Anopheles and poverty, and have limited access to health services. There is a great diversity of indigenous communities in Colombia living in malaria-endemic areas; however, the burden of infection in these populations has not been studied extensively. This study aimed to determine the prevalence of Plasmodium infections in indigenous and non-indigenous communities in two malaria-endemic areas in Colombia.The indigenous population is considered a highly susceptible group to malaria because individuals usually live in areas with high exposure to A community-based cross-sectional survey was conducted in seven villages of Turbo and El Bagre municipalities; three of these villages were indigenous communities. Inhabitants of all ages willing to participate were included. Sociodemographic and clinical data were recorded as well as household information. The parasitological diagnosis was performed by microscopy and nested PCR. The prevalence of microscopy and submicroscopic infection was estimated. An adjusted GEE model was used to explore risk factors associated with the infection.Plasmodium spp. was detected in 30 subjects ; from those, 29 were in the indigenous population, 47% of infections were afebrile, and most of them submicroscopic (10/14). Microscopic and submicroscopic prevalence was 2.5% (CI 95% 1.6–3.9) and 1.7% (CI 95% 0.9–2.9), respectively. In El Bagre, all infections occurred in indigenous participants , and 81% were submicroscopic. By contrast, in Turbo, the highest prevalence occurred in indigenous people , but 88.8% were microscopic. Living in an indigenous population increased the prevalence of infection compared with a non-indigenous population .Among 713 participants, 60.7% were from indigenous communities. Plasmodium infection in indigenous communities. A substantial proportion of asymptomatic and submicroscopic carriers were detected. The identification of these infections, not only in indigenous but also in the non-indigenous population, as well as their associated factors, could help to implement specific malaria strategies for each context.There is a high proportion of  Plasmodium vivax infection [Although significant advances have been made towards malaria elimination in several endemic countries, malaria remains a significant public health problem ; the Wornfection . Particunfection .Anopheles bite exposure, but also because they have high poverty rates and little to no access to diagnostic and treatment services [In the Americas, the indigenous population is considered one of the most vulnerable groups to suffer from malaria. The elevated vulnerability is not solely explained by the fact that individuals live in areas with a high services . Informaservices .Plasmodium spp. caused 46.7% of the total infections [In Colombia, 3.4% of the population is indigenous, and there are around 710 indigenous communities located in 27 departments [fections . Unfortufections . Withoutfections . In 2018fections .Plasmodium prevalence in them.Antioquia was one of the departments with the highest malaria prevalence in Colombia for many years. However, the cases have decreased markedly from 20,511 in 2008 to 4971 in 2017 . This coPlasmodium falciparum and 22% of P. vivax asymptomatic infections can harbor gametocytes [One of the main challenges of malaria control programs is the early diagnosis and treatment, not only for symptomatic but also for asymptomatic infections, which represent a silent reservoir of parasites . Compareetocytes ; similaretocytes .Plasmodium has been previously explored, finding frequencies from 2 to 15% in general population, with most of the infections being submicroscopic [In Colombia, the prevalence of low-density infections by roscopic –22. In proscopic . In the roscopic . Togetheroscopic –27.Plasmodium infections in indigenous and non-indigenous communities in two malaria-endemic areas in Antioquia-Colombia and to explore the associated factors to the Plasmodium infections.Most of the studies about low-density infections in Colombia have been conducted in the general population and pregnant women, but there are not reports in special populations such as indigenous people. This study aimed to determine the prevalence of microscopic and submicroscopic A cross-sectional study was conducted in seven malaria-endemic villages of Turbo and El Bagre municipalities between November 2016 and June 2017. Villages were selected based on the historical records of malaria cases, and the accessibility for field staff. Three of these villages were indigenous communities (Los Aguacates and Los Almendros in El Bagre and Arqua in Turbo).2, an altitude of 2 metres above sea level and an average temperature of 28 °C. The main economic activity is banana production [2, an average temperature of 32 °C and an altitude of 50 metres above sea level [P. vivax was the predominant species [The two towns selected for this study are located in the Antioquia department Fig. . Turbo . Samples were stored at 4 °C until processing at the laboratory of the local hospital in each municipality. The sample was used to perform diagnosis by microscopy using thick and thin blood smears and to measure hemoglobin levels using by a HemoCue . A blood sample was dropped on to Whatman filter paper #3  and later used for molecular diagnosis. Mild anaemia was defined as a hemoglobin level between 10 g/dL and 10.9 g/dL, moderate anaemia between 7 g/dL and 9.9 g/dL and severe anaemia below 7 g/dL following World Health Organization criteria [Field-stained thick and thin blood smears were read in the field by an expert microscopist according to national guidelines  with a lP. vivax cases were treated with chloroquine for 3 days and primaquine (PQ) for 14 days, P. falciparum cases were treated with artemether plus lumefantrine for 3 days [All participants with positive thick smear received anti-malarial treatment according to national guidelines; r 3 days .Plasmodium genus. Positive samples for Plasmodium spp. were further processed using species-specific primers for P. falciparum and P. vivax.DNA was extracted from half a blood spot of the filter cards (approximately 30 µL of blood) using the QIAamp DNA Mini Kit following the manufacturer’s recommendations . Nested PCR was performed as a two-step procedure following the protocol described by Singh et al. with a LOD of 1–6 parasites/μL . AmplifiThis study was reviewed and approved by the Ethics Committee of the Medicine school, Universidad de Antioquia, Colombia (Record 011 dated 28 July 2016). Before to start the fieldwork, permission from community leaders was approved in each village. Signed informed consent was obtained before the interview and blood sampling of all participants. In the case of individuals < 18 years old, an additional informed consent from parents or legal guardians was also obtained.Plasmodium infection was estimated by diagnostic tests (microscopy and nPCR), locality (Turbo and El Bagre), and type of community (indigenous and non-indigenous communities). 95% confidence intervals (CI) are shown for each estimation. A generalized estimating equation (GEE) model [Plasmodium infection (outcome) with 95% confidence intervals for each factor.All data from questionnaires and forms were entered into a Microsoft Access database, and statistical analyses were conducted in STATA 14 . A description of the population was carried out for both, individual  and housing factors . Frequencies were expressed as numbers and percentages for qualitative variables and medians with the interquartile range for the quantitative ones. The prevalence of E) model  was usedPlasmodium infection in both municipalities was higher in indigenous communities than non-indigenous communities (6.69% vs. 0.36%)  than Turbo . The proportion of The characteristics of participants, according to the residence in the indigenous communities, are shown in Table The gender and age distribution was similar in both groups, and the majority of people have lived in an endemic malaria region for more than four years . Most of the indigenous people lived in houses with more than five people (59.1%) and had animals in their homes (97%). Additionally, a high proportion of them did not drain standing water in their homes (93%), and 34% did not have access to electricity.Plasmodium spp. infection was 4.21% (95% CI 2.95–5.96%), 60% of these infections were detected by microscopy, and P. vivax was the predominant species . The prevalence of infection