Plasmodium falciparum and Plasmodium vivax are considered in the malaria diagnostic and treatment policies. However, the existence and prevalence of Plasmodium ovale spp. and Plasmodium malariae in Ethiopia have not been extensively investigated. The objective of this study was to use a multiplex IgG antibody detection assay to evaluate evidence for exposure to any of these four human malaria parasites among asymptomatic individuals.Malaria remains a very important public health problem in Ethiopia. Currently, only Plasmodium antigens: P. falciparum circumsporozoite protein (CSP), P. falciparum apical membrane antigen-1 (AMA1), P. falciparum liver stage antigen-1 (LSA1), and homologs of the merozoite surface protein-1 (MSP1)-19kD antigens that are specific for P. falciparum, P. vivax, P. ovale spp. and P. malariae.Dried blood spots (DBS) were collected from 180 healthy study participants during a 2016 onchocerciasis survey in the Jimma Zone, southwest Ethiopia. IgG antibody reactivity was detected using a multiplex bead assay for seven Plasmodium antigens tested. The most frequent responses were against P. falciparum AMA1  and P. vivax . However, IgG antibodies against P. ovale spp. and P. malariae were detected in 19 (11%) and 13 (7%) of the participants, respectively, providing serological evidence that P. malariae and P. ovale spp., which are rarely reported, may also be endemic in Jimma.One hundred six participants (59%) were IgG seropositive for at least one of the P. malariae and P. ovale spp. are aetiologies of malaria in Ethiopia, which is critical for proper diagnosis and treatment.The findings highlight the informative value of multiplex serology and the need to confirm whether  Plasmodium species causing malaria in Ethiopia are Plasmodium falciparum, accounting for about 60% of cases, and Plasmodium vivax, which accounts for about 40% of cases, with the former being the cause of most severe clinical manifestations and deaths [Plasmodium species.In 2017, an estimated 219 million cases of malaria occurred worldwide, compared with 239 million cases in 2010 and 217 million cases in 2016 . The Fedd deaths , but mayPlasmodium ovale spp. [Plasmodium malariae [P. malariae and P. ovale spp. during the 1980s and 1990s [ale spp. , 8 and Pmalariae  have alsnd 1990s –8. HowevPlasmodium antigen targets used to assess malaria seroprevalence in the Limu Kossa District, Jimma Zone in southwest Ethiopia. Four antigen targets were specific for P. falciparum infection, including indicators of long and short-term infection. Homologs of the P. falciparum merozoite surface protein 1 (MSP-1) for P. vivax, P. ovale spp. and P. malariae were used to characterize species-specific responses [The multiplex bead assay (MBA) can provide accurate and quantitative information about the diversity of exposure to malaria parasites by measuring human antibody levels against several antigens concurrently. In this study, MBA incorporating seven esponses –13.A total of 180 dried blood spot (DBS) samples were collected in October 2016 as part of a community-based onchocerciasis survey in the villages of Arengama 1, Arengama 2 and Konche in Limu Kossa District, Jimma zone, Oromia Region, which is located 400 km in the south west of Addis Ababa. Study participants were selected through convenience sampling among village residents with age 5 years and above who provided consent and assent and participated in an interview and finger prick blood collection.P. falciparum CSP, 60 µg P. falciparum LSA1, 20 µg P. falciparum AMA-1, and 20 µg of each of the four species-specific MSP-1-19kD antigen in 50 mM 2-(N-morpholino) ethanesulfonic acid (MES), 0.85% NaCl, pH 5.0. As a control, for non-specific binding of human IgG, 15 µg GST was coupled to beads a using the same conditions.Seven malaria antigens were coupled to polystyrene beads using 1-ethyl-3-(3-dimethylaminopropyl)  carbodiimide to convert carboxyl groups on the surface of the magnetic microspheres  to reactive esters. The esters readily react with available primary amino groups on the antigens to form covalent amide bonds between antigen and microspheres. The seven recombinant antigens were expressed as Glutathione S-transferases (GST) fusion proteins and were coupled to activated beads in separate reaction tubes (12.5 million beads each) using 30 µg Escherichia coli extract), incubated for 1 h at 37 °C, and then stored at 4 °C overnight. The next day, the assay plate  was pre-wetted with 200 µl phosphate buffered saline pH 7.2 plus 0.05% Tween20 (PBS-T) and evacuated with vacuum. To prepare a bead mix, beads coupled with each of the 7 different malaria antigens were re-suspended by vortexing, and 15 µl of each  of the antigen-coupled beads were transferred to conical tubes containing 5 ml Buffer A . Of this bead mix, 50 µl were transferred to each well (~ 2500 each bead classification per well) of the assay plate using multichannel pipette and washed twice with 100 µl of PBS-T. To detect IgG reactivity, 50 µl of the prepared samples were added in duplicate wells, incubated for 90 min at room temperature on a shaker and then washed three times. After washing, 50 µl of biotinylated mouse anti-human IgG  diluted at 50 ng/well in buffer A was added to each well and incubated for 45 min at room temperature on a shaker. After three washes to remove the excess biotinylated antibodies, 50 µl 1:200 streptavidin-phycoerythrin  solution in buffer A was added per well and incubated at room temperature for 1 h on a shaker. The plate was washed once, 50 µl of buffer A were added to each well and incubated for 30 min at room temperature on a shaker. In the last step, the plate was washed and 125 µl of PBS added to each well, incubated for 2 min with shaking, and followed by immediate reading on the Bio-Rad Bio-Plex 200 System, . Data were acquired with Bio-Plex manager 6.1 Software (Bio-Rad) with a protocol to read plates at High RPI and 100 beads/well per region. The means of the median fluorescence intensity (MFI) minus background from blank wells (MFI-bg) were calculated as the final MFI-bg reading for duplicate wells for each sample. Positivity to the seven antigens for the four Plasmodium species was evaluated using previously determined cut-off values  seropositive persons to P. falciparum AMA1, 52 (29%), to P. falciparum MSP 1-19kD, 16 (9%) to, P. falciparum CSP and 4 (2%) to P. falciparum LSA1  and 11% (20) had evidence of exposure to either P. falciparum CSP and/or P. falciparum LSA1 antigens.Of the 180 enrolled study participants, 121 (67.2%) were male and 59 (32.8%) females with a mean age of 49 years (standard deviation = 14). Of all samples analysed, 106 (59%) were seropositive for IgG antibodies against at least one of the malaria antigens tested. Antibodies reactive with P. falciparum and P. vivax. Additionally 6.7% had evidence for P. falciparum and P. malariae exposure. The serologic evidence of exposure to different Plasmodium species is indicated in Table Fifteen percent (27 of 180) of the study participants had evidence for a lifetime exposure history to both Plasmodium species type and intensive implementation of malaria diagnosis and treatment accordingly is important for the elimination programmes. Malaria in Ethiopia has traditionally considered to be caused primarily by P. falciparum and P. vivax hence these species are targeted in the national malaria diagnosis and treatment strategy; there has been limited information on the importance of P. ovale spp. and P. malariae in Ethiopia. Here, a novel MBA, a bead-based technology that allows multi-analyte profiling, where microsphere beads with unique light spectra are coupled to specific antigens [Malaria elimination is the interruption of local transmission. This is achieved by detecting every infection and managing each case properly . Identifantigens , to detePlasmodium species elicit antibody responses to multiple parasite stages and the presence of these antibodies can be used as indicators of parasite exposure [Plasmodium species exposure types in Jimma area. The information will inform national malaria diagnosis and treatment strategies, for example, by including guidance for supporting diagnosis of all malaria species found in Ethiopia.Human infections with exposure . MBA usiPlasmodium antigens such as AMA1 [P. falciparum, as antibody responses to these antigens decay faster than responses to blood-stage antigens. The MSP-1 antigen, a predominant merozoite surface antigen that is present in all examined Plasmodium species, and the species-specific 19kD fragments (MSP 1-19kD) were useful in this study to determine that all four species were being transmitted. P. falciparum and P. vivax MSP-1 are used extensively as serological biomarkers, and often used for population studies [Antibodies to  as AMA1 , CSP [15 as AMA1 , and LSA as AMA1 , and wer studies , 18 and P. falciparum and P. vivax are considered the main aetiologies of malaria in Ethiopia and the national diagnostic and treatment strategies only cover these two species. However, in this survey antibody evidence in 10.6% and 