Search

Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites. [ABSTRACT FROM AUTHOR]

Background: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. Methods and findings: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual’s treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months’ time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. Conclusions: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5–6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer’s disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease. [ABSTRACT FROM AUTHOR]

Plasmodium relapses are attributed to the activation of dormant liver-stage parasites and are responsible for a significant number of recurring malaria blood-stage infections. While characteristic of human infections caused by P. vivax and P. ovale, their relative contribution to malaria disease burden and transmission remains poorly understood. This is largely because it is difficult to identify ‘bona fide’ relapse infections due to ongoing transmission in most endemic areas. Here, we use the P. cynomolgi–rhesus macaque model of relapsing malaria to demonstrate that clinical immunity can form after a single sporozoite-initiated blood-stage infection and prevent illness during relapses and homologous reinfections. By integrating data from whole blood RNA-sequencing, flow cytometry, P. cynomolgi-specific ELISAs, and opsonic phagocytosis assays, we demonstrate that this immunity is associated with a rapid recall response by memory B cells that expand and produce anti-parasite IgG1 that can mediate parasite clearance of relapsing parasites. The reduction in parasitemia during relapses was mirrored by a reduction in the total number of circulating gametocytes, but importantly, the cumulative proportion of gametocytes increased during relapses. Overall, this study reveals that P. cynomolgi relapse infections can be clinically silent in macaques due to rapid memory B cell responses that help to clear asexual-stage parasites but still carry gametocytes. [ABSTRACT FROM AUTHOR]

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host’s immune system, thus promoting overall parasite survival in the face of host immunity. [ABSTRACT FROM AUTHOR]

Introduction: Asymptomatic carriage of P. falciparum is frequent in areas endemic for malaria and individual diagnosis of clinical malaria attacks is still difficult. We investigated the impact of changes in malaria endemicity on the diagnostic criteria for malaria attacks in an area of seasonal malaria transmission. Methods: We analyzed the longitudinal data collected over 20 years from a daily survey of all inhabitants of Ndiop, a rural community in central Senegal, in a logistic regression model to investigate the relationship between the level of Plasmodium falciparum parasitemia and the risk of fever, with the aim of determining the best parasitemia thresholds for attributing to malaria a fever episode. Results: A total of 34,136 observations recorded from July 1993 to December 2013 from 850 individuals aged from 1 day to 87 years were included. P. falciparum asymptomatic carriage declined from 36% to 1% between 1993 and 2013. A total of 9,819 fever episodes were associated with a positive blood film for P. falciparum. Using age-dependent parasitemia thresholds for attributing to malaria a fever episode, we recorded 6,006 malaria attacks during the study period. Parasitemia thresholds seemed to be lower during the low-to-zero transmission season and tended to decrease with changes in control policies. The number of clinical malaria attacks was overestimated for all age groups throughout the study when all fever episodes associated with P. falciparum parasitemia were defined as malaria attacks. Conclusion: Pyrogenic thresholds are particularly sensitive to changes in malaria epidemiology and are therefore an interesting tool to accurately assess the burden of malaria in the context of declining transmission. [ABSTRACT FROM AUTHOR]

Background: Mass azithromycin distributions have been shown to reduce mortality in preschool children, although the factors mediating this mortality reduction are not clear. This study was performed to determine whether mass distribution of azithromycin, which has modest antimalarial activity, reduces the community burden of malaria.Methods and Findings: In a cluster-randomized trial conducted from 23 November 2014 until 31 July 2017, 30 rural communities in Niger were randomized to 2 years of biannual mass distributions of either azithromycin (20 mg/kg oral suspension) or placebo to children aged 1 to 59 months. Participants, field staff, and investigators were masked to treatment allocation. The primary malaria outcome was the community prevalence of parasitemia on thick blood smear, assessed in a random sample of children from each community at study visits 12 and 24 months after randomization. Analyses were performed in an intention-to-treat fashion. At the baseline visit, a total of 1,695 children were enumerated in the 15 azithromycin communities, and 3,029 children were enumerated in the 15 placebo communities. No communities were lost to follow-up. The mean prevalence of malaria parasitemia at baseline was 8.9% (95% CI 5.1%-15.7%; 52 of 552 children across all communities) in the azithromycin-treated group and 6.7% (95% CI 4.0%-12.6%; 36 of 542 children across all communities) in the placebo-treated group. In the prespecified primary analysis, parasitemia was lower in the azithromycin-treated group at month 12 (mean prevalence 8.8%, 95% CI 5.1%-14.3%; 51 of 551 children across all communities) and month 24 (mean 3.5%, 95% CI 1.9%-5.5%; 21 of 567 children across all communities) than it was in the placebo-treated group at month 12 (mean 15.3%, 95% CI 10.8%-20.6%; 81 of 548 children across all communities) and month 24 (mean 4.8%, 95% CI 3.3%-6.4%; 28 of 592 children across all communities) (P = 0.02). Communities treated with azithromycin had approximately half the odds of parasitemia compared to those treated with placebo (odds ratio [OR] 0.54, 95% CI 0.30 to 0.97). Parasite density was lower in the azithromycin group than the placebo group at 12 and 24 months (square root-transformed outcome; density estimates were 7,540 parasites/μl lower [95% CI -350 to -12,550 parasites/μl; P = 0.02] at a mean parasite density of 17,000, as was observed in the placebo arm). No significant difference in hemoglobin was observed between the 2 treatment groups at 12 and 24 months (mean 0.34 g/dL higher in the azithromycin arm, 95% CI -0.06 to 0.75 g/dL; P = 0.10). No serious adverse events were reported in either group, and among children aged 1 to 5 months, the most commonly reported nonserious adverse events (i.e., diarrhea, vomiting, and rash) were less common in the azithromycin-treated communities. Limitations of the trial include the timing of the treatments and monitoring visits, both of which took place before the peak malaria season, as well as the uncertain generalizability to areas with different malaria transmission dynamics.Conclusions: Mass azithromycin distributions were associated with a reduced prevalence of malaria parasitemia in this trial, suggesting one possible mechanism for the mortality benefit observed with this intervention.Trial Registration: The trial was registered on ClinicalTrials.gov (NCT02048007). [ABSTRACT FROM AUTHOR]

Background: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. Methodology/Principal findings: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti–infected mouse model. The cytotoxicity of compounds was tested on Madin–Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 μM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 μM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 μM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 μM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC–DA and TC–CF were more potent against B. microti infection in mice than their monotherapies. Conclusions/Significance: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies. [ABSTRACT FROM AUTHOR]