was higher in Turbo compared to El Bagre ; most of the infections in Turbo were detected by microscopy  and 93.75% (15/16) had malaria symptoms  than females (2.9%), with an aPR for the male sex of 2.38 (95% CI 1.07–5.31). People who have lived in the endemic region for more than 4 years had a higher prevalence of infection than those living in the endemic region for less time . The infection was also higher in people with previous self-reported malaria as compared to people with no malaria history . Furthermore, living in an indigenous community increased the prevalence e of infection as compared to the non-indigenous population . Regarding household factors, it was found that participants with no electricity services in their households have a higher prevalence of Plasmodium infection . The PR could not be calculated for the association with water and sewage services (the convergence was not achieved in the GEE model); because none of the infected people had access to these services.The analysis of the association of the subjects and household characteristics with Plasmodium infections. A household description of indigenous and non-indigenous communities is detailed in Table Living in an indigenous community as well as some characteristics of the houses were associated with a higher incidence rate of Plasmodium infections in indigenous and non-indigenous communities from Antioquia, Colombia, and its associated factors, to describe the distribution of disease prevalence among heterogeneous populations; this knowledge is necessary to implement proper control strategies for each context [Plasmodium infections it was increased 12-fold in indigenous communities as compared to non-indigenous communities in both municipalities. Even more, all infections in El Bagre were detected in indigenous communities (11/11), and most of them were asymptomatic and submicroscopic (9/11). On the contrary, most of the infections in indigenous communities in Turbo were symptomatic and microscopic (84.2%).This study evaluated the prevalence of microscopic and submicroscopic  context . We founIt is known that malaria transmission in Colombia varies among the endemic regions ; in thisP. vivax and it is well known that the PQ used for the P. vivax treatment can induce haemolytic crises in individuals with glucose 6-phosphate dehydrogenase (G6PD) deficiency. The G6PD deficiency is distributed worldwide, however, its frequency varies among regions and ethnic groups. A previous report in malaria-endemic areas of Colombia located on the Pacific coast, found a frequency of G6PD deficiency of 6.56% [P. vivax infections found in these populations, there is a need for further evaluation of the frequency of G6PD deficiency in malaria-endemic areas in view that the primaquine treatment (14 days) is required for the radical cure.Most of the malaria cases were caused by of 6.56% , studiesPlasmodium exposure, where an anti-disease immunity is first achieved, resulting in a reduction of severe malaria and mortality. Then, an anti-parasitic immunity is slowly acquired and confers protection against high parasitic densities, which in turn protect against the severe disease [Plasmodium infections using a GEE analysis. However, a previous study in Nariño- Colombia showed that having suffered from more than one malaria episode was associated with an increased risk of having asymptomatic infections  [As previously reported, malaria immunity is determined by previous  disease ; this co1.1–5.4) . These dHousehold factors are also associated with malaria risk . It was Taken together, the individual and housing characteristics could help to understand why the indigenous population has a higher prevalence than its counterpart, the non-indigenous population does. Ethnicity is an important determinant of health conditions, influencing the morbidity and mortality rates in different ethnic groups and interfering with access to health services for some populations . In ColoIt is possible to suggest that the diversity of epidemiologic characteristics of malarial infection among the Colombian subpopulations account for an ideal environment for parasite evolution. In this environment, the parasite can interact with susceptible populations from different ethnicities and under different public health interventions . The preFurther studies are needed to evaluate the potential integration of molecular tests into the surveillance programs to promptly detect malaria infection in the community in order to contribute to the control and future elimination strategies. However, due the low prevalence of infection in this region of Colombia, there is also a need to evaluate the costs per assay comparing to conventional test, including equipment, reagents, staff, training, and maintenance in order to evaluate the cost-effectiveness of molecular test for their potential integration into surveillance strategies and explore alternatives as serological surveillance. A previous study in a low transmission setting in Indonesia, suggested that reactive-active detection of cases in the community using molecular test had high costs per individual screened, however, compared to microscopy, molecular test was most cost-effective for the detection of infections . Also, aPlasmodium reservoir in the region. At last, it is possible that other variables which were not considered in the GEE model could explain the associated factors to Plasmodium infections. Future studies are required to improve the knowledge about the risk factors of the Plasmodium infections in indigenous communities. Despite these limitations, these results are useful to understand malaria transmission in studied places and to suggest prevention efforts according to the individual, housing, and environmental characteristics.This study has some limitations. First, due to cross-sectional design, the association with malaria status should be interpreted with caution, as they do not imply causality. Second, it was no possible to analyse the risk factors for asymptomatic infections exclusively due to the low number of this type of infection. This last could, in turn, affect the accuracy of confidence intervals for some of the factors analysed due to the sample size. Third, as mentioned before, the villages in this study were selected based on the historical records of malaria cases, the distance to the urban area, and the accessibility for field staff, and a random selection of the villages included in the study was not performed. The results of this study cannot be extrapolated to the general population; nevertheless, they are useful to exhibit the problems around the asymptomatic infections in the indigenous and non-indigenous people. Fourth, considering that nowadays there are ultra-sensitive molecular tests for the detection of low-density infections, the prevalence in this study could be underestimated due to the limit of detection of the nPCR used, nevertheless, the nPCR used in this study was able to detect 1.6 times more infections than microscopy showing the presence of a significant Plasmodium infections were in indigenous communities. Nevertheless, the infection profile was different for each town. A substantial proportion of asymptomatic and submicroscopic carriers were detected in El Bagre, while most of the symptomatic and microscopic infections were identified in Turbo. These findings provide an understanding of the key characteristics of asymptomatic, submicroscopic, and microscopic infections in the study population: to live in an indigenous community, having had previous malaria episodes and not having access to electricity, sewage system, and water services. The current malaria control efforts could benefit from the implementation of targeted interventions in indigenous villages using molecular tests to identify submicroscopic reservoirs that could be contributing to malaria transmission, although further studies are needed to evaluate the potential integration of molecular test into the surveillance programs to promptly detect malaria infection in the community, especially in high-risk groups. Additionally, the identification of these infections not only in indigenous but also in the non-indigenous populations, as well as demographic, social and household factors related to them, could help to implement specific malaria strategies for each context.This study reveals that in both municipalities, most of the Additional file 1. Additional table.