7.2% of the study participants had IgG responses that were specific for P. malariae and P. ovale spp., respectively. The samples tested came from a convenience sample of the population, and thus may not be representative of the local or national population. Furthermore, because these are antibody responses, these rates may not be accurate estimates of current incidence or prevalence even in the tested population, the results clearly indicates that at some time point, P. malariae and P. ovale spp. have been present in Ethiopia. Additional studies are needed to demonstrate the distribution and prevalence for P. malariae and P. ovale spp. Even though the findings are novel for Ethiopia, both P. ovale spp. and P. malariae were similarly reported in some countries in Africa [Currently, n Africa , 20, 21.P. falciparum and P. vivax MSP 1-19kD antigens was almost equal, with 29 and 28% of the population in Limu Kossa having IgG antibodies to these two antigens, respectively. Fifteen percent of the population had IgG to both of these antigens, confirming that exposure to both species has occurred in Ethiopia.The study also showed the value of MBA serology, as it allows analysing multiple antibodies at one time and indicated antibody exposure to more than one human malaria species in 27% of the study participants. The seroprevalence of P. falciparum and P. vivax infections remain as a primary concern in Ethiopia, the detection of antibodies specific against P. malariae and P. ovale spp. is important [P. malariae and P. ovale by microscopy due to low parasitaemia and resemblance to P. vivax particularly in cases of mixed infection. Furthermore, RDTs have limited ability to detect these species [P. malariae and P. ovale cases can affect patient medical outcomes in particular and the elimination programme in general. The choice of treatment for confirmed P. ovale spp. and P. malariae infection is chloroquine at 25 mg/kg for 3 days [P. ovale spp. and P. malariae are also susceptible to artemisinin-based combination therapy (ACT). Mixed infection of P. falciparum with P. ovale spp. or P. malariae can successfully be treated with ACT drugs [P. ovale spp. and to P. malariae is not studied in Ethiopia.While mportant  because mportant . It is c species , which ar 3 days . P. ovalCT drugs . HoweverP. malariae and P. ovale spp. in the treatment algorithm; for example, microscopy might be recommended in cases where RDTs are negative but symptoms persist. Future studies with larger populations that include microscopy or DNA based methods are needed to unequivocally demonstrate the presence of active P. malariae and P. ovale infections. These findings are important for the Ethiopian programme in their quest to eliminate human malaria.Multiple serological data sets generated using MBA, which might not have otherwise been collected as part of surveillance, is helpful for program decision-making. It is important for the national treatment guidelines to explicitly include The study participants are residents of Konche, Arengama 1 and Arengama 2 villages of Limu Kossa Districts selected based on convenient sampling. Therefore, the result may not be generalizable to the entire district population.P. falciparum and P. vivax having the highest exposure rates. It also revealed that P. malariae and P. ovale spp. were endemic at low levels in the Limu Kossa district of Ethiopia. Therefore, there is a need for robust studies to identify the distribution of Plasmodium species present in Ethiopia. Those findings may impact the national strategies for elimination, including clinical diagnosis and treatment, as well as the priorities for programmatic research.The study showed well-defined evidence of malaria infections among the study participants, with


Malaria is still a significant public health burden in the tropics. Infection with malaria causing parasites results in a wide range of clinical disease presentations, from severe to uncomplicated or mild, and in the poorly understood asymptomatic infections. The complexity of asymptomatic infections is due to the intricate interplay between factors derived from the human host, parasite, and environment. Asymptomatic infections often go undetected and provide a silent natural reservoir that sustains malaria transmission. This creates a major obstacle for malaria control and elimination efforts. Numerous studies have tried to characterize asymptomatic infections, unanimously revealing that host immunity is the underlying factor in the maintenance of these infections and in the risk of developing febrile malaria infections. An in-depth understanding of how host immunity and parasite factors interact to cause malaria disease tolerance is thus required. This review primarily focuses on understanding anti-inflammatory and pro-inflammatory responses to asymptomatic infections in malaria endemic areas, to present the view that it is potentially the shift in host immunity toward an anti-inflammatory profile that maintains asymptomatic infections after multiple exposures to malaria. Conversely, symptomatic infections are skewed toward a pro-inflammatory immune profile. Moreover, we propose that these infections can be better interrogated using next generation sequencing technologies, in particular RNA sequencing (RNA-seq), to investigate the immune system using the transcriptome sampled during a clearly defined asymptomatic infection. Plasmodium that affect humans. P. falciparum is globally the most deadly and is the most prevalent parasite in Africa  and every 10-fold increase in parasite density have been associated with an increased risk of developing febrile malaria, probably due to the underlying reduced host immunity  1 genotypes have been associated with different clinical manifestations of malaria. In Nigerian children, msp1 K1 and MAD20 alleles were associated with asymptomatic malaria and a minimal risk of becoming febrile    and rece (GLuRP) . This te (GLuRP) , 57, is m clones . This tem clones . With inP. falciparum infections over time  proteins such as P. falciparum erythrocyte membrane proteins (PfEMP) and repetitive interspersed family (RIFIN)  . A more  (RIFIN) . Chromat (RIFIN) . It is h (RIFIN) . An oppo (RIFIN) . Pre- an (RIFIN) . This wachildren . The reschildren . This tenscripts , 109. ThAsymptomatic malaria appears to be driven by host immunity, such that following multiple exposures to symptomatic infections an individual's immune system is potentially primed to control symptoms. Initially, at a young age, individuals in malaria endemic areas are likely to have high pro-inflammatory responses to control infection, which also results in the clinical manifestation of disease. With age, due to multiple exposure to infection, the immune system is perhaps trained and the balance shifts to a predominance of anti-inflammatory responses to control the infection with no clinical signs of disease, to a point that infection is tolerated and parasitemia is maintained. Since immunity to malaria is not sterile, there appears to be a trade-off between anti-parasite and anti-disease immunity, with the latter dominating in asymptomatic infections, where the focus is controlling disease rather than clearing parasites. This compromise in host immunity to control disease and not clear parasites is likely driven by the anti-inflammatory immune response. A transcriptomic approach to analyzing the cells and cytokines involved in the process would provide the necessary insight to unraveling the role of both anti- and pro-inflammatory responses to asymptomatic infections.P. falciparum single cell RNA-seq studies of individual immune cells and parasites with good criteria for defining asymptomatic infections hold the key to understanding these chronic and debilitating infections as well as host pathogen interactions in general.Though there is still a gap in our understanding of how the parasite remains in an asymptomatic state in individuals from malaria endemic areas, various studies have implicated genes responsible for gene regulation and chromatin remodeling and it is still unclear how host immunity is involved. High-throughput studies are needed to understand differential gene expression in immune cells that have shown differential activity in asymptomatic vs. symptomatic or uninfected states . RecentlFurthermore, there is a need for a consensus on how asymptomatic infections are defined. Longitudinal cohorts or follow ups over a period of 6 weeks, 4 weeks prior to the asymptomatic case and 2 weeks following the case, with no history or evidence of the individual having taken antimalarials and no evidence of fever 48 h before and after the case, are likely to be the most feasible approach to defining an asymptomatic infection. More sensitive tools for defining the presence of parasitemia to minimize missing sub-patent infections requires methods such as PCR, LAMP, or the newly described hemozoin sensing assay . With a KK and LO-O conceived, drafted, and reviewed the manuscript. KW drafted and reviewed the 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.


