the first hypothesis. Although we did not find any evidence of association between K1 or K2 with Lowland, Highland or non-Papuan ethnicity, it is possible that a common erythrocyte polymorphism(s) defining parasite binding and invasions preference and that this transcends the different ethnic groups, and differentiates K1 and K2. Genomic analysis of these isolates might provide further insights.A further possible explanation for the sympatric co-existence of the K1 and K2 subpopulations is an unidentified biological barrier such as mutually exclusive vector preferences or differential erythrocyte binding specificities. Further investigation of the local P. falciparum substructure in Timika. However, similar substructure has been observed in Cambodia, where it was postulated to support the emergence of drug resistance, highlighting a need for intense surveillance strategies [Although we conducted a thorough characterisation of the subpopulations, we cannot conclusively determine the factors underlying the marked rategies . It is iOur study demonstrates considerable genetic heterogeneity between co-endemic species and distinct substructure within species that can be observed at small spatial scales, and may be reliant on passive as well as active case detection. These findings highlight the need for complementary new surveillance strategies to identify local transmission patterns that cannot be detected with traditional malariometric methods.S1 FigThe plot was generated using ArcGIS software on GPS coordinate data from the individuals identified with malaria parasitaemia. Each dot presents an individual case, with colour-coding by species according to PCR data.(TIFF)Click here for additional data file.S2 FigP. falciparum population. Different MLGs are distinguished with different colours and shapes. Circles, pentagons and diamonds indicate MLGs found in two, three, and more than three individuals respectively.This plot was generated using ArcGIS software on the GPS coordinates for the 22 identical MLGs in the (TIFF)Click here for additional data file.S3 FigP. falciparum case, with colour-coding by subpopulation as defined by STRUCTURE.The plot was generated using ArcGIS software on GPS coordinate data. Each dot presents an individual (TIFF)Click here for additional data file.S4 Figet al., with the addition here of a site label for Timika, Papua Indonesia [P. falciparum parasite rate in the 2–10 year age group (PfPR2–10) within the stable spatial limits of transmission in 2010. The approximate locations of the study sites described here are indicated with black stars.This map is a modified version of ndonesia . The ori(TIF)Click here for additional data file.S5 FigP. vivax population and the other four Indonesian islands.This plot illustrates the limited differentiation between the Timika (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Monitoring and evaluation of entomological, parasitological and clinical data is an important component of malaria control as it is a measure of the success of the interventions. In many studies, clinical data has been used to monitor trends in malaria morbidity and mortality. This study was conducted to demonstrate age dependent prevalence of malaria in the pre- and post-interventions period.A series of cross-sectional malaria parasitological surveys were conducted in Iguhu, western Kenya. Participants were randomly selected school-aged children between 6 and 13 years. The study was conducted between June 2002–December 2003 and January 2012–February 2015. Sexual and asexual parasite prevalence and densities were determined using microscopy. Age-dependence in parasite infections was compared between 2002–2003 and 2012–2015.Plasmodium falciparum had the highest prevalence of 43.5 and 11.5% in the pre- and post-intervention periods. Plasmodium malariae had a prevalence of 2.3 and 0.2%, while Plasmodium ovale had a prevalence of 0.3 and 0.1% during the pre- and post-intervention period, respectively. There was a 73.7% reduction in prevalence of P. falciparum in the post-intervention compared to the pre-intervention period. Plasmodium falciparum parasite density increased by 71.2% between pre- and post-intervention period from (geometric mean of) 554.4–949.2 parasites/µl. Geometric mean gametocytaemia in Iguhu was higher in the post-intervention period (106.4 parasites/µl), when compared to the pre-intervention period (54.1 parasites/µl). Prevalence and density of P. falciparum showed a lower age-dependency during post-intervention period when compared to pre-intervention period.The study provides evidence for reduction of malaria prevalence following the introduction of LLINs and ACT in western Kenya. Fewer people become infected but the few infected may be more infectious as suggested by higher gametocyte densities. The high parasite densities, which were not dependent on age, observed in the post intervention period imply that a more comprehensive integrated malaria management may be required to sustain the current interventions and hence reduce malaria transmission. Monitoring and evaluation of entomological, parasitological and clinical data is important to measure effectiveness of malaria control programmes. Most commonly, clinical data has been used to monitor trends in malaria morbidity and mortality. Active surveillance involving longitudinal cohort