Parasitic infections remain widespread in developing countries and constitute a major public health problem in many parts of sub-Saharan Africa. It is prevalent among children under 5 years and pregnant women; however, studies among the later high risk group is limited in the northern part of Ghana. Here, we evaluated the prevalence and associated factors of parasitic infections among pregnant women at first antenatal care visit in northern Ghana.This was a cross-sectional study conducted at the Department of Obstetrics and Gynecology, Bolgatanga Regional Hospital, Upper East Region-Ghana. A total of 334 consecutive consenting pregnant women were included. Questionnaires were administered to obtain socio-demographic data. Venous blood, stool and urine samples were collected for parasite identification using microscopy. Factors associated with parasitic infections were evaluated using regression models. Statistical analysis was performed using R.P. falciparum malaria (21.6%) and schistosomiasis (0.6%). Polyparasitic infection was identified in 6.6% of the population. Increasing age  was associated with lower odds whiles presence of domestic animals , being in the second trimester of pregnancy , having no formal education  and basic education as the highest educational level  were independent predictors of increased odds of giardiasis. Similarly, having no formal education  was independently associated with higher odds of P. falciparum malaria. The use of insecticide treated net (ITN)  and mosquito repellent  were independent predictors of lower odds of P. falciparum malaria.Parasitic infections identified were giardiasis (30.5%), P. falciparum malaria are common among pregnant women in northern Ghana. The major associated factors of giardiasis are lack of or low level of formal education, the presence of domestic animals and being in the second trimester of pregnancy. Increasing age confers protection against giardiasis. Likewise, lack of formal education is an associated factor for P. falciparum malaria among pregnant women in northern Ghana. The use of ITN and mosquito repellents reduce the risk of P. falciparum malaria. Given the possible role of parasitic infections in adverse pregnancy outcomes, our findings highlight the need for regular screening and treatment of infected women in the northern parts of Ghana. Public health education and improving socio-economic status could help reduce the risk of parasitic infections among pregnant women in the region.Giardiasis and  Parasitic infections are a considered global public health problem. These infections are more prevalent in developing countries. The predominant parasitic infections in Africa are intestinal parasitic infections (IPIs) and malaria , 2.IPIs are conditions in which a parasite infects the gastrointestinal tract of humans. A recent report by the World Health Organization (WHO) indicates that approximately 1.5 billion people, accounting for about 24% of the global population, are infected with intestinal parasites , malaria [21.6% (95% CI: 16.9–27.2)] and schistosomiasis [0.6% (95% CI: 0.1–2.3)]  Only P. entified .Using univariate logistic regression analysis, increasing age  was associated with lower odds of giardiasis whereas being in the second trimester of pregnancy at first ANC visit , presence of domestic animals , having no formal education  and basic education as the highest educational level  were associated with increased odds of giardiasis . After aP. falciparum malaria , p<0.0001] and ITN  to be associated with lower odds whereas residing in rural areas  and no formal education  were associated with increased odds of  malaria . Adjusti malaria .Schistosoma mansoni). This notwithstanding, the prevalence of giardiasis was 30.5%. A much higher prevalence of 80% was reported by an earlier study in Accra-Ghana [Several studies on IPIs have been conducted in Ghana; however, these studies are predominantly among the pediatric population. Although a number of studies on IPIs have been conducted among pregnant women, the northern parts of the country is often overlooked. IPIs are caused by either helminths, protozoa or both; however, in this study among pregnant women from northern Ghana, we did not detect helminth infection. This could be due to the periodic national mass drug administration for helminth infestations and bilharzia conducted by the Ghana Health Service in an effort to reduce the burden of neglected tropical diseases in the country. It is thus not surprising that the prevalence of schistosomiasis was also very low Click here for additional data file.


Plasmodium vivax transmission or eliminate dormant P. vivax liver-stage parasites will be essential for malaria elimination. Development of these interventions has been hindered by the lack of P. vivax in vitro culture and could be accelerated by a safe and reproducible clinical model in malaria-naive individuals.Interventions that interrupt P. vivax isolate. Participants  were inoculated with P. vivax–infected red blood cells to initiate infection, and were treated with artemether-lumefantrine (Study 1) or chloroquine (Study 2). Primary endpoints were safety and infectivity of the new isolate. In Study 2, transmission to mosquitoes was also evaluated using mosquito feeding assays, and sporozoite viability was assessed using in vitro cultured hepatocytes.Healthy, malaria-naive adults were enrolled in 2 studies to assess the safety, infectivity, and transmissibility of a new Anopheles mosquitoes at peak gametocytemia. Mosquito infection rates reached 97% following membrane feeding with gametocyte-enriched blood, and sporozoites developed into liver-stage schizonts in culture.Parasitemia and gametocytemia developed in all participants and was cleared by antimalarial treatment. Adverse events were mostly mild or moderate and none were serious. Sixty-nine percent of participants (11/16) were infectious to P. vivax gametocytes from humans to mosquitoes, and have established an experimental model that will accelerate the development of interventions targeting multiple stages of the P. vivax life cycle.We have demonstrated the safe, reproducible, and efficient transmission of ACTRN12614000930684 and ACTRN12616000174482. National Health and Medical Research Council Program Grant 1132975 (Study 1). Bill and Melinda Gates Foundation (OPP1111147) (Study 2). Plasmodium vivax is the most globally widespread human malaria parasite, and the predominant cause of malaria outside of Africa  appear early during blood-stage infection before the onset of symptoms, which increases the likelihood of transmission before treatment. P. vivax produces hypnozoites, which are dormant liver-stage parasites that cause relapses months to years after initial infection. Hypnozoites are reported to account for up to 80% of all P. vivax infections  could interrupt transmission from primary infections, relapses, and also asymptomatic infections that remain undiagnosed and transmissible for a prolonged period. A TBV would reduce morbidity and mortality by preventing both new clinical infections and hypnozoite formation  or volunteer infection studies (VIS), have been established where malaria infections are initiated either by sporozoite inoculation or by the induced blood-stage malaria (IBSM) model (P. vivax transmission from humans to mosquitoes. The IBSM model uses cryopreserved and characterized P. vivax–infected red blood cells (RBCs) to initiate infection. There have been only 2 previous P. vivax IBSM studies (both conducted at our center), in which a total of 8 adults were infected with a P. vivax isolate from the Solomon Islands; however, efficient transmission to mosquito was not achieved  model . To dateachieved , 15. Theent ref. , as wellent ref. ). Here, n = 2) undertaken from October 8, 2014, to January 8, 2015, and Study 2 (n = 24) undertaken from February 