The patient was discharged home and, upon clinic follow-up, did not have any further complications associated with either her disease or therapy. A review on the treatment of severe malaria in all trimesters of pregnancy supports the WHO recommendation for intravenous artesunate as the drug of choice. This case illustrates the importance of recognizing malaria in pregnant women from endemic countries and shows that artesunate compounds can be used safely in pregnancy, particularly with high parasitemia.Human malaria has arguably affected more of human history than any other pathogen. Pregnant women have a higher risk of developing severe malaria as well as the risk of severe complications. We present a case of severe malaria in a pregnant patient from sub-Saharan Africa who was treated successfully with artesunate. A 28-year-old Nigerian woman with a 20-week intrauterine pregnancy presented with a five-day history of fever and diffuse joint pains. Evaluation of peripheral thin blood smear demonstrated a parasitemia of 9.8%. The patient was admitted to the intensive care unit, and oral clindamycin/quinine was initiated until intravenous artesunate was obtained. The patient completed four doses of IV artesunate, and after the 4 Plasmodium cycles through female Anopheles mosquitos, which then feed on humans and spread the infection, and can in some individuals occur as an asymptomatic infection [Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale have more of an indolent course of infection, Plasmodium falciparum has a more fulminant pathogenic response and accounts for the majority of deaths from human malaria, especially in those who are less than five years of age in Africa [Human malaria, which had over 200 million documented infected cases in 2016, has arguably affected more of human history than any other pathogen. The genus n Africa . Pregnann Africa . In 2013n Africa . In the n Africa . Here, wStreptococcus pyogenes throat infection and discharged with cephalexin for treatment. Her symptoms worsened, and she returned three days later to the ED for evaluation. On physical exam, she was afebrile, tachycardic, tachypneic, and hypotensive. Pertinent initial labs demonstrated hypoproliferative anemia , thrombocytopenia , D-dimer of 10.04 μg/mL, and LDH 341 U/L. Total bilirubin was 1.2 mg/dl. Complete blood count led to the evaluation of peripheral smear in which numerous inclusion bodies were noted  with a five-day history of fever, diffuse joint pain, night sweats, vomiting, and malaise. She had arrived in the US to visit her family about nine days before her admission. She was initially diagnosed with cystitis and re noted . On furtd smears  demonstral smear . The patP. falciparum malaria in both the second and third trimesters the same as for nonpregnant adults. A review on the treatment of severe malaria in all trimesters of pregnancy supports the WHO recommendation for intravenous artesunate as the drug of choice [It should be noted that pregnant women in Nigeria (and other parts of sub-Saharan Africa) are usually given intermittent preventive treatment for malaria. Our patient was unable to tolerate prophylactic trimethoprim/sulfamethoxazole secondary to chronic emesis; this is of great importance as pregnant women are noted to have an increase in gametocytemia (mainly in the placenta) and thus have an increased risk of transmitting malaria to others who live in the same vicinity due to this increased gametocyte reservoir . The WHOf choice .P. falciparum causes intrauterine growth retardation, premature delivery, and low birth weight and thus increases newborn mortality; it also increases maternal morbidity and mortality. Peripheral smears may be negative due to sequestration within the placenta. Selective accumulation of infected erythrocytes in the placenta involves their interaction with syncytiotrophoblastic chondratin sulfate A. In regard to complete blood counts, severe malaria is known to cause both anemia and thrombocytopenia [Malaria due to ytopenia . Malariaytopenia . Thromboytopenia . Thromboytopenia , 11.The CDC guidelines for treatment of severe malaria include parenteral quinidine gluconate plus either doxycycline, tetracycline, or clindamycin. Artesunate-based parenteral therapy followed by an oral course  can also be used . In the This case illustrates the importance of recognizing malaria in pregnant women from endemic countries and that artesunate compounds can be used safely in pregnancy particularly in individuals with high parasitemia.


P. falciparum and non-falciparum malaria.The aim was to assess factors affecting disease severity in imported We reviewed medical records from 2793/3260 (85.7%) of all episodes notified in Sweden between 1995 and 2015 and performed multivariable logistic regression.P. falciparum (9.4%), P. vivax (7.7%), P. ovale (5.3%), P. malariae (3.3%), and mixed P. falciparum episodes (21.1%). Factors associated with severe P. falciparum malaria were age <5 years and >40 years, origin in nonendemic country, pregnancy, HIV, region of diagnosis, and health care delay. Moreover, oral treatment of P. falciparum episodes with parasitemia ≥2% without severe signs at presentation was associated with progress to severe malaria with selected criteria. In non-falciparum, age >60 years, health care delay and endemic origin were identified as risk factors for severe disease. Among patients originating in endemic countries, a higher risk for severe malaria, both P. falciparum and non-falciparum, was observed among newly arrived migrants.Severe malaria according to WHO 2015 criteria was found in P. falciparum and non-falciparum episodes. Current WHO criteria for severe malaria may need optimization to better guide the management of malaria of different species in travelers and migrants in nonendemic areas.Severe malaria was observed in  P. falciparum and non-falciparum infections that need to be considered in the management of malaria in travelers and migrants.We investigated risk factors for severe malaria in approximately 2700 cases of imported malaria. We identified variables associated with severity of  Plasmodium falciparum is the leading cause of severe malaria , witWHO 2000 , 15. HeaΧ2 test, or the Fisher exact test when the latter was computationally possible. The Mann-Whitney U test was used for comparing the distribution of numeric variables between 2 groups and the Kruskal-Wallis test among multiple groups. Univariable logistic regression was used to test potential risk factors for severe malaria, and variables with a P value less than .2 were included in the multivariable logistic regression model. The variables with a P values less than .05 were kept in the multivariable model. The Wald test was used to test the regression coefficients. As individual patients could appear more than once in the dataset, the standard errors were estimated with the cluster-robust sandwich estimator. P values less than .05 were considered statistically significant.The analyses were performed with Stata version 14.2 . In the descriptive analyses, the categorical variables were summarized by proportions, and the numerical variables were summarized by medians and interquartile ranges (IQR). In comparative analyses, categorical variables were analyzed using the Pearson The study was approved by the Ethical Review Board in Stockholm, Sweden .During 1995 to 2015, 3099 malaria cases were reported to the Public Health Agency of Sweden; additionally, 161 unreported episodes were identified through hospital data. Medical records were available for 2793 (85.7%) episodes, with missing records from all regions. After exclusion of relapse and recrudescence episodes (n = 116) and reported episodes without microbiology confirmed parasites or only gametocytes without symptoms after recent treatment abroad (n = 24), 2653 episodes were included in the analysis .P. vivax in newly arrived Eritrean migrants  didP. falciparum, although severe forms are described in all species [P. ovale and P. malariae are more rare [P. falciparum. Nonetheless, the proportion of severe P. vivax fulfilling the WHO criteria but also criteria for poor prognosis was notable, especially among newly arrived Eritreans arriving 2014 and 2015. A smaller subset of the severe episodes was caused by P. ovale and P. malariae (using the severe P. vivax criteria), some also fulfilled criteria prognostic of unfavorable outcome and were admitted to ICU, of which there have been only scarce previous reports [Severe malaria is well recognized to be caused predominantly by  species , 20, 21.ore rare , 22, andP. falciparum infections than in P. falciparum monoinfections. The effect of simultaneous presence of another species of Plasmodium is not fully elucidated [Furthermore, severe malaria was more common in mixed ucidated ; here, oP. vivax, both multiorgan failure and death have been reported in endemic areas [P. vivax episodes in Sweden, we could not see the same level of severity and most episodes fulfilled only 1 criterion for severe P. vivax malaria, dominated by severe anemia or hyperbilirubinemia. Severe anemia could be debilitating and might require blood transfusions. Hyperbilirubinemia has been described as a common feature of P. falciparum and P. vivax malaria, appearing as a sign of erythrocyte destruction or liver dysfunction [In severe ic areas , 24. Amofunction . Howeverfunction , 26, 27.function , 15 wereP. falciparum malaria. The overall malaria mortality in Sweden is, however, not known and our data only reflect the diagnosed cases. A study in the United States suggested that 15% of the lethal malaria cases were diagnosed at autopsy [The case fatality rate in our study corresponds to previous studies in nonendemic settings , 20, and autopsy , 28.P. falciparum, health care delay was associated with severe malaria, criteria prognostic of unfavorable outcome, and ICU admission. Even in non-falciparum episodes, health care delay of 3 days or more affected disease severity significantly. Health care delay was most common in young children and patients born in nonendemic countries. This further emphasizes the necessity to enquire about travel history and promptly investigate for malaria in febrile patients returning from malaria-endemic countries, which is a challenge in a country such as Sweden where malaria is a relatively rare disease.Delayed diagnosis and antimalarial treatment are well-known risk factors for severe malaria and death , 10. OurP. falciparum malaria. This effect remained after adjusting for age, patient origin, health care delay, and even patient delay. Because most severe episodes fulfilled WHO criteria already at hospital admission, differences in the management of malaria in hospitals is unlikely to explain this effect. Rather, some differences in health care delay not mentioned in the medical chart may have