studies are used in obtaining parasitological data. While hospital-based passive surveillance captures only symptomatic cases, a field based active surveillance system captures trends in both symptomatic and asymptomatic cases and is thus less biased.The major malaria intervention strategies in Kenya include vector control using indoor residual spraying (IRS) and long-lasting insecticide-treated nets (LLINs) –3, and ePlasmodium parasite profile during pre- and post-intervention periods in western Kenya.A reduction in transmission can result in altered or reduced immune acquisition which would increase the population vulnerability to unstable transmission, epidemics and severe disease . An age This study is based on a long-term Plasmodium parasitological surveillance programme starting in June 2002 to date. Snapshots of this data was taken between June 2002 and December 2003, and January 2012–February 2015, this being the pre- and post-intervention period. The data in the two time periods was aggregated. The relative change in parasite profile was determined in log transformed nonlinear data.The study was conducted in Iguhu, western Kenya (1430–1580 m above sea level)   compared to the pre-intervention period  554.4–949.2 parasites/µl   , however it did not show any age-dependence in the post intervention period (R2 = 0.0001 and R2 = 0.13)  during the pre-intervention period and (R2 = 0.01) in the post intervention period (Fig. Prevalence and density of iod Fig. . ParasitP. falciparum before the introduction of the use of insecticide-treated bed nets in 2006 and a reduction of prevalence post-intervention. During the pre-intervention period, there was also a reported resistance in the drug of choice to treat the malaria parasite, SP [Findings from this study indicate that there was relatively high prevalence of site, SP . Bed netsite, SP  to 80% isite, SP .The introduction of the interventions in the study site reduced exposure to the parasite hence the prevalence was reduced. However, in 2015 despite the low parasite prevalence across the study sites, parasite densities doubled. This clearly shows a reduced ability to suppress parasitaemias. A small reservoir of parasitaemias may sustain transmission in a community due to their high infectiousness . IndividP. falciparum gametocytes in asymptomatic carriers increases the infectiousness of humans to mosquitoes as they are not likely to seek medical treatment and serve as reservoirs for continuous transmission of the parasite [The presence of parasite . The useparasite , 20. In parasite , and henparasite . As asymParasitaemia is proportional to the duration of exposure to malaria transmission. Thus, older individuals have lower parasitaemia compared to younger individuals such as children in the same environment , 20. TheThere is a limitation with the accuracy of parasite densities. In this study, standard WHO procedures were used to measure parasite densities. However, due to the limitation of human ability, parasite density estimates may be with errors. For example, if microscopists look longer and more carefully, they may count more sexual and/or asexual parasites. Double readings method which allowed per slide to be read by two trained microscopists was used, and further quality control, where 20% of the slides were randomly selected for further verification by senior external microscopists. These measures minimized the parasite counting errors. In the future, quantitative PCR may be used to more accurately estimate parasite density.The study provides evidence of reduction of malaria prevalence following the introduction of LLINs and ACT in western Kenya. Fewer people become infected but the few infected may be more infectious as suggested by higher gametocyte densities. The high parasite densities observed, which were not dependent on age, imply that a more comprehensive integrated malaria management may be required to sustain the current interventions and hence reduce malaria prevalence. This may include both additional vector control measures such as larviciding, and parasite transmission control, such as judicious use of single low dose primaquine to reduce gametocyte in the highlands where transmission is low. It may also involve identification of malaria transmission foci that can be targeted with additional interventions such as larviciding and IRS.Continuous monitoring of the parasitological profile is recommended to detect further changes in asexual and sexual parasite prevalence and density so as to inform malaria control programme on what additional steps need to be taken to further reduce the disease prevalence. In general, reduction in malaria transmission and disease prevalence may result in reduced functional immunity and increase the vulnerability of the population to severe disease in case interventions are interrupted. The method described here of using age-related parasite densities provide a simple, accurate, reliable and reproducible field technique for indicating important changes in population based parasitological profiles. The development of functional immune assays would greatly help in providing information on trends in immune status of populations with reduced exposure to malaria. This data would justify the continued use of entomological interventions even as the disease moves towards elimination and eradication.