22, 2016, to May 21, 2017  were reported: 12 attributed to malaria , 1 deemed possibly related to artemether-lumefantrine (somnolence), and 1 not related to malaria or artemether-lumefantrine (headache 49 days after treatment)  or moderate  in severity. In Study 2, 355 AEs were reported (9/L), chills, elevated alanine aminotransferase (peak 6.9 × ULN), and arthralgia. No serious AEs were reported in either trial.All participants were inoculated with an estimated 564 viable eatment) . Most AEreported . A totalAll 26 participants developed blood-stage parasitemia. In Study 1, parasites were first detected by 18S quantitative PCR (18S qPCR) on day 5 in both participants. Parasitemia peaked at 21,836 parasites/mL and 8949 parasites/mL on the day of treatment (day 8), and was completely cleared following treatment with artemether-lumefantrine . In Studpvs25 transcripts/mL to gametocytes/mL, the peak gametocyte levels were 5.5% (median) of the peak asexual parasite levels, and gametocytemia correlated with asexual parasitemia (P < 0.0001) (Gametocytes were first detected (above 10 gametocytes/mL) on day 6 in Study 1  and betw 0.0001) . The couP < 0.0001) compared with participants in cohort 1 . Participants were not infectious on days 6 and 7 (0/8), and only one participant was infectious on day 8 (1/8). The rate of mosquito infection was highest on day 10 (P = 0.04), and membrane feeding with serum replacement  also resulted in significantly higher mosquito infection rates than membrane feeding with whole blood (P = 0.02) (P < 0.0001)  of participants were infectious to mosquitoes ( = 0.02) . Success 0.0001) .To increase mosquito infection rates in this model, we enriched gametocytes over a percoll gradient either approximately 10-fold or approximately 40-fold to increase the density of gametocytes offered to mosquitoes in the membrane feeding assays . Very hiP. vivax malaria infection in humans, thereby establishing a new clinical model for evaluating P. vivax transmission–blocking interventions. Moreover, we have demonstrated the potential to exploit this model to produce viable clonal sporozoites capable of hepatocyte infection that could be used to evaluate interventions targeting P. vivax liver-stage parasites.We have demonstrated, for what we believe is the first time, the safe, reproducible, and efficient transmission of gametocytes during experimental P. vivax HMP013 inoculum was safe and well-tolerated. The isolate was generated from a donor with blood group O (RhD positive), overcoming the need to match study volunteers’ blood group to that of the inoculum. The number and severity of AEs were in line with safety outcomes from published malaria IBSM trials, 2 of which used P. vivax  the ability to readily carry out IBSM studies in nonendemic countries, (b) prior knowledge of P. vivax genotype and drug sensitivity, (c) ability to carry out multiple studies with the same strain and dose, and (d) simplified trial design and conduct because all participants develop blood-stage parasitemia simultaneously.A limitation of this study is the small sample size; further studies are needed to determine the true variability in P. vivax gametocytes from healthy nonimmune participants to mosquitoes during experimental human malaria infection. This experimental model can be used for early clinical evaluation of drug and vaccine candidates, and could provide a source of sporozoites for the evaluation of P. vivax liver stages. This model will further our understanding of the biology of all stages of P. vivax infection and provide critical information for malaria control and elimination agendas.In conclusion, we have demonstrated the safe, reproducible, efficient transmission of n = 6) and cohort 2b (n = 2), conducted separately , and a phase 1b human-to-mosquito transmission study Study 2). Healthy, malaria-naive males and nonpregnant, nonlactating females aged between 18 and 55 years were eligible to participate. Study 1 was conducted with 2 participants inoculated 24 hours apart. Study 2 was undertaken as 3 cohorts of 8 participants. Due to recruitment limitations, cohort 2 was performed as cohort 2a  returning to Australia from India who presented with malaria-related symptoms. Informed consent was obtained , and 200 mL of blood was collected. The patient tested negative for blood-borne pathogens using a Red Cross donation protocol and the RBCs were cryopreserved as previously described  by 18S qPCR  twice-das25 mRNA  from days25 mRNA . Curativs25 mRNA . All pars25 mRNA  and 2.Anopheles stephensi mosquitoes via DFAs (2 per participant), DMFAs with whole venous blood in lithium heparin anticoagulant (2–3 per participant), or membrane feeding assays with serum replacement (MFA-SR)  or on days 9 and 10 (cohorts 2 and 3). All feeding assays were performed before drug treatment was initiated. Gametocytes were fed to (MFA-SR) . Explora(MFA-SR) . We dete(MFA-SR) , 22. Mic(MFA-SR) . Salivar(MFA-SR) . Sporozo(MFA-SR) .P. vivax isolate in healthy, malaria-naive adults. Safety endpoint measures were the frequency and severity of AEs, and results of clinical laboratory tests, physical examinations, vital sign assessments, and electrocardiographs. Infectivity endpoint measures were parasitemia and gametocytemia growth profiles determined by 18S qPCR and pvs25 qRT-PCR. A secondary endpoint in Study 2 was transmissibility of P. vivax gametocytes from humans to mosquitoes. Successful transmission was defined as at least 1 oocyst-positive mosquito per feeding assay, measured by 18S qPCR. Additional primary and secondary objectives were to characterize the pharmacokinetic-pharmacodynamic relationship between chloroquine concentration and clearance of blood-stage parasites. These will be reported separately.Primary endpoints were the safety (both studies) and infectivity (Study 1) of the Data collected for the primary and secondary objectives for this study will be available with other supporting documents  after publication upon request with a data transfer agreement. Direct inquiries to the corresponding author. All methodologies are presented in this manuscript or the P. vivax isolate in the IBSM model. The first-in-human pilot study (Study 1) required only 2 participants. Study 2 was designed to assess the parasite-clearing activity of chloroquine. Normative data on log parasite clearance rate was used in sample size estimation from 18 IBSM studies involving 102 individuals with mean decay rate of 0.063 log parasites per hour and SD of 0.019. It was determined that a sample size of 20 participants has 80% power to identify a difference of 20% in mean decay rate compared with a reference standard as significant at 5% 2-sided significance based on a 1-sample t test. Statistical analysis was performed using GraphPad Prism version 8.2.1 (infectivity endpoints), and R version 3.3.3 . The D’Agostino-Pearson normality test was used to determine if continuous data were normally distributed. When comparing 2 groups of nonparametric data, the Mann-Whitney test was used. More than 2 groups of nonparametric data were compared by Kruskal-Wallis test with Dunn’s multiple comparison test. P less than 0.05 was considered statistically significant.Both trials were designed to assess the in vivo safety of the Both studies were approved by the QIMR Berghofer Human Research Ethics Committee. Study 2 was also approved by the Australian Defence Human Research Ethics Committee. All participants met the eligibility criteria  and gaveKAC, JSM, and JJM conceived the study and designed the experiment. JSM, SE, AO, and SC provided clinical oversight. KAC, CYTW, MA, HM, GJR, and MR performed molecular and entomology experiments. VMA, LL, TBS, and KAC were responsible for liver-stage assays. KAC, DK, and EB analyzed data. KAC wrote the manuscript. All authors reviewed the manuscript and approved the final version.