contributed to the higher morbidity.Health care presentation outside the 2 main cities in Sweden, Stockholm and Gothenburg, was an independent risk factor for severe P. falciparum progressing to severe malaria during oral treatment had a higher parasitemia compared to episodes without deterioration. Even though the number of episodes progressing to severe malaria is small, our findings suggest that high parasitemia is a warning sign, also acknowledged in the previous WHO 2010 definition of severe malaria where parasitemia >2% was specified as a criterion for severe malaria in nonimmune individuals and >5% in the expected immune [Episodes of d immune . In the d immune , 31 and d immune .P. falciparum cases, severe malaria and death is generally recognized to be less frequent in this group [P. falciparum among African immigrants increased with time of residency in Sweden, confirming our previous findings within a subset of Stockholm patients [P. falciparum and non-falciparum episodes in newly arrived migrants from sub-Saharan Africa were even higher. This was most pronounced in non-falciparum, where over 50% of the severe episodes occurred in newly arrived migrants, possibly caused by prolonged symptom duration or insufficient treatment for prevention of relapse infections as well as factors associated with migration, including poor health care, malnutrition, and stress [Although patients born in malaria-endemic countries account for a proportionally high number of the imported is group , 33–35, is group . Howeveris group , 38. Herpatients . Interesd stress , 39–41. P. falciparum may have contributed to the high rates of severe malaria observed in non-falciparum infections [The strength of this study is the large dataset and nationwide coverage, containing detailed clinical data from the majority of malaria episodes reported to the national surveillance system during 2 decades. The limitations are inherent in the retrospective design and data relying on medical records with varying level of detail. Moreover, neither single nor mixed infections were systematically confirmed by PCR, thus underdiagnosis of mixed infections with fections .Our study contributes to the understanding of severity of imported malaria in different species and patient categories. Further studies are needed to establish effective severity criteria, adjusted to different species, populations, and settings that in turn could contribute to an improved management of malaria. Moreover, the findings of increased risk of severe malaria irrespective of species in newly arrived migrants highlights the need for increased attention to malaria in migrants.P. falciparum and non-falciparum species. Moreover, we identified newly arrived migrants as a new risk group for severe malaria. The WHO criteria for severe P. falciparum and non-falciparum malaria did not effectively reflect disease severity in this nonendemic setting, and improved criteria are needed to better guide the management of imported malaria in travelers and migrants.In this nationwide clinical assessment of imported malaria in Sweden over 2 decades, we observed severe malaria, also with the most severe signs, caused by both The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiz292_suppl_Supplementary_Figure_1aClick here for additional data file.jiz292_suppl_Supplementary_Figure_1bClick here for additional data file.jiz292_suppl_Supplementary_Figure_1cClick here for additional data file.jiz292_suppl_supplementary_Table_1Click here for additional data file.jiz292_suppl_Supplementary_Figure_LegendClick here for additional data file.


Plasmodium parasites. Plasmodium falciparum merozoites, the invasive form of the blood-stage parasite, invade human erythrocytes in a complex but rapid process. This multi-step progression involves interactions between parasite and human host proteins. Here we show that antibodies against a vaccine antigen, PfGAMA, co-immunoprecipitate with PfMSP10. This interaction was validated as direct by surface plasmon resonance analysis. We then demonstrate that antibodies against PfMSP10 have growth inhibitory activity against cultured parasites, with the region PfMSP10 R1 that is critical for its interaction with PfGAMA being the key target. We also observe that the PfMSP10 R1 region is highly conserved among African field isolates. Lastly, we show that high levels of antibodies against PfMSP10 R1 associate with reduced risk to clinical malaria in children resident in a malaria endemic region in northern Uganda. Put together, these findings provide for the first time the functional context of the important role of PfGAMA/PfMSP10 interaction in erythrocyte invasion and unveil a novel asexual blood-stage malaria vaccine target for attenuating P. falciparum merozoite invasion.Clinical manifestation of malaria is mainly due to intra-erythrocytic development of  P. falciparum malaria is a serious global health and economic burden, with an estimated 219 million cases and 435,000 related deaths in 2017 alone  of a malaria endemic region in Northern Uganda who were enrolled and prospectively monitored for symptomatic malaria episodes for a year. The study, which has been extensively reported . All efforts were made to minimize animal suffering.25-G715); ecto-PfMSP10 (D29-Q507); PfMSP10 region (R) 1, amino acid D29-N188; R2, N110-E268; R3, I189-V348; R4, E269-R428; and R5, N349-Q507. Briefly, wheat codon optimized PfGAMA  and dissociation (kd) rate constants were obtained by global fitting in a 1:1 binding model equation. Measurement conditions were optimized so that the contribution of mass transport to the observed KD values was negligible.All SPR experiments were performed using a Biacore X100 instrument  according to the manufacturer's instructions . After sTotal rabbit IgGs to PfMSP10, PfMSP10 truncates, EBA175, and His-GST for GIA were purified from respective rabbit antisera with HiTrap protein G-Sepharose columns  according to the manufacturer's protocol. GIA was performed as per an established protocol  and those below (n = 33). Kaplan–Meier plots were used to generate curves for time-to-first clinical episode. Analyses were performed either by GraphPad Prism  or R software .The Kruskal–Wallis test was used to test the significance of differences in EBA values between groups followed by the Bonferroni correction test. Antibody associations with age were examined by Kruskal–Wallis with the Bonferroni–Dunn P. falciparum utilizes several self and host proteins to recognize and invade erythrocytes  inhibited growth of the 3D7 parasite strain by 26 ± 2% (mean ± SE), significantly higher than that of the negative control  induced a GIA activity of 11 ± 3% which was significantly higher than that of the negative control, anti-His-GST antibodies , suggesting that the region essential for invasion of erythrocyte is different from the region involved in erythrocyte binding.Secondly, we sought to investigate if PfMSP10 interacts with the erythrocyte surface. Using ecto-PfMSP10 as well as the five regions, we performed erythrocyte binding assays (EBA). We observed that recombinant ecto-PfMSP10 as well as PfMSP10 R2 bound to the erythrocyte surface . The erymsp10 sequences of field isolates from different sites in West and East Africa (n = 110) deposited in PlasmoDB (http://plasmodb.org/), we calculated nucleotide diversity (π) and Tajima's D, indicative of diversifying selection. While the full length pfmsp10 is moderately conserved, both metrics did not show any peaks in nucleotide sequence diversity suggesting an absence of balancing selection of R1  protein.Thirdly, we sought to determine the genetic polymorphism of this gene in malaria endemic populations. Based on the These results suggest that the N-terminal region of PfMSP10 is important for erythrocyte invasion and development.in vitro, and we recently observed that PfMSP10 is recognized by sera from African children . The breadth of antibody responses appeared to differ according to PfMSP10 region; specifically, regions in close proximity to the less conserved C-terminal (R4 and 5) had higher seropositivity (>70%) than the relatively more conserved N-terminal R1 and R2 regions which had seropositivity ranging between 22 and 63% in different age groups  (29-N188), corresponding to the PfGAMA binding region, is a key target of anti-PfMSP10 antibodies with GIA activity, and protective antibodies in natural P. falciparum infection, making it an ideal target for malaria vaccine studies.Since anti-PfMSP10 antibodies inhibited merozoite invasion children . For alle groups . Consiste groups . Furtherf blood) . HoweverP. falciparum merozoites is an intricate and rapid process that is mediated by numerous parasite and host proteins . More specifically, the KD value of the N-terminal PfGAMA/PfMSP10 R1 was comparable to that of ecto-PfMSP10 (KD = 6.5 × 10−7 M), suggesting that PfMSP10 R1 is the key interacting region. Consistently, IFA analysis of PfMSP10 R1 and PfGAMA validated that both proteins co-localize on the surface of free merozoites. Like other GPI anchored proteins, the PfGAMA/PfMSP10 interaction could be enriched by lipid raft-like membrane on the merozoite surface   . In our 0 mg/ml) . Antibod0 mg/ml) , and thu0 mg/ml) . However0 mg/ml) . TherefoConsistent with previous studies , we obseLike other known merozoite surface proteins, PfMSP10 may be a target of subtilisin-like serine protease (SUB1) mediated proteolytic processing to produce the final functional fragments , 31. Basmsp10 gene, especially R1, is conserved among field isolates and is comparable to adsl, a highly conserved housekeeping gene , a short, conserved and naturally immunogenic region corresponding to the PfGAMA binding region, as an important component of the merozoite invasion machinery. Although additional supportive data will be required, based on this study, the region warrants further evaluation for possible inclusion in multi-component blood-stage malaria vaccine strategies.In conclusion, targeting relatively small parts of a protein presents an opportunity to develop an optimal vaccine against malaria. These interacting regions as in the case of PfGAMA/PfMSP10 R1 are more likely conserved to preserve critical protein functions. The data presented here demonstrate that PfMSP10 R1 , Lacor Hospital (LHIREC 023/09/13), and Uganda National Council for Science and Technology . Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. The animal study was reviewed and approved by Kitayama Labes Co., Ltd. . All animal immunizations were commercially conducted at Kitayama Labes Co., Ltd. .TT and ET conceived and designed experiments. HN, BK, KJ, MM, TA, NP, TE, and TH conducted experiments. HN, BK, TT, and ET analyzed the data. BK, NP, TT, and ET wrote the manuscript. All authors discussed and edited the 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.