Background: In Vietnam, a rapid decline of P. falciparum malaria cases has been documented in the past years, the number of Plasmodium falciparum malaria cases has rapidly decreased passing from 19.638 in 2012 to 4.073 cases in 2016. Concomitantly, the spread of artemisinin resistance markers is raising concern on the future efficacy of the ACTs. An evaluation of the clinical impact of the artemisinin resistance markers is therefore of interest. Methods: The clinical effectiveness of dihydroartemisinin-piperaquine therapy (DHA-PPQ) has been evaluated in three districts characterized by different rates of ART resistance markers: K13(C580Y) mutation and delayed parasite clearance on day 3 (DPC3). Patients were stratified in 3 groups a) no markers, b) one marker (suspected resistance), c) co-presence of both markers (confirmed resistance). In the studied areas, the clinical effectiveness of DHA-PPQ has been estimated as malaria recrudescence within 60 days. Results: The rate of K13(C580Y) ranged from 75.8% in Krong Pa to 1.2% in Huong Hoa district. DPC3 prevalence was higher in Krong Pa than in Huong Hoa (86.2% vs 39.3%). In the two districts, the prevalence of confirmed resistance was found in 69.0% and 1.2% of patients, respectively. In Thuan Bac district, we found intermediate prevalence of confirmed resistance. Treatment failure was not evidenced in any district. PPQ resistance was not evidenced. Confirmed resistance was associated to the persistence of parasites on day 28 and to 3.4-fold higher parasite density at diagnosis. The effectiveness of malaria control strategies was very high in the studied districts. Conclusion: No treatment failure has been observed in presence of high prevalence of ART resistance and in absence of PPQ resistance. K13(C580Y) was strongly associated to higher parasitemia at admission, on days 3 and 28. Slower parasite clearance was also observed in younger patients. [ABSTRACT FROM AUTHOR]

The aggregated distributions of host–parasite systems require several different infection parameters to characterize them. We advise readers how to choose infection indices with clear and distinct biological interpretations, and recommend statistical tests to compare them across samples. A user-friendly and free software is available online to overcome technical difficulties. [ABSTRACT FROM AUTHOR]

Currently, malaria rapid diagnostic tests (RDTs) are widely used for malaria diagnosis, but test performance and the factors that lead to failure of Plasmodium ovale detection are not well understood. In this study, three pLDH-based RDTs were evaluated using cases in China that originated in Africa. The sensitivity of Wondfo Pf/Pan, CareStart pLDH PAN and SD BIOLINE Pf/Pan in P. ovale detection was 70, 55 and 18%, respectively. CareStart was worse at detecting P. o. curtisi (36.5%) than at detecting P. o. wallikeri (75.0%), and SD could not detect P. o. curtisi. The overall detection ratio of all three RDTs decreased with parasite density and pLDH concentration. Wondfo, CareStart and SD detected only 75.0, 78.1 and 46.9% of the P. ovale cases, respectively, even when the parasitemia were higher than 5000 parasites/μL. Subspecies of P. ovale should be considered while to improve RDT quality for P. ovale diagnosis to achieve the goal of malaria elimination. [ABSTRACT FROM AUTHOR]

Clinical and model studies indicate that low nitric oxide (NO) bioavailability due in part to profound hypoargininemia contributes to cerebral malaria (CM) pathogenesis. Protection against CM pathogenesis may be achieved by altering the diet before infection with Plasmodium falciparum infection (nutraceutical) or by administering adjunctive therapy that decreases CM mortality (adjunctive therapy). This hypothesis was tested by administering citrulline or arginine in experimental CM (eCM). We report that citrulline injected as prophylaxis immediately post infection (PI) protected virtually all mice by ameliorating (i) hypoargininemia, (ii) urea cycle impairment, and (iii) disruption of blood brain barrier. Citrulline prophylaxis inhibited plasma arginase activity. Parasitemia was similar in citrulline- and vehicle control-groups, indicating that protection from pathogenesis was not due to decreased parasitemia. Both citrulline and arginine administered from day 1 PI in the drinking water significantly protected mice from eCM. These observations collectively indicate that increasing dietary citrulline or arginine decreases eCM mortality. Citrulline injected ip on day 4 PI with quinine-injected ip on day 6 PI partially protected mice from eCM; citrulline plus scavenging of superoxide with pegylated superoxide dismutase and pegylated catalase protected all recipients from eCM. These findings indicate that ameliorating hypoargininemia with citrulline plus superoxide scavenging decreases eCM mortality. [ABSTRACT FROM AUTHOR]

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC. [ABSTRACT FROM AUTHOR]

The article offers information on the Chagas disease, or American trypanosomiasis, which is caused by the protozoan parasite Trypanosoma cruzi, and has limited treatment options with two drugs available Benznidazole and Nifurtimox. It states that therapeutic vaccination has been proposed for the control of T. cruzi infection. It mentions that preconceptional vaccine aimed at preventing future congenital transmission of the parasite would be good for control of the disease.

The development of a sterilizing vaccine against malaria remains one of the highest priorities for global health research. While sporozoite vaccines targeting the pre-erythrocytic stage show great promise, it has not been possible to maintain efficacy long-term, likely due to an inability of these vaccines to maintain effector memory T cell responses in the liver. Vaccines based on human cytomegalovirus (HCMV) might overcome this limitation since vectors based on rhesus CMV (RhCMV), the homologous virus in rhesus macaques (RM), elicit and indefinitely maintain high frequency, non-exhausted effector memory T cells in extralymphoid tissues, including the liver. Moreover, RhCMV strain 68–1 elicits CD8+ T cells broadly recognizing unconventional epitopes exclusively restricted by MHC-II and MHC-E. To evaluate the potential of these unique immune responses to protect against malaria, we expressed four Plasmodium knowlesi (Pk) antigens (CSP, AMA1, SSP2/TRAP, MSP1c) in RhCMV 68–1 or in Rh189-deleted 68–1, which additionally elicits canonical MHC-Ia-restricted CD8+ T cells. Upon inoculation of RM with either of these Pk Ag expressing RhCMV vaccines, we obtained T cell responses to each of the four Pk antigens. Upon challenge with Pk sporozoites we observed a delayed appearance of blood stage parasites in vaccinated RM consistent with a 75–80% reduction of parasite release from the liver. Moreover, the Rh189-deleted RhCMV/Pk vectors elicited sterile protection in one RM. Once in the blood, parasite growth was not affected. In contrast to T cell responses induced by Pk infection, RhCMV vectors maintained sustained T cell responses to all four malaria antigens in the liver post-challenge. The delayed appearance of blood stage parasites is thus likely due to a T cell-mediated inhibition of liver stage parasite development. As such, this vaccine approach can be used to efficiently test new T cell antigens, improve current vaccines targeting the liver stage and complement vaccines targeting erythrocytic antigens. [ABSTRACT FROM AUTHOR]

Background: Despite the improvements in diagnostic tools for detection of Trypanosoma cruzi in human blood samples, the isolation of parasite from bloodstream in the chronic phase of Chagas disease is challenging. Thus, there is an increasing interest in the development of strategies that allow an accurate monitoring of the parasite load in bloodstream of Chagas disease patients. Given that, the comparison of a classical diagnostic method such as blood culture and multiplex quantitative real-time PCR (qPCR) was few explored so far. Therefore, this study aimed to compare the detection and quantification of T. cruzi load in the circulating blood of patients with chronic Chagas disease, using blood culture and qPCR techniques. Methods⁄Principal findings: The multiplex real-time quantitative PCR assay (qPCR) based on TaqMan technology was evaluated in 135 blood samples from 91 patients with chronic Chagas disease presenting indeterminate (asymptomatic, n = 23) and cardiac (chronic cardiomyopathy, n = 68) forms, in comparison with the classical blood culture (BC) technique. The total positivity of qPCR and BC was 58.5% and 49.6%, respectively. The median parasite load of all positive patients was 1.18 [0.39–4.23] par. eq.⁄mL, ranging from 0.01 to 116.10 par. eq.⁄mL. We did not find significant differences between T. cruzi load with age and distinct clinical manifestations of patients. Conclusions/Significance: Our data suggest that qPCR can be an auxiliary tool for studies that require T. cruzi isolation from the bloodstream of patients with chronic Chagas disease, after the establishment of a parasite load cut-off that guarantees a relative success rate of parasite isolation using BC technique. [ABSTRACT FROM AUTHOR]