Plasmodium falciparum infections. Yet, variable detection of certain targeted motifs, low parasitaemia, but also deletion of pfhrp2 gene or its homologue pfhrp3, may result in false-negative RDT leading to misdiagnosis and delayed treatment. This study aimed at investigating the prevalence, and understanding the possible causes, of P. falciparum RDT-negative infections at Montpellier Academic Hospital, France.Rapid diagnostic tests (RDTs) detecting the histidine-rich protein 2 (PfHRP2) have a central position for the management of P. falciparum RDT infections were screened for pfhrp2 or pfhrp3 deletion; and exons 2 were sequenced to show a putative genetic diversity impairing PfHRP2 detection.The prevalence of falsely-negative RDT results reported before and after the introduction of a loop-mediated isothermal amplification (LAMP) assay, as part as the malaria screening strategy in January 2017, was analysed. Negative P. falciparum negative RDTs from January 2006 to December 2018 was low (3/446). Whereas no cases were reported from 2006 to 2016 (0/373), period during which the malaria diagnostic screen was based on microscopy and RDT, prevalence increased up to 4.1% (3/73) between 2017 and 2018, when molecular detection was implemented for primary screening. Neither pfhrp2/3 deletion nor major variation in the frequency of repetitive epitopes could explain these false-negative RDT results.The overall prevalence of pfhrp2 and pfhrp3 genes in three P. falciparum RDT-negative infections and reviews the possible reasons for non-detection of HRP2/3 antigens in a non-endemic setting. It highlights the emergence of falsely negative rapid diagnostic tests in a non-endemic setting and draws attention on the risk of missing malaria cases with low parasitaemia infections using the RDT plus microscopy-based strategy currently recommended by French authorities. The relevance of a novel diagnostic scheme based upon a LAMP assay is discussed.This paper demonstrates the presence of  Plasmodium falciparum infection and a significant increase in severe cases between 1996 and 2016 [P. falciparum [Malaria remains a major public health issue in tropical regions and accounts for a significant burden in non-endemic areas. Imported malaria indeed represents one of the most prevalent infectious diseases among travelers and migrants in industrialized countries, France being the most impacted European country . A crosslciparum . BiologiPlasmodium parasites but may be time-consuming for initial screening, especially in non-endemic countries where samples are often negative, and relies upon highly trained personnel [P. falciparum which are encoded by an abundant secreted antigen, the histidine-rich protein 2 (PfHRP2). Its homologue, PfHRP3, shares significant sequence homologies and, as such, may be recognized by monoclonal antibodies raised against PfHRP2 [P. falciparum infections than those targeting pan-Plasmodium aldolase or lactate dehydrogenase (pLDH) [pfhrp2 and/or pfhrp3  , 9. A griewed in ). Genetiiewed in , 11, 12.iewed in . In thiscreening –18.Plasmodium and PfHRP2 antigens. Both techniques were used at the Parasitology-Mycology Department of Montpellier Academic Hospital for all suspected malaria cases until December 2016, when a novel strategy, based upon a LAMP assay and a PfHRP2-based RDT, was introduced for primary diagnosis of malaria. This allowed detecting falsely-negative RDT results, of which the cause was investigated in this study.Since 2017, according to the French National Authority for Health  and the P. falciparum infections over 13 years, from January 2006 to December 2018, when two diagnostic schemes for malaria screening were used. The study population included all cases of P. falciparum imported malaria diagnosed at the Parasitology-Mycology Department of Montpellier Academic Hospital, France.The aim of the study was to investigate the prevalence and possible causes of RDT-negative i.e. any febrile patient with a history of travel to malaria-endemic areas), both using antigenic detection by a RDT, were applied in the laboratory , which targets pan-aldolase and PfHRP2 antigens, was used from January 2006 to August 2009. SD Bioline Malaria Ag Pf/Pan , detecting pLDH and PfHRP2 antigens, was used from September 2009 to December 2018.Thin films were stained with May-Grünwald Giemsa and were examined using oil immersion magnification (1000×) for at least 20 min before being considered negative. Parasites density was estimated as the percentage of infected red blood cells.Thick smears were stained with Giemsa . All the® Malaria kit (Meridian®), targeting a pan-Plasmodium mitochondrial DNA sequence, according to the manufacturer’s instructions.For LAMP assays, samples were processed using the Alethiai.e. positive pan LAMP and negative RDT) were further analysed for molecular and antigenic testing at the Microbiology Laboratory of Nîmes Academic Hospital (France). Samples were retested 2 days after patient sampling by another RDT used for routine practice: the BinaxNOW RDT  targeting pan aldolase and PfHRP2 antigens. For molecular screening, DNA was extracted from 200µL of whole blood using EZ1® DNA Blood 200 µL kits (QIAGEN®) on the Biorobot® EZ1 workstation, according to the manufacturer’s instructions. Two in-house qPCR methods, one distinguishing Plasmodium species by specific melting curves of the 18S rRNA [P. falciparum-specific cox1 gene [P. falciparum identification.All samples with conflicting results  and pfhrp3 (PF3D7_1372200) were retrieved from PlasmoDB database (http://www.PlasmoDB.org). Pair of primers specific to the 5′ and 3′ ends of exon 2 of pfhrp2  (expected size: 816 bp) and pfhrp3  (expected size: 698 bp) were used to assess gene deletion following previous recommendations [pfhrp2 and pfhrp3 was performed using PfuII polymerase (Agilent) under the following cycling conditions: 94 °C for 2 min followed by 35 cycles of 94 °C for 20 s, 54 °C for 20 s, 62 °C for 90 s and 62° for 7 min. PCR products were purified using spin columns (QIAGEN®) and sent for Sanger sequencing (Eurofins Genomic®). Nucleotide sequences were translated into corresponding amino acids and aligned against the Pf3D7 reference genome using NPS@  software and ESPript 3.0 program for data assembling. Frequency of repetitive histidine and alanine motifs (i.e. AHHAHHAAD and AHHAAD) were assessed and sensitivities were predicted according to Baker’s model [Samples positive for ndations . Amplifi’s model .P. falciparum positive samples were diagnosed at the Parasitology Department of the Academic Hospital of Montpellier from January 2006 to December 2018. Almost all patients originated from African countries  between January 2017 and December 2018, i.e. after the introduction of the novel scheme for primary diagnosis of malaria. The three patients presented fever with history of recent travelling from endemic countries: Cameroun, Ivory Coast or Gabon in June 2017, August 2018 and December 2018, respectively. Non-detection of PfHRP2 in these three samples was confirmed by two RDTs from different brands (i.e. SD Bioline and BinaxNOW). Parasite densities in RDT-negative samples were estimated on thin and thick blood smears and ranged from < 0.001 to 0.05% or ~ 5 parasites/µL to ~ 800 parasites/µL, respectively , P. falpfhrp2 is the major source of repetitive motifs detected by PfHRP2-based RDTs; however, variations in the frequency of targeted repeats may influence accurate binding of specific antibodies [pfhrp2 . Using the second diagnostic scheme, three cases of P. falciparum false-negative RDTs were detected, while no case had ever been reported when RDTs were combined solely with microscopy techniques. Amplifications of pfhrp2 and pfhrp3 in parasite isolates allowed ruling out gene deletion as the cause of non-detection by RDTs. Although this study presents some limitations due to its retrospective nature, it allowed reconsidering the ability of PfHRP2-based RDTs to detect all P. falciparum infections in the laboratory and may thus be valuable for the community.The aim of the present study was to investigate the prevalence and understanding the possible causes of RDT-negative PfHRP2-based RDTs. First, results may be operator-dependent and rely on good product design and manufacturing quality [i.e. the percentage of malaria samples in the panel giving a positive result) of more than 90% when tested on P. falciparum parasites at 200 parasites/µL [®vs 100% for parasitaemia > 500 parasites/µL [Various factors may influence the performances of  quality . All falsites/µL . Second,sites/µL ). Detectsites/µL , e.g. 75sites/µL . Here, tP. falciparum parasites lacking pfhrp2 and pfhrp3 genes in Peru [pfhrp2 or pfhrp3 may have spread in a broader range of endemic regions, impairing clinical case management and control efforts for malaria elimination [pfhrp2 nor pfhrp3 genes was found in all three