The World Health Organization estimates that about 25 million pregnant mothers are currently at risk for malaria, and that malaria accounts for over 10,000 maternal and 200,000 neonatal deaths per year. The current hypothesis of early life programming supports the premise that many developmental delay and disorders may have their origin In-utero. Therefore, the current study aimed at evaluating the possible impact of experimental malaria exposure In-utero on neurobehavioral profile in mice offspring.5 infected red blood cells. Pregnant mice (both infected and uninfected) were allowed to deliver and the offspring were monitored up to postnatal day 42 when anxiety-like, Obsessive-Compulsive Disorder (OCD), and locomotor activity were evaluated using elevated plus maze, marble burying, and Open Field Test, respectively.Pregnant mice were intraperitoneally infected on gestational day 13 with 1.02×10The current study showed that maternal infection with Plasmodium berghei resulted in an interesting behavior in offspring characterized by increased anxiety-like and OCD behaviors. Locomotor activity was however not affected.It may be concluded that In-utero exposure to experimental malaria in mice causes behavioral changes. In-Utero plasmodium berghei infection induces anxiety-like behavior in the offspring.In-Utero plasmodium berghei infection induces obsessive-compulsive disorder in the offspring.In-Utero plasmodium berghei infection does not have any effect on locomotor activity in the offspring.The World Health Organization estimates that about 25 million pregnant mothers are currently at risk of inflicting malaria. Also, malaria accounts for over 10000 maternal and 200000 neonatal deaths per year mainly in regions where malaria infection is very high. First-time pregnant women could develop the clinical signs of malaria whereas females who had given birth before with greater immunity may be asymptomatic and therefore not seek treatment, leading to a greater placental parasite load. It could result in significant problems for the developing fetus, especially the brain of the fetus. Therefore, we used experimental malaria in pregnant animals to see how malaria will affect the behavior of offspring. We found out that baby of pregnant animals that had malaria exposure in the uterus, presented with different behavioral problems like anxiety, obsessive-compulsive disorder when the babies were 42 days old. These behavioral changes probably implicate that asymptomatic pregnant mother may potentially expose her fetus to the chronic disease.1.The World Health Organization reported that about roughly 25 million pregnant mothers are at risk for malaria, which accounts for more than 10,000 maternal and 200,000 neonatal deaths each year . These fExpecting mothers are three times more at risk for acute disease due to malaria infection in comparison with their non-expecting counterparts, and also have a mortality rate of nearly 50% due to acute disease . It is hInterestingly, extreme level of placental infection is observed in gravid females with the maximum degree of immunity resulting in moderate maternal manifestation as well as an inordinate growth in fetal complications . TherefoLBW, as a consequence of preterm delivery or fetal growth restriction, is associated with high risk of developmental setback in children . In addiRecent studies thus suggest variables that interfere with the closely controlled In-utero environment may alter the normal nervous system developmental processes . CurrentEpidemiological studies also associate maternal infections with greater threat of developmental setback, schizophrenia, autism, and periventricular leukomalacia; a major cause of cognitive impairment in LBW babies . Murine 2.2.1.The BALB/c mice were obtained from National Institute of Health, Pakistan, and maintained in conventional housing at COMSATS Institute of Information Technology. Infection experiments were conducted in adult females with 10–12 weeks of age. Plasmodium berghei was a gift from Dr. Aina . The iRBC used in the experimental infections were obtained from in vivo passage in BALB/c mice, when the percentage of iRBC was roughly 10%. Thin blood smears were collected daily and stained with Giemsa to monitor parasitemia. All animals were fed with normal rat chow and all procedures conformed to the national guidelines on animal experimentation and welfare and were approved by Comsats Institute of Information Technology.2.2.Presence of vaginal plug and increase of body weight were collectively utilized to determine the time of pregnancy, according to Freyre et al.  two fema2.3.5 iRBC, and parasitemia was recorded every day. The model employed in the current study was predicated on a formerly supported murine model of Malaria in Pregnancy (MIP), which reproduced key pathogenic factors of MIP  were permitted to deliver and their offspring were monitored up to the Postnatal Day (PND) 42. Since P. berghei is deadly in BALB/c mice, foster mothers were utilized in the newborn post-natal follow-up studies. Consequently, progenies both from the infected mothers and control ones were additionally transferred to the foster mothers to prevent weight bias due to differential maternal nourishment. The progenies were weighed each day.Pregnant mice were Intraperitoneally (IP) inoculated on GD13 with 1.02×10s of MIP . A lower2.4.2.4.1.Each mouse was separately positioned in the middle of a white acrylic cage (30×30×15 cm) and was allowed to explore it for 30 minutes. In this time period, total distance travelled, number of rearing , and episodes of grooming were recorded. At the conclusion of testing, the amount of fecal boli was determined and the cage was cleaned using a 10% ethanol solution. In this protocol, the locomotor activity was signified by total distance travelled in the cage, while the vertical display was assigned by the number of rearing. In regards to defecation in male mouse, under certain conditions, this parameter seemed to signify not an emotional reaction but a kind of scent marking .2.4.2.Both in laboratory and natural environments, mice use accessible bedding material to cover obnoxious sources of distress existing in their home surroundings . Within 2.4.3.Elevated plus maze reflects a conflict between the rodents' preference for protected areas and their innate motivation to explore novel environments . An appaElevated plus maze reflects a battle between the rodents` desire for secure areas and their inborn motivation to search novel surroundings . The app2.5.All results are presented as a Means±Standard Error of Mean (SEM). Intergroup comparisons were evaluated with the Student unpaired-test. Differences were considered statistically significant when P<0.05. GraphPad Prism 5  was used for all statistical analyses.3.3.1.It was observed that exposure to P. beghei during pregnancy from GD13 led to gradual increase in the percentage of iRBCs as shown in 3.2.In this test, no significant difference was observed in locomotor (P=0.6803) and vertical activities (P=0.6671) between the control and experimental groups. Likewise, there was also no difference in episodes of grooming (P=0.4007). Nevertheless, the mice of P. berghei-exposed group had higher number of fecal pellets , and in Elevated Plus Maze Test (reduced percentage of time in open arms and increased time in closed arm). It is logical that the above observations could be a result of distortion of the neuromodulatory systems within the cerebral cortex known to influence behavior. An obvious anxiogenic effect of In-utero malaria infection is that it provides the opportunity to assume that it is associated with a rapid decrease in the amount of serotonin (5-HT) in different cerebral structures. It was previously observed that the case of the cerebral serotonergic system firmly corresponded with the level of stress in rodents and humans .Grooming activity non-significantly reduced in the experimental group compared with that of the control. Previous studies showed that selective agonists of dopamine D1 and D5 receptors evoked the escalation of grooming behavior, while D2 receptor agonists considerably curtailed this type of behavioral activity . Thus, aThe current study results also showed that experimental malaria infection in mice offspring resulted in increased marble burying, which is a test to evaluate OCD in rodents. It might also reflect the elements of defensive behavior; rodents try to stop predators such as snakes from penetrating their burrows by digging soil and kicking it toward the intruder. Defensive burying is a renowned paradigm to measure anxiety . The burTherefore, the current study results demonstrated that experimental In-utero malaria infection possessed a significant modulatory behavioral potential. Experimental In-utero malaria infection resulted in anxiogenic, escape, and OCD behaviors in mice offspring. These behavioral shifts probably show that the implications of asymptomatic pregnant mother may potentially expose her fetus to the chronic disease. It is therefore imperative that all pregnant mothers in malaria endemic regions be screened for malaria parasite and given safe anti-malaria drugs such as prophylaxis. At the same time, the probable mechanisms underlying how In-utero malaria infection affects the cerebral modulatory systems  may need specific thorough investigations.The study protocol was approved by Ethics Committee of Department of Pharmacy, CIIT, Abbottabad Campus .