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases. [ABSTRACT FROM AUTHOR]

Background: The effectiveness of anti-parasite treatment with benznidazole in the chronic Chagas disease (ChD) remains uncertain. We evaluated, using data from the NIH-sponsored SaMi-Trop prospective cohort study, if previous treatment with benznidazole is associated with lower mortality, less advanced cardiac disease and lower parasitemia in patients with chronic ChD. Methods: The study enrolled 1,959 ChD patients and abnormal electrocardiogram (ECG) from in 21 remote towns in Brazil. A total of 1,813 patients were evaluated at baseline and after two years of follow-up. Those who received at least one course of benznidazole were classified as treated group (TrG = 493) and those who were never treated as control group (CG = 1,320). The primary outcome was death after two-year follow-up; the secondary outcomes were presence at the baseline of major ChD-associated ECG abnormalities, NT-ProBNP levels suggestive of heart failure, and PCR positivity. Results: Mortality after two years was 6.3%; it was lower in the TrG (2.8%) than the CG (7.6%); adjusted OR: 0.37 (95%CI: 0.21;0.63). The ECG abnormalities typical for ChD and high age-adjusted NT-ProBNP levels suggestive of heart failure were lower in the TrG than the CG, OR: 0.35 [CI: 0.23;0.53]. The TrG had significantly lower rates of PCR positivity, OR: 0.35 [CI: 0.27;0.45]. Conclusion: Patients previously treated with benznidazole had significantly reduced parasitemia, a lower prevalence of markers of severe cardiomyopathy, and lower mortality after two years of follow-up. If used in the early phases, benznidazole treatment may improve clinical and parasitological outcomes in patients with chronic ChD. Trial registration: ClinicalTrials.gov, Trial registration: . [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells provide the first line of defense against malaria parasite infection. However, the molecular mechanisms through which NK cells are activated by parasites are largely unknown, so is the molecular basis underlying the variation in NK cell responses to malaria infection in the human population. Here, we compared transcriptional profiles of responding and non-responding NK cells following exposure to Plasmodium-infected red blood cells (iRBCs) and identified MDA5, a RIG-I-like receptor involved in sensing cytosolic RNAs, to be differentially expressed. Knockout of MDA5 in responding human NK cells by CRISPR/cas9 abolished NK cell activation, IFN-γ secretion, lysis of iRBCs. Similarly, inhibition of TBK1/IKKε, an effector molecule downstream of MDA5, also inhibited activation of responding NK cells. Conversely, activation of MDA5 by liposome-packaged poly I:C restored non-responding NK cells to lyse iRBCs. We further show that microvesicles containing large parasite RNAs from iRBCs activated NK cells by fusing with NK cells. These findings suggest that NK cells are activated through the MDA5 pathway by parasite RNAs that are delivered to the cytoplasm of NK cells by microvesicles from iRBCs. The difference in MDA5 expression between responding and non-responding NK cells following exposure to iRBCs likely contributes to the variation in NK cell responses to malaria infection in the human population. [ABSTRACT FROM AUTHOR]

Background: Drugs currently used for the treatment of Chagas’ disease, nifurtimox and benznidazole, have a limited effectiveness and toxic side effects. With the aim of finding new therapeutic approaches, in vitro and in vivo anti-Trypanosoma cruzi activity of vitamin C alone and combined with benznidazole were investigated. Methodology/Principal findings: The trypanocidal activity on epimastigote and trypomastigote forms was evaluated by counting parasites in a Neubauer chamber after treatment with the compounds. For the amastigote stage, transgenic parasites expressing β-galactosidase were used and quantified by measuring the β-galactosidase activity. The cytotoxicity of compounds was tested on Vero cells. The redox state of the parasite was evaluated by determining the reduced thiol levels (spectrophotometric assay) and the intracellular oxidative state (by flow cytometry). The in vivo trypanocidal activity was evaluated on a murine model of Chagas’ disease. The trypanocidal activity of vitamin C and benznidazole was similar for the three parasite forms. When combining both drugs, vitamin C did not induce any change in the antiparasitic activity of benznidazole on trypomastigotes; however, on mammal cells, vitamin C diminished the cytotoxicity degree of benznidazole. Two mechanisms of action may be postulated for vitamin C: a lethal pro-oxidant effect on the parasite when used alone, and an antioxidant effect, when combined with benznidazole. A similar behavior was observed on infected mice; i.e., parasite counts in infected mice treated with vitamin C were lower than that of the control group. Animals treated with benznidazole presented lower parasitemia levels, as compared with those treated with vitamin C alone. Again, vitamin C did not cause any effect on the antiparasitic profile of benznidazole. Even though a combined treatment was employed, the antioxidant effect of vitamin C on the host was evidenced; a 100% survival was observed and the weight loss occurring during the acute phase of the infection was reduced. Conclusions/Significance: Based on these results, the combination of vitamin C with benznidazole could be considered as an alternative treatment for Chagas’ disease. These preliminary results encourage further research to improve the treatment of Chagas’ disease. [ABSTRACT FROM AUTHOR]

The quantity of the intra-erythrocytic deoxyhemoglobin S (Hb S) affects the level of protection against malaria and also the sickling phenomenon. This study reports on significantly lower concentration of Hb S in females than males. Data came from 350 children, aged 12–47 months who participated in a phase 2b malaria vaccine trial. Hemoglobinopathy and G6PD deficiency typing was necessary to ascertain equal representation of these malaria protective traits across the vaccine cohorts. Hemoglobin types (HbAA, HbAS) and % Hb S were evaluated by HPLC. Alpha thalassemia (alpha-thal) and G6PD genotypes were evaluated by PCR. The overall prevalence for HbAS was 20%, 46% for 3 alpha genes and 10% for 2 alpha genes and 14% for G6PD A-. More females of HbAS/αα/αα genotype had low Hb S than males and had mean % Hb S of 37.5% ± 5.4 SD, compared to 42.0% ± 2.5 SD in males of same genotype (P = 0.018). Consistent with reduction of the malaria protective Hb S in females, parasite load in females was nearly twice that of males but the difference was not statistically significant. The X-chromosome linked G6PD deficiency did not influence the level of Hb S. We conclude that, the low Hb S in these females explains the resultant higher malaria parasite load. We speculate that the low Hb S in females could also explain observations suggesting that the sickling phenomenon tends to be less severe in females than males. [ABSTRACT FROM AUTHOR]