samples, consistent with the low prevalence of such deletions [pfhrp2 gene was found highly polymorphic. A binary logistic model used to predict RDT detection sensitivity [pfhrp2 does not appear to be the cause of the increasing rate of P. falciparum negative RDT results in the laboratory. The possibility of low expression of HRP2/3 antigens cannot be ruled out in the absence of quantitative analysis at the protein level [Since the first demonstration of  in Peru , other s in Peru –34. The  in Peru . In thismination . Howevereletions . Alike peletions , 38–44 tsitivity  revealedsitivity . Moreovesitivity . Taken tin level . Anti-HRin level . In addii.e. after the implementation of a new malaria diagnostic scheme based upon a RDT in combination with a LAMP assay. A hypothesis to explain the emergence of falsely negative RDTs would be a reduction in quality of the tests at the stage of manufacture since 2017 but this has not been reported. One could also hypothesize that the introduction of a molecular method, more sensitive than microscopy and antigenic tests [The observed overall prevalence of falsely negative RDTs is low (0.67%), although similar to that found in France at a national scale between 2012 and 2017 57/6118; 0.93%) . It is interesting to relate the emergence of RDT false-negative results to the introduction of a novel strategy for the biological diagnosis of malaria in the laboratory. While no false-negative results had been identified over an 11-years period (2006–2016), all cases were reported between January 2017 and December 2018, ic tests –18, has ; 0.93% (ic tests . The Freic tests . Indeed,ic tests .P. falciparum RDT-negative results in a non-endemic setting, this study reviews the possible reasons for non-detection of HRP2/3 antigens and highlights the absence of gene deletion in P. falciparum infections diagnosed since 2006. It draws attention on the risk of missing malaria cases with low parasitaemia infections using the diagnostic strategy currently recommended by French authorities. In this context, this study may lay stepping-stones towards recommendations including molecular detection for malaria diagnosis in non-endemic countries.Through the analysis of


Plasmodium falciparum infection and accelerate towards elimination. Questions remain about their effectiveness and evidence from different transmission settings is needed.Population-wide interventions using malaria testing and treatment might decrease the reservoir of A pilot quasi-experimental study to evaluate a package of population-wide test and treat interventions was conducted in six health facility catchment areas (HFCA) in the districts of Kanel, Linguère, and Ranérou . Seven adjacent HFCAs were selected as comparison. Villages within the intervention HFCAs were stratified according to the 2013 incidences of passively detected malaria cases, and those with an incidence ≥ 15 cases/1000/year were targeted for a mass test and treat (MTAT) in September 2014. All households were visited, all consenting individuals were tested with a rapid diagnostic test (RDT), and, if positive, treated with dihydroartemisinin-piperaquine. This was followed by weekly screening, testing and treatment of fever cases (PECADOM++) until the end of the transmission season in January 2015. Villages with lower incidence received only PECADOM++ or case investigation. To evaluate the impact of the interventions over that transmission season, the incidence of passively detected, RDT-confirmed malaria cases was compared between the intervention and comparison groups with a difference-in-difference analysis using negative binomial regression with random effects on HFCA.During MTAT, 89% (2225/2503) of households were visited and 86%  of individuals were tested, for a combined 77% effective coverage. Among those tested, 291 (1.5%) were RDT positive (range 0–10.8 by village), of whom 82% were < 20 years old and 70% were afebrile. During the PECADOM++ 40,002 visits were conducted to find 2784 individuals reporting fever, with an RDT positivity of 6.5% (170/2612). The combination of interventions resulted in an estimated 38% larger decrease in malaria case incidence in the intervention compared to the comparison group . The cost of the MTAT was $14.3 per person.It was operationally feasible to conduct MTAT and PECADOM++ with high coverage, although PECADOM++ was not an efficient strategy to complement MTAT. The modest impact of the intervention package suggests a need for alternative or complementary strategies. With an increasing international focus on malaria elimination, there is a need for interventions that are effective at reducing transmission in different settings . AlthougPlasmodium falciparum infection by microscopy in children under 5 years old was 5.7%, which decreased to 0.4% in 2017. There is a gradient of transmission intensity, with a gradient in the prevalence from 7.3% in the south-east of the country to near zero in parts of the north [Programme National de Lutte contre le Paludisme  shifted its strategy from control to elimination and partnered with stakeholders to conduct implementation research on potential elimination strategies in different transmission settings to inform the national malaria strategic plan.Senegal has greatly reduced its malaria burden in the last decade through scale up of control tools . In 2008Population-wide drug administration strategies are being evaluated as tools to decrease the reservoir of infection and accelerate towards elimination, but more data on their impact and feasibility are needed. Mass test and treat (MTAT) has been proposed as one population-wide drug administration intervention for transmission reduction . This apThe PATH Malaria Control and Elimination Partnership in Africa (MACEPA), in collaboration with the PNLP, implemented a package of interventions in northern Senegal, including an MTAT followed by a weekly fever screen, test and treat campaign or case investigation. The goal of this work was to evaluate its impact, feasibility and cost and to provide evidence to inform the national malaria elimination strategy.Plasmodium falciparum is responsible for most of the infections and the main malaria vectors are Anopheles gambiae, Anopheles arabiensis, and Anopheles funestus [The study was conducted in Kanel, Linguère and Ranérou districts, in Matam and Louga regions in northern Senegal Fig.  from Sepfunestus , 12. Malfunestus . First-lfunestus .Fig. 1MaThe study used a quasi-experimental design with non-random selection of the intervention and comparison groups. Six health facility catchment areas (HFCA) were purposefully selected by the PNLP to receive the intervention based on the 2013 annual incidence of malaria cases. Seven HFCAs from the same districts were selected as a comparison group for the study. Characteristics of intervention and comparison areas are presented in Additional file P. falciparum malaria cases  during the 2014 to 2015 transmission season. Secondary objectives were characterizing demographic and spatial patterns of infection to guide the strategy toward elimination; describing the operational feasibility of implementing different parasitaemia-clearing strategies; and estimating the costs of the interventions.The primary objective was to evaluate whether a combination of malaria parasitaemia-clearing strategies could substantially decrease malaria incidence in low transmission areas in Linguère, Ranérou, and Kanel districts. The primary endpoint was the incidence of passively detected, RDT-confirmed A census was done at the outset of the study and households within the intervention and comparison areas were visited and asked to complete a brief survey including number of individuals in the household, number of sleeping spaces and number of bed nets. Household GPS coordinates were collected as part of this process and were used to locate houses during the interventions. All efforts were made to achieve full coverage and, if households that had been missed during the census were found during the interventions, these were added to the census database.Case investigation was conducted for all malaria cases (index cases) detected at the health posts or by community health workers in stratum 1 intervention villages. The household of the index case was visited, and FTAT was conducted there. In the five closest neighboring households within a 100-m radius, focal screen test and treat (FSTAT) was conducted: all individuals were screened for risk factors  and those with at least one risk factor were tested with an RDT and treated if positive. Because a high number of secondary cases were found during the FTAT and FSTAT within the same area, these were considered an outbreak. A focal drug administration was conducted as a response in the affected villages: DHAp was administered to all individuals regardless of infection status, using the same inclusion