Access to long-lasting insecticidal nets (LLINs) has increased and malaria has decreased globally, but malaria transmission remains high in parts of sub-Saharan Africa and insecticide resistance threatens current progress. Eave tubes are a new tool for the targeted delivery of insecticides against mosquitoes attempting to enter houses. The primary objective of this trial is to test whether screening plus eave tubes (SET) provides protection against malaria, on top of universal coverage with LLINs in an area of intense pyrethroid resistance. The trial will also assess acceptability and cost-effectiveness of the intervention.A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of SET on clinical malaria incidence in children living in central Côte d’Ivoire. Forty villages will be selected based on population size and the proportion of houses suitable for modification with SET. Using restricted randomization, half the villages will be assigned to the treatment arm (SET + LLINs) and the remainder will be assigned to the control arm (LLINs only). In both arms, LLINs will be distributed and in the treatment arm, householders will be offered SET.Anopheles mosquitoes will be examined for parity status and tested for sporozoite infection.Fifty children aged six months to eight years old will be enrolled from randomly selected households in each of the 40 villages. Cohorts will be cleared of malaria parasites at the start of the study and one year after recruitment, and will be monitored for clinical malaria case incidence by active case detection over two years. Mosquito densities will be assessed using CDC light traps and human landing catches and a subset of Acceptability of SET will be monitored using surveys and focus groups. Cost-effectiveness analysis will measure the incremental cost per case averted and per disability-adjusted life year (DALY) averted of adding SET to LLINs. Economic and financial costs will be estimated from societal and provider perspective using standard economic evaluation methods.This study will be the first evaluation of the epidemiological impact of SET. Trial findings will show whether SET is a viable, cost-effective technology for malaria control in Côte d’Ivoire and possibly elsewhere.ISRCTN18145556, registered on 01 February 2017 – retrospectively registered. The incidence of malaria worldwide declined by 41% between 2000 and 2015 –8.Anopheles gambiae mosquitoes) captured indoors, and a 7% reduction in anemia in children living in houses with screening and closed eaves, compared to unmodified houses [Although house modifications to prevent mosquito entry are not a new malaria control tool , 10, thed houses . A recend houses .An. gambiae in sub-Saharan Africa [Open eaves, which are found in many traditional African houses, are a particularly important source of attractive cues for malaria mosquitoes, and represent the key entry point for n Africa –18. It wn Africa –22. EaveThe eave tube consists of a section of pipe fitted into a closed eave, with a screened insert that is placed inside the tube. The insert is treated with an electrostatic coating, which holds powder formulations of insecticides. This insecticide delivery method can be highly effective, even against resistant mosquitoes . KillingThe aim of the cluster randomized controlled trial (CRT) presented here is to determine whether screening plus eave tubes, the SET intervention, provides protection against malaria, on top of the current best practice of universal coverage of LLINs (one bednet per two people). LLINs are the primary tool used for malaria vector control globally  and therTo assess whether SET (screening plus eave tubes) and LLINs  reduces the incidence of clinical malaria in children between the ages of six months and ten years, compared to universal coverage of LLINs alone.Assess whether SET + LLINs reduces the incidence of malaria infection in children between the ages of six months and ten years, compared to LLINs alone.Assess whether SET + LLINs reduces the prevalence of anemia in children between the ages of six months and five years, compared to LLINs alone.Assess whether SET + LLINs is associated with a rise in symptoms of respiratory infections in children between the ages of six months and ten years, compared to LLINs alone.Assess the impact of SET + LLINs on indoor and outdoor densities of malaria vectors, compared to LLINs alone.Assess the impact of SET + LLINs on entomological inoculation rate , compared to LLINs alone.Assess the impact of SET + LLINs on parity in the malaria vector population, compared to LLINs alone.Monitor levels of insecticide resistance in villages that receive SET + LLINs, compared to villages that receive LLINs alone.Assess end users’ perceptions of and willingness to adopt SET in the treatment and control arms, and predictors of those perceptions and actions.Assess malaria prevention behavior in households with SET versus LLINs alone, as well as within the treatment and control arms.Assess willingness to maintain SET and identified key factors that influence willingness to maintain.Measure attitudes, emotions, knowledge, and beliefs relating to malaria and malaria control interventions, including SET and LLINs.Measure the incremental cost-effectiveness of adding SET to LLINs from the societal and provider perspective under trial conditions.Establish the relative contribution to costs of the distinct programmatic elements , and identify the inputs that contribute the most to overall costs.Estimate the potential cost of providing SET at larger scale over an extended period of 3 and 5 years under operational scenarios.The study site is situated in the Gbêkê region in central Côte d’Ivoire. It is highly malaria endemic with year-round transmission, peaking during the rainy season (May through October) –27. FortCommunity consent to participate in the study will be obtained through meetings with village leaders and inhabitants. A census will be carried out prior to the start of the study. LLIN distribution numbers will be calculated based on data from the census. Consent for the house modifications will be obtained from individual homeowners during door-to-door visits, following the randomization of villages to trial arms.Each candidate village is associated with a health center that is responsible for providing medical care to a group of villages. In cases where the health center is not situated in the village, a Community Health Worker (CHW) is charged with providing routine care for uncomplicated malaria, diarrhea, and mild respiratory infections in children. Study team members will visit each of the health centers associated with a trial village, to inform the health center officials of the trial activities. CHWs in the trial villages will be trained in the trial procedures.Eligible children will be randomly selected for recruitment into the active case detection (ACD) cohort in each village. Recruitment will be limited to children aged eight or younger, to avoid children aging out during the two year monitoring period. Study team members will seek informed consent from the parents or caregivers of the children before enrolling the child in the cohort. Children aged eight will be asked to assent, in addition to parental consent. Children in the treatment arm will be recruited for the cohort irrespective of whether they live in a house that has been modified with SET to avoid bias related to house structure.The study design is summarized in Fig. During the first visit and one year after recruitment, all children in the ACD cohort will be cleared of malarial parasites. Children in the cohort will be monitored for two years to determine clinical malaria incidence, malaria infection incidence, and prevalence of acute respiratory infections. Twice a year, at the start and end of the rainy season, all children in the cohort aged five years or younger will be tested for anemia. If the child leaves the village permanently or the parents withdraw their consent, another child will be recruited into the cohort, preferably of similar age and in the same household.Indoor mosquito density will be monitored every other month in 12 randomly selected houses in each village using CDC light traps. Indoor and outdoor mosquito density and exposure to infectious bites will be assessed using human landing catches (HLC) every month in four randomly selected houses per village. Four villages in each study arm will be selected for insecticide resistance monitoring. The villages will be selected based on the presence of permanent breeding sites. Mosquitoes from these eight villages will be sampled once a year over four years .Questionnaires will be administered to consenting participants in six study villages  at the start and end of the monitoring period, and in two study villages  at the midpoint of the monitoring period. Ethnographic studies will take place in three of the villages in the trial arm during the installation of the SET intervention. Focus groups also will be conducted midway through the trial in two villages from the treatment arm and one village from the control arm. Data from the social science activities will inform the economic evaluation, but no primary cost data will be collected from households.Allocation of villages to study arms will be restricted to randomizations that limit the difference between the mean values in each study arm of the following: malaria prevalence; socioeconomic status; village size, and proportion of houses suitable for SET. Data on these variables by cluster will be obtained through a pre-trial cross-sectional survey. Data on any additional potentially confounding ecological factors not included in the randomization will be collected and corrected for in the analysis.Given the nature of the intervention, it is impossible to conduct this study in a fully blinded manner but those parts of the data collection that can be blinded will be. Observer bias will be reduced where feasible. All laboratory work with samples will be blinded where possible. Standard light traps that do not rely on the ability of the fieldworker to collect specimens will be used to reduce mosquito collector bias, in addition to HLCs. All analyses will be conducted on blinded data.House modification. Any open eaves or gaps in the walls will be sealed with concrete. Windows will be screened and damaged doors will be repaired with wood.Eaves tube installation. A 20 cm long piece of PVC pipe with a diameter of 15 cm (the eave tube) will be