Malaria-induced acute kidney injury (MAKI) is a life-threatening complication of severe malaria. Here, we investigated the potential role of the angiotensin II (Ang II)/AT1 receptor pathway in the development of MAKI. We used C57BL/6 mice infected by Plasmodium berghei ANKA (PbA-infected mice), a well-known murine model of severe malaria. The animals were treated with 20 mg/kg/day losartan, an antagonist of AT1 receptor, or captopril, an angiotensin-converting enzyme inhibitor. We observed an increase in the levels of plasma creatinine and blood urea nitrogen associated with a significant decrease in creatinine clearance, a marker of glomerular flow rate, and glomerular hypercellularity, indicating glomerular injury. PbA-infected mice also presented proteinuria and a high level of urinary γ-glutamyltransferase activity associated with an increase in collagen deposition and interstitial space, showing tubule-interstitial injury. PbA-infected mice were also found to have increased fractional excretion of sodium (FENa+) coupled with decreased cortical (Na++K+)ATPase activity. These injuries were associated with an increase in pro-inflammatory cytokines, such as tumor necrosis factor alpha, interleukin-6, interleukin-17, and interferon gamma, in the renal cortex of PbA-infected mice. All modifications of these structural, biochemical, and functional parameters observed in PbA-infected mice were avoided with simultaneous treatment with losartan or captopril. Our data allow us to postulate that the Ang II/AT1 receptor pathway mediates an increase in renal pro-inflammatory cytokines, which in turn leads to the glomerular and tubular injuries observed in MAKI. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi is a genetically heterogeneous group of organisms that cause Chagas disease. It has been long suspected that the clinical outcome of the disease and response to therapeutic agents are, at least in part, related to the genetic characteristics of the parasite. Herein, we sought to validate the significance of the genotype of T. cruzi isolates recovered from patients with different clinical forms of Chagas disease living in Argentina on their biological behaviour and susceptibility to drugs. Genotype identification of the newly established isolates confirmed the reported predominance of TcV, with a minor frequency of TcI. Epimastigote sensitivity assays demonstrated marked dissimilar responses to benznidazole, nifurtimox, pentamidine and dihydroartemisinin in vitro. Two TcV isolates exhibiting divergent response to benznidazole in epimastigote assays were further tested for the expression of anti-oxidant proteins. Benznidazole-resistant BOL-FC10A epimastigotes had decreased expression of Old Yellow Enzyme and cytosolic superoxide dismutase, and overexpression of mitochondrial superoxide dismutase and tryparedoxin- 1, compared to benznidazole-susceptible AR-SE23C parasites. Drug sensitivity assays on intracellular amastigotes and trypomastigotes reproduced the higher susceptibility of AR-SE23C over BOL-FC10A parasites to benznidazole observed in epimastigotes assays. However, the susceptibility/resistance profile of amastigotes and trypomastigotes to nifurtimox, pentamidine and dihydroartemisinin varied markedly with respect to that of epimastigotes. C3H/He mice infected with AR-SE23C trypomastigotes had higher levels of parasitemia and mortality rate during the acute phase of infection compared to mice infected with BOL-FC10A trypomastigotes. Treatment of infected mice with benznidazole or nifurtimox was efficient to reduce patent parasitemia induced by either isolate. Nevertheless, qPCR performed at 70 dpi revealed parasite DNA in the blood of mice infected with AR-SE23C but not in BOL-FC10A infected mice. These results demonstrate high level of intra-type diversity which may represent an important obstacle for the testing of chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Protective immunity to blood-stage malaria is attributed to Plasmodium-specific IgG and effector-memory T helper 1 (Th1) cells. However, mice lacking the costimulatory receptor CD28 (CD28KO) maintain chronic parasitemia at low levels and do not succumb to infection, suggesting that other immune responses contribute to parasite control. We report here that CD28KO mice develop long-lasting non-sterile immunity and survive lethal parasite challenge. This protection correlated with a progressive increase of anti-parasite IgM serum levels during chronic infection. Serum IgM from chronically infected CD28KO mice recognize erythrocytes infected with mature parasites, and effectively control Plasmodium infection by promoting parasite lysis and uptake. These antibodies also recognize autoantigens and antigens from other pathogens. Chronically infected CD28KO mice have high numbers of IgM+ plasmocytes and experienced B cells, exhibiting a germinal-center independent Fas+GL7-CD38+CD73- phenotype. These cells are also present in chronically infected C57BL/6 mice although in lower numbers. Finally, IgM+ experienced B cells from cured C57BL/6 and CD28KO mice proliferate and produce anti-parasite IgM in response to infected erythrocytes. This study demonstrates that CD28 deficiency results in the generation of germinal-center independent IgM+ experienced B cells and the production of protective IgM during experimental malaria, providing evidence for an additional mechanism by which the immune system controls Plasmodium infection. [ABSTRACT FROM AUTHOR]

Long-distance migrations influence the physiology, behavior, and fitness of migratory animals throughout their annual cycles, and fundamentally alter their interactions with parasites. Several hypotheses relating migratory behavior to the likelihood of parasitism have entered the literature, making conflicting, testable predictions. To assess how migratory behavior of hosts is associated with parasitism, we compared haemosporidian parasite infections between two closely related populations of a common North American sparrow, the dark-eyed junco, that co-occur in shared habitats during the non-breeding season. One population is sedentary and winters and breeds in the Appalachian Mountains. The other population is migratory and is found in seasonal sympatry with the sedentary population from October through April, but then flies (≥ 900 km) northwards to breed. The populations were sampled in the wild on the shared montane habitat at the beginning of winter and again after confining them in a captive common environment until the spring. We found significantly higher prevalence of haemosporidian parasite infections in the sedentary population. Among infected juncos, we found no difference in parasite densities (parasitemias) between the sedentary and migrant populations and no evidence for winter dormancy of the parasites. Our results suggest that long-distance migration may reduce the prevalence of parasite infections at the population level. Our results are inconsistent with the migratory exposure hypothesis, which posits that long-distance migration increases exposure of hosts to diverse parasites, and with the migratory susceptibility hypothesis, which posits that trade-offs between immune function and migration increase host susceptibility to parasites. However, our results are consistent with the migratory culling hypothesis, which posits that heavily infected animals are less likely to survive long-distance migration, and with the migratory escape hypothesis, which posits that long-distance migration allows host populations to seasonally escape areas of high infection risk. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a leading cause of childhood mortality in sub-Saharan Africa. Identifying patients who are at risk for severe manifestations at presentation still remains challenging. This study examines whether a semi-quantitative test on C-Reactive Protein (CRP) could be useful for rapidly predicting the presence or absence of malarial parasitemia in febrile children. Method: Data were collected from children with fever or a history of fever at the Agogo Presbyterian Hospital in the Ashanti Region of Ghana. Haematological measurements, microscopic detection of plasmodium species and semi-quantitative CRP measurements with a membrane–based immunoassay for whole blood were performed. CRP was classified as positive when the measured level was ≥ 10 mg/l. Results: During 548 visits, thick blood film results could be obtained from 541 patients, 270 (49.3%) yielded parasitemia with Plasmodium spp. Whereas malaria parasites were detected in only a few patients (7.1%) with normal CRP levels (< 10mg/l), more than a half of patients with an increased CRP concentration (≥ 10 mg/l) were parasite positive (OR 14.5 [CI 4.4–47.6], p<0.001). Patients with increased CRP levels had more than an eight-fold likelihood for parasitemia after correction for other parameters (adjusted OR 8.7 [CI 2.5–30.5], p<0.001). Sensitivity, specificity as well as positive predictive and negative predictive values of CRP for malaria were 99.3% (CI 96.2%-100%), 9.2% (CI 6.4%-12.8%), 31.7% (CI 27.4%-36.1%) and 97.0% (CI 84.2%-99.9%), respectively. Conclusion: The semi-quantitative method of measuring CRP is cheap, rapid and easy to perform but not useful in predicting parasitemia and malaria. However, due to its high negative predictive value, it could have a role in identifying those patients unlikely to be presenting with clinical malaria. [ABSTRACT FROM AUTHOR]