and exclusion criteria described below for the MTAT.Prise en charge à domicile” or case management at the household level, and PECADOM++ was an enhanced PECADOM consisting of weekly mass fever screen, test and treat along with reactive focal test and treat (FTAT). It was implemented in all households in intervention villages in strata 2 and 3 for 16 weeks from October 2014 until January 2015. All households received a weekly visit to screen for reported fever in the previous week. All individuals reporting fever in the previous week received an RDT, and RDT-positive individuals received DHAp. Additionally, in households with a positive RDT, FTAT was done and all individuals received an RDT, and RDT-positive individuals received appropriate treatment.PECADOM stands for “All households in stratum 3 intervention villages were targeted for the MTAT. All households were visited by the study team and all individuals older than 2 months of age were invited to participate. Informed consent was obtained from the head of household prior to requesting any information from the household and individual written consent was then obtained from all participating members or from their parents/guardians in the case of children under the age of eighteen. A household questionnaire was then administered to the household head , and an individual questionnaire was administered to each household member . Information was collected using standardized questionnaires programmed in ODK Collect (an Open Data Kit tool) on smartphones. Three attempts were made to locate individuals who were absent at the time of the survey .® rapid diagnostic test (RDT). Patients who tested positive for malaria were treated with DHAp, unless there were contra-indications. DHAp was chosen based on the longer half-life and prophylactic effect. Since there are contraindications for administering DHAp to women within the first trimester of pregnancy, reproductive age women who were eligible for treatment were asked about their pregnancy status and the timing of their last menstrual period. RDT positive women were not treated and were referred to a clinic if they were within the first trimester of pregnancy or if they were uncertain about their pregnancy status. RDT positive women in the second or third trimester of pregnancy and infants between 2 and 6 months of age were treated with artemether-lumefantrine (AL) instead of DHAp, according to national guidelines. RDT-positive individuals were also referred to a health facility for treatment if they had certain contraindications including allergies to artemisinin drugs, had recently taken artemisinin drugs or other contraindicated drugs, or had previous heart palpitations.All consenting individuals were tested for malaria using a Premier Medical First ResponseDuring all three interventions, all cases receiving DHAp were followed up one week later to assess adherence to treatment (reported and through observation of blister) and adverse events through a standardized questionnaire.Field visits were conducted by teams composed of a field worker and a community health worker. Data were entered using handheld Android phones programmed with ODK Collect custom-designed questionnaires. Data was stored on the Android phone and then uploaded daily to a central ODK server. Internal data checks were set up to prevent missing data and identify inconsistent entries. Data analysis was done using Stata 13.1 according to a pre-defined analysis plan.P. falciparum) as the dependent variable. To control for spatial clustering of malaria cases, a fixed effect at the HFCA level and a random effect at the village level were included. Analysis was stratified according to age group: under 10 years of age and 10 years or older, as some of the risk factors in adults (occupation and education) were not relevant for young children. Additionally, separation into two groups allowed for exploration of differences in risk factors for children compared to adults.Risk factor analysis for RDT positivity during the MTAT was conducted using univariable and multivariable logistic regression with RDT result  of the individuals were tested, of whom 3.5% (7) had a positive RDT. Forty neighbouring households with 628 individuals were also visited to conduct FSTAT, where 54 (8.6%) had at least one FSTAT risk factor, and 11 (20.4%) had a positive RDT. Seventeen of the 18 secondary cases were found in three villages of the same area, which was considered an outbreak. A focal drug administration was done as a response in twenty households, during which 405 individuals received DHAp on weeks 48 and 49. The mean number of days between index case detection and the start of the household visits was 1.3.PECADOM++ was conducted during 16 weeks from October 2014 to January 2015 in all households in intervention strata 2 and 3 villages. Of the 3577 households registered during the census in the target areas, 93% (3307) were approached for PECADOM++ at least once, with a total of 40,002 visits and a median of 12 visits per household  were not present at the time of the survey and 36 (0.2%) refused. As a result, 18,992 individuals received an RDT, an individual coverage rate of 86%. The combination of household and individual coverage resulted in 77% effective coverage of RDT testing. Each team interviewed and tested 25 individuals per day on average. The study profile is shown in Fig. Among the population reached, 45%  of household members were male and 44%  were younger than ten years of age. Individuals who were absent at the time of the survey were more likely to be male (72% (2271/3142)) and more likely to be over the age of ten (81% (2531/3141)). Among individuals reached, bed net usage was high, with 65% reporting use of a net the previous night. Only 2.6%  of individuals reported travel outside of the district in the previous two weeks, 3.4%  had fever (axillary temperate ≥ 37.5 °C) or reported fever in the previous 24 h and 6.0%  had fever or reported fever in the previous 7 days.Of the 18,992 individuals tested as part of the MTAT, 291 were RDT positive, yielding an overall prevalence of 1.5% (range 0%- 10.8% by village). The prevalence of RDT positivity by age group and sex is shown in Fig. Within the RDT positive group, 261 individuals (90%) were treated with DHAp, 2 (0.7%) were treated with AL, 22 (8%) were referred and 6 (2%) were not treated for other reasons, such as refusal. Among the 208 (80%) individuals that were treated with DHAp and found during the follow up visit, 203 (98%) completed full treatment (according to self-report or observation of the blister pack during the follow-up visit) and 5 (2%) did not complete the course. Among those followed up, 23 (11.1%) had one or multiple adverse events, all of which were mild, and included vomiting (19 cases (9.1%)), fever (8 cases (3.8%)), and itching (1 case (0.5%)).Among the RDT-positive individuals, 82% (238/291) were younger than 20 years of age, 70% (204/291) were afebrile (no measured fever or reported fever in previous 24 h) and 2% (5/291) had travel history in the previous month. The percentage of those who were afebrile was not significantly different by age group, being 73% (33/45) in children younger than 5 years old, 70% (69/98) in children 5–9 years old, 69% (47/68) in children 10–14 years old, 63% (17/27) in children 15–19 years old and 72% (38/53) in adults aged > 20 years.Results of the univariable and multivariable logistic regression to assess risk factors for RDT positivity is presented separately for children < 10 years of age Table  and oldeRDT positivity showed evidence of clustering at the household level, with 43% of the total infections being in households with at least one other infection and 90% of households not having any RDT positives.Figure In a sensitivity analysis, the significance of the effect decreased when including the RDT-positive febrile individuals found during the MTAT and PECADOM in the outcome . Since the coefficient on average bed net ownership was the opposite as expected, an additional sensitivity analysis was run omitting this variable from the regression, and found that the interaction term remained similar to the original analysis .Additional file The cost for MTAT was $14.3 per person tested. The bulk of the cost was incurred during implementation, which was 85% of the total and was largely made up of supervision costs  and transportation costs . The cost for PECADOM++ was lower, at $3.1 per person. Supervisor and transportation costs were a lower share of costs than in the MTAT and made up 18% and 22% of total costs, respectively, while training costs made up the largest share at 33%. Case investigation was by far the cheapest intervention at $0.4 per person. Training (29%), transportation (28%), and supervision (25%) were again the dominant cost categories. The salaries of enumerators and community health workers