installed at 1.5 - 2 m intervals into the outer walls of occupied rooms (e.g. bedrooms and living rooms but not storage rooms). The target location will be approximately 20 cm below the roof, but this may need to be adapted depending on different house designs.Maintenance. The condition of the SET intervention will be monitored through village ‘walk-throughs’ every two months, when any damage to window screening, eave tubes, doors, or walls will be recorded and repaired.Insecticide treatment and retreatment. The eave tube inserts will be treated using a commercially available, 10% wettable powder formulation of the pyrethroid insecticide beta-cyfluthrin . Inserts in the study will be replaced with freshly treated inserts if bioassay mortality falls below 70%.The SET intervention consists of four elements:An. gambiae s.l. mosquitoes will be allowed to recruit freely to the insert for one hour and knock down and mortality will be assessed 24 h post exposure.Persistence of the insecticide will be monitored by monthly bioassays with local, field-collected mosquitoes. These bioassays will consist of an eave tube with the insert placed at the end of the tube. A bottle filled with hot water and covered with a worn sock will be placed behind the insert to act as a host cue. Local, field-collected 2–5 day old female LLINs  will be distributed at universal coverage to each household in all of the study villages. Côte d’Ivoire’s national malaria control program (NMCP) guidelines will be followed to encourage consistent and correct use of the LLINs. During the routine visits to villages for epidemiological and entomological monitoring, trial staff will administer standardized questionnaires, including questions on the presence of LLINs in the sleeping areas and their use by the household.Children enrolled in the ACD cohort will receive routine visits from the study team (a trained nurse and the CHW) every two weeks (rainy season) or every month (dry season).At the first visit, additional data will be collected on household level socioeconomic indicators, methods of vector control used in the household, and sleeping habits. Children five years of age or younger will have a blood sample taken for immediate measurement of hemoglobin levels using a spectrophotometer . All children will receive a three-day course of a first-line antimalarial recommended by the NMCP in Côte d’Ivoire (artesunate-amodiaquine or artemether-lumefantrine), to clear any existing malaria parasite infection. The study team will observe adherence for the first dose and parents or guardians will be instructed to complete the treatment at home. The CHW will be instructed to check compliance and the parents or guardians will be asked to bring the empty drug packet to the following visit. A thick blood smear will be taken on the next visit (two weeks later) to confirm parasite clearance.At all visits, the child’s axillary temperature will be recorded. If the child is febrile (axillary temperature ≥ 37.5 °C), or has a history of fever in the past 48 h, or if the parents report that the child is sick, a health exam will be carried out and a record will be made of the child’s symptoms, pulse, and respiratory rate. A blood sample will be taken from febrile children by finger prick for a malaria rapid diagnostic test . A thick blood smear and a blood spot on filter paper will also be taken for subsequent confirmation of the infection. If the mRDT is positive and the study nurse diagnoses uncomplicated malaria, the child will be treated with a first-line antimalarial (artesunate-amodiaquine) for three days, according to the guidelines of the NMCP. Treatment for malaria will be provided free of charge through this system. If the child exhibits any symptoms of severe illness, he or she will be sent immediately to the closest health clinic for treatment. Once a month, a thick blood smear and blood spot will be taken from all children in the cohort to monitor for asymptomatic parasite infections. During these visits, a record will be made of recent travel, and whether the child slept under an LLIN the preceding night.Four times during the course of the monitoring period, at the start and end of the rainy season, all children in the cohort will be checked for respiratory symptoms  and children five years of age or younger will have a blood sample taken for immediate measurement of hemoglobin levels using a spectrophotometer. One year after the initial parasite clearance, at the start of the next rainy season, all of the cohort children will again be cleared of malaria parasites with a three-day course of front-line antimalarials (artesunate-amodiaquine), confirmed by thick blood smear on the following visit.To capture any malaria cases that occur between cohort visits, the parents or guardians will be given a card at the initial visit, identifying the child as a participant in the study, with instructions to present this card whenever the child receives medical treatment between visits. The back of the trial card will provide space to record the date of visit and the diagnosis.An. gambiae s.l. and all An. funestus and An. nili females will be dissected to determine parity. Both species are known to be vectors of malaria in Côte d’Ivoire [An. gambiae s.l. is the numerically dominant vector in the trial area [An. gambiae s.l. females to species, and to measure the prevalence of kdr and ace-1 resistance mutations.Each month, mosquitoes will be sampled using human landing catches (HLC) both indoors and outdoors for one night at four randomly selected houses in each of the 40 study villages. Starting at 18:00, one capturer will sit inside of the house in the living room area and one will sit outside of the house. These capturers will collect mosquitoes on their bare feet and legs using small glass tubes, which will then be plugged with cotton and brought back to the laboratory. A supervisor will pass by every hour to ensure that the capturer is awake and following the correct capture protocol. At 01:00, a second team will take over and continue the captures until 08:00. When the HLC samples are brought back to the lab, the mosquitoes will be identified using a species key based on morphological traits . A subsed’Ivoire –31, howeial area . After dIn addition to the HLC sampling, CDC light traps will be placed every other month in 12 randomly selected houses for one night in each village. These traps will be set in the sleeping area at 18:00 and removed the following morning at 08:00. The study team members will record the number of people who slept in the room during the trap night, the number of LLINs installed in the sleeping area, and whether the people reported sleeping under the available LLINs during the trap night. The mosquitoes captured in the traps will be brought back to the laboratory for morphological species identification.An. gambiae larvae from eight study villages (four per arm). These field-collected larvae will be reared to adulthood in the insectary at IPR, and bioassays will be conducted with 2–5 day old females. WHO insecticide susceptibility bioassays will be conducted using diagnostic concentrations of the active ingredient in the LLINs (deltamethrin) and the eave tubes (beta-cyflutherin). CDC bottle bioassays will also be done with both actives, to measure resistance intensity. A susceptible An. gambiae lab strain (Kisumu) will be tested as a control in these bioassays. After the bioassays, surviving mosquitoes will be put in RNAlater and stored at -80 °C for subsequent microarray analysis to monitor expression of genes potentially involved in metabolic resistance.To monitor resistance in the study area throughout the course of the trial, larval dips will be used to collect During the trial, data will be collected on perceptions of and willingness to adopt the SET intervention, knowledge relating to the SET intervention, and attitudes and emotions relating to malaria and new malaria prevention interventions. These data will be collected using three complimentary methodologies: (1) questionnaires administered to homeowners and heads of household, (2) ethnographic studies , and (3) focus groups.The questionnaires will be administered before the installation of the intervention and at the end of the monitoring period in the six villages designated for social science activities , eaves tube installation, SET maintenance, or treatment and retreatment of inserts with insecticide. Where resources (e.g. staff) are shared between more than one element, costs will be allocated using a suitable proxy. Costs for research activities will be excluded. Financial costs will be obtained from project expenditure records. Economic costs  will be identified from project records and via social science activities, with a value for donated resources being imputed from market rates. Capital costs will be annualized over their useful life  and annualized at a discount rate of 3% in the economic costing.The numbers of malaria cases averted in the SET + LLIN arm compared to the LLIN only arm will be used to calculate disability adjusted life years (DALYs) averted using standard methods . If the A Trial Steering Committee (TSC) will be established to provide oversight of the study. TSC members will be independent of the trial and its institutions, and have the necessary expertise to monitor study progress and participant safety. The TSC will be responsible for monitoring the progress of the trial, adherence to protocol, patient safety, and for consideration of new information. The TSC will also have access to the study data at regular intervals to determine if additional interim analyses of trial data should be undertaken, and assess any safety issues that may arise during the study. The TSC can make the decision to stop the study at any point.Day-to-day management of the trial is the responsibility of the chief investigator and co-investigators. They will maintain appropriate medical and research records, in compliance with Good Clinical Practice (GCP), and all regulatory and institutional requirements. Standard Operating Procedures (SOPs) will be developed in accordance with Good Clinical Practice (GCP) and Good Field Entomology Practice (GFEP). An independent consultant will review study protocols, SOPs, and QA reports. A study monitor will conduct audits at key phases of the study.The LLINs distributed as part of this trial (Permanet 2.0) have been fully evaluated by the WHO Pesticide Evaluation Scheme (WHOPES) and are routinely