Background: Intermittent preventive treatment of malaria in pregnancy (IPTp) with dihydroartemisinin-piperaquine (IPTp-DP) has been shown to reduce the burden of malaria during pregnancy compared to sulfadoxine-pyrimethamine (IPTp-SP). However, limited data exist on how IPTp regimens impact malaria risk during infancy. We conducted a double-blinded randomized controlled trial (RCT) to test the hypothesis that children born to mothers given IPTp-DP would have a lower incidence of malaria during infancy compared to children born to mothers who received IPTp-SP.Methods and Findings: We compared malaria metrics among children in Tororo, Uganda, born to women randomized to IPTp-SP given every 8 weeks (SP8w, n = 100), IPTp-DP every 8 weeks (DP8w, n = 44), or IPTp-DP every 4 weeks (DP4w, n = 47). After birth, children were given chemoprevention with DP every 12 weeks from 8 weeks to 2 years of age. The primary outcome was incidence of malaria during the first 2 years of life. Secondary outcomes included time to malaria from birth and time to parasitemia following each dose of DP given during infancy. Results are reported after adjustment for clustering (twin gestation) and potential confounders (maternal age, gravidity, and maternal parasitemia status at enrolment).The study took place between June 2014 and May 2017. Compared to children whose mothers were randomized to IPTp-SP8w (0.24 episodes per person year [PPY]), the incidence of malaria was higher in children born to mothers who received IPTp-DP4w (0.42 episodes PPY, adjusted incidence rate ratio [aIRR] 1.92; 95% CI 1.00-3.65, p = 0.049) and nonsignificantly higher in children born to mothers who received IPT-DP8w (0.30 episodes PPY, aIRR 1.44; 95% CI 0.68-3.05, p = 0.34). However, these associations were modified by infant sex. Female children whose mothers were randomized to IPTp-DP4w had an apparently 4-fold higher incidence of malaria compared to female children whose mothers were randomized to IPTp-SP8w (0.65 versus 0.20 episodes PPY, aIRR 4.39, 95% CI 1.87-10.3, p = 0.001), but no significant association was observed in male children (0.20 versus 0.28 episodes PPY, aIRR 0.66, 95% CI 0.25-1.75, p = 0.42). Nonsignificant increases in malaria incidence were observed among female, but not male, children born to mothers who received DP8w versus SP8w. In exploratory analyses, levels of malaria-specific antibodies in cord blood were similar between IPTp groups and sex. However, female children whose mothers were randomized to IPTp-DP4w had lower mean piperaquine (PQ) levels during infancy compared to female children whose mothers received IPTp-SP8w (coef 0.81, 95% CI 0.65-1.00, p = 0.048) and male children whose mothers received IPTp-DP4w (coef 0.72, 95% CI 0.57-0.91, p = 0.006). There were no significant sex-specific differences in PQ levels among children whose mothers were randomized to IPTp-SP8w or IPTp-DP8w. The main limitations were small sample size and childhood provision of DP every 12 weeks in infancy.Conclusions: Contrary to our hypothesis, preventing malaria in pregnancy with IPTp-DP in the context of chemoprevention with DP during infancy does not lead to a reduced incidence of malaria in childhood; in this setting, it may be associated with an increased incidence of malaria in females. Future studies are needed to better understand the biological mechanisms of in utero drug exposure on drug metabolism and how this may affect the dosing of antimalarial drugs for treatment and prevention during infancy.Trial Registration: ClinicalTrials.gov number NCT02163447. [ABSTRACT FROM AUTHOR]

Malaria continues to be one of mankind’s most devastating diseases despite the many and varied efforts to combat it. Indispensable for malaria elimination and eventual eradication is the development of effective vaccines. Controlled human malaria infection (CHMI) is an invaluable tool for vaccine efficacy assessment and investigation of early immunological and molecular responses against Plasmodium falciparum infection. Here, we investigated gene expression changes following CHMI using RNA-Seq. Peripheral blood samples were collected in Bagamoyo, Tanzania, from ten adults who were injected intradermally (ID) with 2.5x104 aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria® PfSPZ Challenge). A total of 2,758 genes were identified as differentially expressed following CHMI. Transcriptional changes were most pronounced on day 5 after inoculation, during the clinically silent liver phase. A secondary analysis, grouping the volunteers according to their prepatent period duration, identified 265 genes whose expression levels were linked to time of blood stage parasitemia detection. Gene modules associated with these 265 genes were linked to regulation of transcription, cell cycle, phosphatidylinositol signaling and erythrocyte development. Our study showed that in malaria pre-exposed volunteers, parasite prepatent period in each individual is linked to magnitude and timing of early gene expression changes after ID CHMI. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum infections presenting either as symptomatic or asymptomatic may contain sexual stage parasites (gametocytes) that are crucial to malaria transmission. In this study, the prevalence of microscopic and submicroscopic asexual and gametocyte parasite stages were assessed in asymptomatic children from two communities in southern Ghana. Eighty children aged twelve years and below, none of whom exhibited signs of clinical malaria living in Obom and Cape Coast were sampled twice, one during the rainy (July 2015) and subsequently during the dry (January 2016) season. Venous blood was used to prepare thick and thin blood smears, spot a rapid malaria diagnostic test (PfHRP2 RDT) as well as prepare filter paper blood spots. Blood cell pellets were preserved in Trizol for RNA extraction. Polymerase chain reaction (PCR) and semi-quantitative real time reverse transcriptase PCR (qRT-PCR) were used to determine submicroscopic parasite prevalence. In both sites 87% (95% CI: 78–96) of the asymptomatic individuals surveyed were parasites positive during the 6 month study period. The prevalence of asexual and gametocyte stage parasites in the rainy season were both significantly higher in Obom than in Cape Coast (P < 0.001). Submicroscopic gametocyte prevalence was highest in the rainy season in Obom but in the dry season in Cape Coast. Parasite prevalence determined by PCR was similar to that determined by qRT-PCR in Obom but significantly lower than that determined by qRT-PCR in Cape Coast. Communities with varying parasite prevalence exhibit seasonal variations in the prevalence of gametocyte carriers. Submicroscopic asymptomatic parasite and gametocyte carriage is very high in southern Ghana, even during the dry season in communities with low microscopic parasite prevalence and likely to be missed during national surveillance exercises. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum PfRH5 protein binds Ripr, CyRPA and Pf113 to form a complex that is essential for merozoite invasion of erythrocytes. The inter-genomic conservation of the PfRH5 complex proteins makes them attractive blood stage vaccine candidates. However, little is known about how antibodies to PfRH5, CyRPA and Pf113 are acquired and maintained in naturally exposed populations, and the role of PfRH5 complex proteins in naturally acquired immunity. To provide such data, we studied 206 Ghanaian children between the ages of 1–12 years, who were symptomatic, asymptomatic or aparasitemic and healthy. Plasma levels of antigen-specific IgG and IgG subclasses were measured by ELISA at several time points during acute disease and convalescence. On the day of admission with acute P. falciparum malaria, the prevalence of antibodies to PfRH5-complex proteins was low compared to other merozoite antigens (EBA175, GLURP-R0 and GLURP-R2). At convalescence, the levels of RH5-complex-specific IgG were reduced, with the decay of PfRH5-specific IgG being slower than the decay of IgG specific for CyRPA and Pf113. No correlation between IgG levels and protection against P. falciparum malaria was observed for any of the PfRH5 complex proteins. From this we conclude that specific IgG was induced against proteins from the PfRH5-complex during acute P. falciparum malaria, but the prevalence was low and the IgG levels decayed rapidly after treatment. These data indicate that the levels of IgG specific for PfRH5-complex proteins in natural infections in Ghanaian children were markers of recent exposure only. [ABSTRACT FROM AUTHOR]

Protection at the peak of Plasmodium chabaudi blood-stage malaria infection is provided by CD4 T cells. We have shown that an increase in Th1 cells also correlates with protection during the persistent phase of malaria; however, it is unclear how these T cells are maintained. Persistent malaria infection promotes protection and generates both effector T cells (Teff), and effector memory T cells (Tem). We have previously defined new CD4 Teff (IL-7Rα-) subsets from Early (TeffEarly, CD62LhiCD27+) to Late (TeffLate, CD62LloCD27-) activation states. Here, we tested these effector and memory T cell subsets for their ability to survive and protect in vivo. We found that both polyclonal and P. chabaudi Merozoite Surface Protein-1 (MSP-1)-specific B5 TCR transgenic Tem survive better than Teff. Surprisingly, as Tem are associated with antigen persistence, Tem survive well even after clearance of infection. As previously shown during T cell contraction, TeffEarly, which can generate Tem, also survive better than other Teff subsets in uninfected recipients. Two other Tem survival mechanisms identified here are that low-level chronic infection promotes Tem both by driving their proliferation, and by programming production of Tem from Tcm. Protective CD4 T cell phenotypes have not been precisely determined in malaria, or other persistent infections. Therefore, we tested purified memory (Tmem) and Teff subsets in protection from peak pathology and parasitemia in immunocompromised recipient mice. Strikingly, among Tmem (IL-7Rαhi) subsets, only TemLate (CD62LloCD27-) reduced peak parasitemia (19%), though the dominant memory subset is TemEarly, which is not protective. In contrast, all Teff subsets reduced peak parasitemia by more than half, and mature Teff can generate Tem, though less. In summary, we have elucidated four mechanisms of Tem maintenance, and identified two long-lived T cell subsets (TemLate, TeffEarly) that may represent correlates of protection or a target for longer-lived vaccine-induced protection against malaria blood-stages. [ABSTRACT FROM AUTHOR]

Malaria ranks among the most important infectious diseases worldwide and affects mostly people living in tropical countries. Mechanisms involved in disease progression are still not fully understood and specific treatments that might interfere with cerebral malaria (CM) are limited. Here we show that administration of doxycycline (DOX) prevented experimental CM (ECM) in Plasmodium berghei ANKA (PbA)-infected C57BL/6 wildtype (WT) mice in an IL-10-independent manner. DOX-treated mice showed an intact blood-brain barrier (BBB) and attenuated brain inflammation. Importantly, if WT mice were infected with a 20-fold increased parasite load, they could be still protected from ECM if they received DOX from day 4–6 post infection, despite similar parasitemia compared to control-infected mice that did not receive DOX and developed ECM. Infiltration of T cells and cytotoxic responses were reduced in brains of DOX-treated mice. Analysis of brain tissue by RNA-array revealed reduced expression of chemokines and tumour necrosis factor (TNF) in brains of DOX-treated mice. Furthermore, DOX-administration resulted in brains of the mice in reduced expression of matrix metalloproteinase 2 (MMP2) and granzyme B, which are both factors associated with ECM pathology. Systemic interferon gamma production was reduced and activated peripheral T cells accumulated in the spleen in DOX-treated mice. Our results suggest that DOX targeted inflammatory processes in the central nervous system (CNS) and prevented ECM by impaired brain access of effector T cells in addition to its anti-parasitic effect, thereby expanding the understanding of molecular events that underlie DOX-mediated therapeutic interventions. [ABSTRACT FROM AUTHOR]

Artemisinin resistance is a major threat to malaria control efforts. Resistance is characterized by an increase in the Plasmodium falciparum parasite clearance half-life following treatment with artemisinin-based combination therapies (ACTs) and an increase in the percentage of surviving parasites. The remarkably short blood half-life of artemisinin derivatives may contribute to drug-resistance, possibly through factors including sub-lethal plasma concentrations and inadequate exposure. Here we selected for a new strain of artemisinin resistant parasites, termed the artemisinin resistant strain 1 (ARS1), by treating P. falciparum Palo Alto (PA) cultures with sub-lethal concentrations of dihydroartemisinin (DHA). The resistance phenotype was maintained for over 1 year through monthly maintenance treatments with low doses of 2.5 nM DHA. There was a moderate increase in the DHA IC50 in ARS1 when compared with parental strain PA after 72 h of drug exposure (from 0.68 nM to 2 nM DHA). In addition, ARS1 survived treatment physiologically relevant DHA concentrations (700 nM) observed in patients. Furthermore, we confirmed a lack of cross-resistance against a panel of antimalarials commonly used as partner drugs in ACTs. Finally, ARS1 did not contain Pfk13 propeller domain mutations associated with ART resistance in the Greater Mekong Region. With a stable growth rate, ARS1 represents a valuable tool for the development of new antimalarial compounds and studies to further elucidate the mechanisms of ART resistance. [ABSTRACT FROM AUTHOR]

Chagas disease caused by Trypanosoma cruzi is a neglected disease that affects about 7 million people in Latin America, recently emerging on other continents due to migration. As infection in mice is characterized by depletion of plasma L-arginine, the effect on infection outcome was tested in mice with or without L-arginine supplementation and treatment with 1400W, a specific inhibitor of inducible nitric oxide synthase (iNOS). We found that levels of L-arginine and citrulline were reduced in the heart and plasma of infected mice, whereas levels of asymmetric dimethylarginine, an endogenous iNOS inhibitor, were higher. Moreover, L-arginine supplementation decreased parasitemia and heart parasite burden, improving clinical score and survival. Nitric oxide production in heart tissue and plasma was increased by L-arginine supplementation, while pharmacological inhibition of iNOS yielded an increase in parasitemia and worse clinical score. Interestingly, electrocardiograms improved in mice supplemented with L-arginine, suggesting that it modulates infection and heart function and is thus a potential biomarker of pathology. More importantly, L-arginine may be useful for treating T. cruzi infection, either alone or in combination with other antiparasitic drugs. [ABSTRACT FROM AUTHOR]

Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. Methodology/Principal findings: Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. Conclusions/Significance: The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life. [ABSTRACT FROM AUTHOR]

Severe malaria: Even with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death. Sevuparin in phase I study: The drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated. Sevuparin in phase I/II clinical study: A phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study. Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Malaria continues to be a devastating disease, largely caused by Plasmodium falciparum infection. We investigated the effects of opioid and cannabinoid receptor antagonists on the growth of intraerythrocytic P. falciparum. The delta opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) and the cannabinoid receptor antagonists rimonaband and SR144528 caused growth arrest of the parasite. Notably BNTX and the established antimalarial drug dihydroartemisinin induced prominent pyknosis in parasite cells after a short period of incubation. We compared genome-wide transcriptome profiles in P. falciparum with different degrees of pyknosis in response to drug treatment, and identified 11 transcripts potentially associated with the evoking of pyknosis, of which three, including glutathione reductase (PfGR), triose phosphate transporter (PfoTPT), and a conserved Plasmodium membrane protein, showed markedly different gene expression levels in accordance with the degree of pyknosis. Furthermore, the use of specific inhibitors confirmed PfGR but not PfoTPT as a possible factor contributing to the development of pyknosis. A reduction in total glutathione levels was also detected in association with increased pyknosis. These results further our understanding of the mechanisms responsible for P. falciparum development and the antimalarial activity of dihydroartemisinin, and provide useful information for the development of novel antimalarial agents. [ABSTRACT FROM AUTHOR]