distributed by the NMCP in Côte d’Ivoire.The beta-cyfluthrin used on the eave tube inserts is a pyrethroid insecticide, which typically have low mammalian toxicity and good safety profiles. The trial will use a commercially available formulation of the insecticide. The inserts will be machine treated to minimize contact with the insecticide dust. All personnel responsible for treating and installing treated inserts will be provided with the appropriate personal protective equipment (PPE).In addition to safety considerations relating to the use of insecticides, concerns have previously been raised with regards to house modifications and the possibility that they might increase risk of acute respiratory infections due to changes in the indoor environment . To moniHLC capturers will be vaccinated against yellow fever free of charge if they do not already have proof of yellow fever vaccination. If they become sick with malaria at any point during the trial, they will be provided treatment free of charge at the nearest health facility.Participants are free to withdraw from the study at any point for any reason. Participants include children in the ACD cohort, households where entomological monitoring is conducted, and capturers conducting HLCs. For children in the ACD cohort, the reason for premature termination or withdrawal will be documented – e.g., SAE, withdrawal of consent, loss to follow up. If the child withdraws from the cohort for any of these reasons, another child will be selected to replace them, preferably in the same age group and from the same household but if that is not possible, then from another randomly selected household.The primary endpoint of the trial will be the incidence of clinical malaria determined by active case detection in children six months to ten years of age. Clinical malaria will be defined as an axillary temperature of ≥37.5 °C combined with a positive mRDT. The secondary clinical endpoints will be: (1) incidence of asymptomatic malaria infection detected by microscopy and/or PCR, (2) prevalence of anemia in children five years of age or younger (measured four times during the study), and (3) incidence of the following symptoms of respiratory infection: cough, nasal discharge, rales, elevated age specific respiratory rate, and chest indrawing.An. gambiae s.l., An. funestus, and An. nili) captured per person per night both indoors and outdoors by HLC, and the mean number of malaria vectors captured indoors per trap per night by CDC light trap. The secondary endpoints will be: (a) the entomological inoculation rate (EIR) for An. gambiae s.l., An. funestus, and An. nili calculated as the mean number of sporozoite infective bites/person/year, (b) parity of An. gambiae s.l., An. funestus, and An. nili, (c) the prevalence  and strength (LD50 and resistance ratios) of insecticide resistance to deltamethrin and beta-cyfluthrin measured before, during, and after the trial period in An. gambiae s.l.The primary entomological endpoints will be the mean number of malaria vectors  = 0.84. For 80% power to detect a 50% drop in mean mosquito densities of 23 mosquitoes at the 5% significant level, 7 households need to be sampled in each of the 40 villages. To capture the substantial seasonal changes and to accommodate available resources, 4 randomly selected households will be sampled over one night in each village every month, powered to detect a difference for every two months of pooled data.Pilot studies in semi-field systems in Tanzania and Kenya suggest an up to 80% reduction in mosquito entry into houses with eave tubes and screening , 21; a mAn. gambiae, a log SD of 1.7 and ICC = 0.229. To detect a 50% reduction mosquito density with 80% power at the 5% significance level requires 12 households per cluster. In order to capture seasonal variation in mosquito density, collections will be performed every two months.CDC light traps consistently caught fewer mosquitoes than the HLC during the baseline survey, with a mean nightly catch of 2.5 Clinical data will be collected by study nurses on Android tablets using standardized data entry forms in the Open Data Kit (ODK) Collect app and sent directly to a secure electronic server. Routine data checks will be performed to identify incomplete, missing, inaccurate, or inconsistent data, which will be rectified by the appropriate supervisor. Field workers will not have access to the data once it has been collected. Children in the ACD cohorts will be assigned a unique code, which will be used as an anonymous identifier in the datasets and to label clinical samples. Access to the data will be password protected and restricted to only authorized study investigators and data management staff. Any hardcopy documents will be stored in locked filing cabinets and accessible only to authorized study personnel. Documents will be held for at least five years at the host institution (IPR).Protective efficacy against clinical malaria and malaria infection will be determined by comparing incidence rates of malaria infection and clinical malaria between arms. Following any treatment for malaria, a child will not be considered at risk for four weeks and this period will therefore be censored from follow-up. Overnight stays outside of the village will be recorded with a short questionnaire administered during the cohort visits. If travel outside of the village is common, it will be included as a covariate in the analysis.Primary intention to treat analysis will be comparison of the incidence of clinical malaria episodes in the two intervention groups. All analysis will use a mixed effects model to test the difference in incidence rate between the two arms, allowing for repeated measurements on the same individual, and within-cluster correlation of responses. The effect of year and possible confounders such as age of child, gender, antimalarial drug use, baseline infection status, etc. will be analyzed in separate as per protocol analysis.Survival analysis will be used to compare time to first infections, adjusting for confounding factors using a Cox proportional hazards model, again using methods that allow for within-village correlation of responses.Differences in mean mosquito density (for indoor and outdoor catches) between the two study arms will be analyzed using Poisson regression or negative binomial models, accounting for within-village correlation of responses. Sporozoite rates will be compared between the two arms and between indoor and outdoor caught mosquitoes, using logistic regression models. EIR will be calculated for each study night and compared between study arms using negative binomial regression.Pre-intervention willingness to adopt the SET intervention will be compared to actual consent by logistic regressions. The predictors of willingness to adopt will be tested with structural equation modeling. Profiles of perceptions of the SET intervention will be identified using latent class analysis. Ethnographic and focus group information will be evaluated using thematic analysis.The economic and financial cost of the SET intervention will be presented as total and disaggregated by program element to illustrate the relative share of each element to program costs. Costs will also be presented according to cost categories  to illustrate cost drivers. Costs will be converted into cost per house and cost per person receiving the intervention per year, to facilitate comparison with other malaria vector control interventions (e.g. IRS). Alternative program scenarios  will be presented to estimate operational implementation costs. Uncertainty and sensitivity analysis will be conducted.The numbers of malaria cases averted in the SET + LLIN arm, compared to the LLIN only arm, will be used to calculate disability adjusted life years (DALYs) averted using standard methods . If the An interim analysis of malaria incidence and transmission potential will be done after one year of monitoring. The purpose of the interim analysis is to inform planning of future trials. The current protocol will remain unchanged regardless of interim results. The funder will not have access to any data, only summary results. P-values will be adjusted appropriately in the final analysis to account for the interim analysis.Past success in reducing the global malaria burden has highlighted both the importance of vector control in preventing transmission, and the challenges that remain in meeting the targets set out by the WHO Global Technical Strategy (GTS) for Malaria 2016–2030 . This leThe aim of this CRT is to evaluate whether SET provides additional protection against malaria, compared to LLINs alone. Based on previous house improvement trials , 13, 14,The decision was made to use a pyrethroid insecticide for this CRT, despite intense pyrethroid resistance in the study area. The rationale for this decision was two-fold: first, pyrethroids are a relatively safe class of insecticides with low mammalian toxicity and second, of all the candidate actives evaluated in preliminary testing, the beta-cyfluthrin formulation had the highest bioassay mortality for the longest period of time when tested with local, field collected, pyrethroid resistant mosquitoes . The efficacy of a pyrethroid against resistant mosquitoes is not surprising, given that the delivery method (i.e. a powder formulation on electrostatic netting) has previously been shown to be effective against resistant mosquitoes . AlthougGiven that SET requires permanent modifications to the house structure, both cost and user acceptability are a concern. Several social science and economic activities have been planned in parallel with the epidemiological and entomological monitoring to address these issues. One specific concern is that closing open eaves could reduce airflow within the house, which could alter indoor temperature and humidity, and thus negatively impact the comfort of those living in the house. It is worth noting that the majority of houses in this area already have closed eaves, in many cases with ventilation holes built in at eave level Fig. , similarIf the SET intervention is effective at reducing malaria, acceptable to the local population, and cost-effective as a malaria prevention intervention, the question becomes how to implement the intervention at scale. It has been argued that house improvements on their own will likely have to be implemented outside of the existing health sector, which provides funding for LLIN distributions and IRS campaigns . However