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical. [ABSTRACT FROM AUTHOR]

A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to Plasmodium infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage Plasmodium chabaudi malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during P. chabaudi infection and thus in disease outcome. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax malaria (Pv-malaria) is still considered a neglected disease despite an alarming number of individuals being infected annually. Malaria pathogenesis occurs with the onset of the vector-parasite-host interaction through the binding of pathogen-associated molecular patterns (PAMPs) and receptors of innate immunity, such as toll-like receptors (TLRs). The triggering of the signaling cascade produces an elevated inflammatory response. Genetic polymorphisms in TLRs are involved in susceptibility or resistance to infection, and the identification of genes involved with Pv-malaria response is important to elucidate the pathogenesis of the disease and may contribute to the formulation of control and elimination tools. Methodology/Principal findings: A retrospective case-control study was conducted in an intense transmission area of Pv-malaria in the state of Amazonas, Brazil. Genetic polymorphisms (SNPs) in different TLRs, TIRAP, and CD14 were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 325 patients infected with P. vivax and 274 healthy individuals without malaria history in the prior 12 months from the same endemic area. Parasite load was determined by qPCR. Simple and multiple logistic/linear regressions were performed to investigate association between the polymorphisms and the occurrence of Pv-malaria and parasitemia. The C/T (TLR5 R392StopCodon) and T/T (TLR9 -1486C/T) genotypes appear to be risk factors for infection by P. vivax (TLR5: C/C vs. C/T [OR: 2.116, 95% CI: 1.054–4.452, p = 0.031]; TLR9: C/C vs. T/T [OR: 1.919, 95% CI: 1.159–3.177, p = 0.010]; respectively). Fever (COEF = 7599.46, 95% CI = 3063.80–12135.12, p = 0.001) and the C/C genotype of TLR9 -1237C/T (COEF = 17006.63, 95% CI = 3472.83–30540.44, p = 0.014) were independently associated with increased parasitemia in patients with Pv-malaria. Conclusions: Variants of TLRs may predispose individuals to infection by P. vivax. The TLR5 R392StopCodon and TLR9 -1486C/T variants are associated with susceptibility to Pv-malaria. Furthermore, the TLR9 variant -1237C/C correlates with high parasitemia. [ABSTRACT FROM AUTHOR]

Backgrounds: Malaria is a leading cause of imported febrile illnesses in pediatric travelers, but few studies have addressed severe imported pediatric malaria. We aimed to determine the risk factors and the features of imported pediatric severe malaria. Methods: We conducted a retrospective, descriptive study using the French National Reference Center for Imported Malaria database, in children aged 0–15 years who were hospitalized with a falciparum malaria from January 1st 1996 to December 31th 2005. Uncomplicated and severe cases of falciparum malaria were compared to identify risk factors for severe cases. In the hospitals that reported more than five severe cases during the study period, we evaluated severe cases for prognostic factors and assessed the accuracy WHO criteria for predicting severity. Given the rarity of deaths, adverse outcomes were defined as requiring major therapeutic procedures (MTPs)—e.g., sedation, mechanical ventilation, nasal oxygen therapy, blood transfusions, hemodialysis, fluid resuscitation—or pediatric intensive care unit (PICU) admission. Results: Of 4150 pediatric malaria cases included in the study, 3299 were uncomplicated and 851 (20.5%) were severe. Only one death was recorded during this period. Predictors for severe falciparum malaria were: age <2 years (OR = 3.2, 95% CI = 2.5–4.0, p <0.0001) and a travel in the Sahelian region (OR = 1.7, 95% CI = 1.3–2.0, p = 0.0001). Of 422 severe malaria cases, a stay in a Sahelian region, lack of chemoprophylaxis, age <2 years or thrombocytopenia <100 x 10^3/mm^3 predicted adverse outcomes. Except for the hyperparasitemia threshold of 4%, the main WHO 2000 criteria for severe malaria reliably predicted adverse outcomes. In our study, the threshold of parasitemia most predictive of a poor outcome was 8%. Conclusion: In imported pediatric malaria, children younger than 2 years deserve particular attention. The main WHO 2000 criteria for severity are accurate, except for the threshold of hyperparasitemia, which should be revised. [ABSTRACT FROM AUTHOR]

Cerebral malaria (CM) is the most severe manifestation of human malaria yet is still poorly understood. Mouse models have been developed to address the subject. However, their relevance to mimic human pathogenesis is largely debated. Here we study an alternative cerebral malaria model with an experimental Plasmodium berghei Keyberg 173 (K173) infection in Sprague Dawley rats. As in Human, not all infected subjects showed cerebral malaria, with 45% of the rats exhibiting Experimental Cerebral Malaria (ECM) symptoms while the majority (55%) of the remaining rats developed severe anemia and hyperparasitemia (NoECM). These results allow, within the same population, a comparison of the noxious effects of the infection between ECM and severe malaria without ECM. Among the ECM rats, 77.8% died between day 5 and day 12 post-infection, while the remaining rats were spontaneously cured of neurological signs within 24–48 hours. The clinical ECM signs observed were paresis quickly evolving to limb paralysis, global paralysis associated with respiratory distress, and coma. The red blood cell (RBC) count remained normal but a drastic decrease of platelet count and an increase of white blood cell numbers were noted. ECM rats also showed a decrease of glucose and total CO2 levels and an increase of creatinine levels compared to control rats or rats with no ECM. Assessment of the blood-brain barrier revealed loss of integrity, and interestingly histopathological analysis highlighted cyto-adherence and sequestration of infected RBCs in brain vessels from ECM rats only. Overall, this ECM rat model showed numerous clinical and histopathological features similar to Human CM and appears to be a promising model to achieve further understanding the CM pathophysiology in Humans and to evaluate the activity of specific antimalarial drugs in avoiding/limiting cerebral damages from malaria. [ABSTRACT FROM AUTHOR]

Increasing resistance by malaria parasites to currently used antimalarials across the developing world warrants timely detection and classification so that appropriate drug combinations can be administered before clinical complications arise. However, this is often challenged by low levels of infection (referred to as parasitemia) and presence of predominantly young parasitic forms in the patients’ peripheral blood. Herein, we developed a simple, inexpensive and portable image-based cytometer that detects and numerically counts Plasmodium falciparum infected red blood cells (iRBCs) from Giemsa-stained smears derived from infected blood. Our cytometer is able to classify all parasitic subpopulations by quantifying the area occupied by the parasites within iRBCs, with high specificity, sensitivity and negligible false positives (~ 0.0025%). Moreover, we demonstrate the application of our image-based cytometer in testing anti-malarial efficacy against a commercial flow cytometer and demonstrate comparable results between the two methods. Collectively, these results highlight the possibility to use our image-based cytometer as a cheap, rapid and accurate alternative for antimalarial testing without compromising on efficiency and minimal processing time. With appropriate filters applied into the algorithm, to rule out leukocytes and reticulocytes, our cytometer may also be used for field diagnosis of malaria. [ABSTRACT FROM AUTHOR]

Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite’s genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method. [ABSTRACT FROM AUTHOR]

Pyruvate kinase (PKLR) deficiency protects mice and humans against blood-stage malaria. Although mouse strain AcB62 carries a malaria-protective PklrI90N genetic mutation, it is phenotypically susceptible to blood stage malaria induced by infection with Plasmodium chabaudi AS, suggesting a genetic modifier of the PklrI90N protective effect. Linkage analysis in a F2 cross between AcB62 (PklrI90N) and another PK deficient strain CBA/Pk (PklrG338D) maps this modifier (designated Char10) to chromosome 9 (LOD = 10.8, 95% Bayesian CI = 50.7–75Mb). To study the mechanistic basis of the Char10 effect, we generated an incipient congenic line (Char10C) that harbors the Char10 chromosome 9 segment from AcB62 fixed on the genetic background of CBA/Pk. The Char10 effect is shown to be highly penetrant as the Char10C line recapitulates the AcB62 phenotype, displaying high parasitemia following P. chabaudi infection, compared to CBA/Pk. Char10C mice also display a reduction in anemia phenotypes associated with the PklrG338D mutation including decreased splenomegaly, decreased circulating reticulocytes, increased density of mature erythrocytes, increased hematocrit, as well as decreased iron overload in kidney and liver and decreased serum iron. Erythroid lineage analyses indicate that the number of total TER119+ cells as well as the numbers of the different CD71+/CD44+ erythroblast sub-populations were all found to be lower in Char10C spleen compared to CBA/Pk. Char10C mice also displayed lower number of CFU-E per spleen compared to CBA/Pk. Taken together, these results indicate that the Char10 locus modulates the severity of pyruvate kinase deficiency by regulating erythroid responses in the presence of PK-deficiency associated haemolytic anemia. [ABSTRACT FROM AUTHOR]

A devastating complication of Plasmodium falciparum infection is cerebral malaria, in which vascular leakage and cerebral swelling lead to coma and often death. P. falciparum produces a protein called histidine-rich protein II (HRPII) that accumulates to high levels in the bloodstream of patients and serves as a diagnostic and prognostic marker for falciparum malaria. Using a human cerebral microvascular endothelial barrier model, we previously found that HRPII activates the endothelial cell inflammasome, resulting in decreased integrity of tight junctions and increased endothelial barrier permeability. Here, we report that intravenous administration of HRPII induced blood-brain barrier leakage in uninfected mice. Furthermore, HRPII infusion in P. berghei-infected mice increased early mortality from experimental cerebral malaria. These data support the hypothesis that HRPII is a virulence factor that contributes to cerebral malaria by compromising the integrity of the blood-brain barrier. [ABSTRACT FROM AUTHOR]

In Plasmodium falciparum holoendemic transmission regions of western Kenya, life-threatening pediatric malaria manifests primarily as severe malarial anemia (SMA, Hb≤6.0 g/dL with any density parasitemia). To determine the role that CD4+ T-cell-driven inflammatory responses have in the pathogenesis of SMA, peripheral CD4+ T-cell populations and their intracellular production of pro-inflammatory cytokines (IFN-γ and IL-17) were characterized in children aged 12–36 months of age stratified into two groups: non-severe malarial anemia (non-SMA, Hb≥6.0 g/dL, n = 50) and SMA (n = 39). In addition, circulating IFN-γ and IL-17 were measured as part of a Cytokine 25-plex Antibody Bead Kit, Human (BioSource™ International). Children with SMA had higher overall proportions of circulating lymphocytes (P = 0.003) and elevated proportions of lymphocytes expressing IFN-γ (P = 0.014) and comparable IL-17 (P = 0.101). In addition, SMA was characterized by decreased memory-like T-cells (CD4+CD45RA-) expressing IL-17 (P = 0.009) and lower mean fluorescence intensity in memory-like CD4+ T-cells for both IFN-γ (P = 0.063) and IL-17 (P = 0.006). Circulating concentrations of IFN-γ were higher in children with SMA (P = 0.009), while IL-17 levels were comparable between the groups (P = 0.164). Furthermore, circulating levels of IFN-γ were negatively correlated with IL-17 levels in both groups of children (SMA: r = -0.610, P = 0.007; and non-SMA: r = -0.516, P = 0.001), while production of both cytokines by lymphocytes were positively correlated (SMA: r = 0.349, P = 0.037; and non-SMA: r = 0.475, P = 0.001). In addition, this correlation was only maintained by the memory-like CD4+ T cells (r = 0.365, P = 0.002) but not the naïve-like CD4+ T cells. However, circulating levels of IFN-γ were only associated with naïve-like CD4+ T cells producing IFN-γ (r = 0.547, P = 0.028), while circulating levels of IL-17 were not associated with any of the cell populations. Taken together, these results suggest that enhanced severity of malarial anemia is associated with higher overall levels of circulating lymphocytes, enhanced intracellular production of IFN-γ by peripheral lymphocytes and high circulating IFN-γ levels. In addition, the observed inverse relationship between the circulating levels of IFN-γ and IL-17 together with the reduction in the levels of memory-like CD4+ T cells expressing IL-17 in children with SMA may suggest possible relocation of these cells in the deeper tissues for their pathological effect. [ABSTRACT FROM AUTHOR]

Background: In order to meet the Sustainable Development Goal to decrease maternal mortality, increased access to obstetric interventions such as Caesarean sections (CS) is of critical importance. As a result of women’s limited access to routine and emergency obstetric services in Nigeria, the country is a major contributor to the global burden of maternal mortality. In this analysis, we aim to establish rates of CS and determine socioeconomic or medical risk factors associated with having a CS in Enugu, southeast Nigeria. Methods: Data for this study originated from the Healthy Beginning Initiative study. Participant characteristics were obtained from 2300 women at baseline via a semi-structured questionnaire. Only women between the ages of 17–45 who had singleton deliveries were retained for this analysis. Post-delivery questionnaires were used to ascertain mode-of-delivery. Crude and adjusted logistic regressions with Caesarean as the main outcome are presented. Results: In this sample, 7.22% women had a CS. Compared to women who lived in an urban setting, those who lived in a rural setting had a significant reduction in the odds of having a CS (aOR: 0.58; 0.38–0.89). Significantly higher odds of having a CS were seen among those with high peripheral malaria parasitemia compared to those with low parasitemia (aOR: 1.54; 1.04–2.28). Conclusion: This study revealed that contrary to the increasing trend in use of CS in low-income countries, women in this region of Nigeria had limited access to this intervention. Increasing age and socioeconomic proxies for income and access to care (e.g., having a tertiary-level education, full-time employment, and urban residence) were shown to be key determinants of access to CS. Further research is needed to ascertain the obstetric conditions under which women in this region receive CS, and to further elucidate the role of socioeconomic factors in accessing CS. [ABSTRACT FROM AUTHOR]

Background: In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria. Methods: A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d’Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 Plasmodium falciparum (PfAMA1, PfMSP4, PfMSP1, PfEMP1-DBL1α1-PF13, PfLSA1-41, PfLSA3-NR2, PfGLURP and PfCSP), one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA. Results: High prevalence (7–93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10−3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations. Conclusion: The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity. [ABSTRACT FROM AUTHOR]