was a small fraction of the costs, less than 20% for all interventions. Costing details for MTAT, PECADOM++ and case investigation are shown in Additional files The study demonstrated the feasibility of implementing a large-scale MTAT in 4 weeks, followed by PECADOM++ and case investigation during the rest of the transmission season in a low to moderate malaria transmission setting. The MTAT achieved a household coverage of 89% and a within household coverage of 87% for a combined effective coverage of 77% for RDT testing, which is lower than the 88% reported in the Zambia MTAT [In the study area, the MTAT approach resulted in a very small proportion of the population receiving antimalarial treatment, as only 291 (1.5%) of the 18,992 individuals tested were positive. However, in an area with such low malaria transmission intensity, a substantial number of infections may have been low density and thus not detectable by RDTs . The useDuring PECADOM++, 40,002 visits were needed to find 2784 individuals with reported fever and treat a total of 289 individuals. This screening programme was time and labour intensive considering the number of cases found. In southern Senegal, this approach yielded a much higher RDT positivity rate, with 62% of the tested febrile individuals having a positive RDT . Thus, pThe risk factor analysis also showed that, as expected, fever and having taken an anti-malarial in the previous month were risk factors for RDT positivity in both age groups, whereas bed net use the previous night was unexpectedly not significant. Having at least one other RDT-positive in the household was also a risk factor in both age groups, which, together with the fact that 43% of the total infections were in households with at least one other infection and 90% of households did not have any RDT positives, indicates clustering of infections at the household level. On the other hand, during the PECADOM++, only 17.3% of infections occurred in households that had other RDT positives during the study period, showing that infections do not always occur in the same households over a transmission season and that strategies that try to target hotspots only will miss a large proportion of the infections. In children ≥ 10 years old and adults, having had primary education or Arabic school was found to be protective relative to no education, whereas secondary education was a risk factor, which could be due to higher educated individuals travelling more to higher transmission areas or other behavioral differences.The combination of interventions resulted in an estimated 38% (95% CI 16–55%) larger decrease in malaria case incidence in the intervention group relative to that in the comparison group. This is a significant reduction and suggests that MTAT in combination with PECADOM++ and case investigation offer a feasible parasite reduction strategy. However, this reduction in incidence was moderate and it was achieved with intensive effort. Both MTAT and PECADOM++ required supervision, transportation, training field teams and conducting large-scale individual testing at significant expense. Nevertheless, this impact is higher than that found in previous MTAT studies, where no follow up interventions like PECADOM++ or case investigation were done after the MTAT. A study conducted in Zambia found a 53% decrease on the prevalence of infection with a marginal effect on outpatient malaria case incidence . Two othThe costs for these interventions must also be considered. The cost of $13.9 per person reached is notably higher than the cost of other malaria control interventions . HoweverThe quasi-experimental design and impact evaluation analysis have a few caveats. First, while the difference-in-differences framework allows for comparison between groups with different baseline levels and analysis suggests that the parallel trends assumption holds true in this case, the intervention group was selected based on the higher intensity of malaria transmission in prior years, a programmatic decision which complicated the estimation of impact. Second, this study featured a small number of clusters with selection done at the HFCA level. Third, while reductions in rainfall and NDVI during the intervention period were associated with declines in malaria incidence in both groups, these changes in rainfall and NDVI might have had a different effect on malaria transmission in the intervention than in the comparison areas, given the baseline differences. Fourth, the study used passively collected data on malaria case incidence, assuming that the interventions did not affect care-seeking behaviour and tested this assumption in a sensitivity analysis. When adding the febrile RDT positives found during the MTAT and PECADOM++ in the outcome, the effect of the intervention was smaller and became non-significant. This sensitivity analysis assumed that all febrile individuals would have sought care, however, other studies in the north of Senegal show that only 60% of febrile children younger than 5 years seek care from a health facility or community health worker . TherefoIt is operationally feasible to conduct MTAT and PECADOM++ with a high coverage. PECADOM++ was not an efficient strategy to complement the MTAT, as maintaining a high coverage every week required substantial resources to find and treat a small number of infections. MTAT, implemented at the beginning of the transmission season and followed with PECADOM++ and case investigation, showed only a modest impact. This study suggests alternative or additional strategies are needed to eliminate the parasite reservoir.Additional file 1. Distribution of independent and dependent variables.Additional file 2. Weekly incident malaria cases per 1000 population during the high transmission seasons (Sept. to January) in a) intervention and b) comparison health facility catchment areas*.Additional file 3. PECADOM++ profile.Additional file 4. Weekly PECADOM++ household coverage by health facility catchment area.Additional file 5. Incident malaria cases over high malaria transmission seasons (Sept. to January) of 2012–2013*, 2013–2014 and 2014–2015 in intervention vs comparison group to assess parallel trends assumption in the difference-in-difference analysis.Additional file 6. MTAT costsAdditional file 7. PECADOM++ costs.Additional file 8. Case investigation costs.


Virulence gathers papers with a different perspective – understanding the response of the mammalian host to infection with the Plasmodium parasite. Taken together, this collection of writing is based on an implicit understanding of the human body as a fortress against invasion by the marauding parasite. Innate defenses such as fever, phagocytosis, and systemic inflammation, and the pathogen-elicited adaptive responses of cellular immunity and antibody are the walls, ramparts, boiling oil, and fortified gates that protect us from malaria, as from other infections. In this series, our panel of expert authors examine how these defenses work, the parasite’s strategies for eluding them, what happens when they fail and progression to severe disease occurs, and how biomarkers of the body’s defense systems can be valuable diagnostic indicators of morbidity and prognosis. This knowledge is of great value in the continued effort to develop improvements in the management of severe malaria, by directing defenses down beneficial pathways to minimize pathology, and in the design of effective vaccines against Plasmodium parasites.Much attention has been directed toward a recent slowing of progress in malaria control worldwide, with particular emphasis on growing evidence which suggests that the twin pillars of malaria reduction – chemotherapy and anti-mosquito measures – are both less effective than previously. This special issue of P. falciparum infection. Parasite-derived ligands on the surface of the infected erythrocyte mediate receptor binding, enabling sequestration of maturing blood-stage asexual parasites away from splenic filtration. This binding results in endothelial activation and damage to the vessels, and this injury is a key feature of severe life-threatening malaria (SM). This is an example of harmful host responses which can lead to increased pathology. However, only a minority of infections progress to SM, and so the host is usually able to manage this interaction. Clara Erice and Kevin Kain [The endothelium of the microvasculature of the human host is a crucial tissue in vin Kain  explore vin Kain  also traPlasmodium species, and has been examined closely in experimental rodent infections, where its potential to contribute to immunopathology and SM is well known. Lisa Drewry and John Harty [Inflammation is a feature of illness caused by all hn Harty  guide ushn Harty  rather phn Harty  develop hn Harty .Plasmodium species other than P. falciparum. Anupkumar Anvikar, Sam Wassmer, and colleagues [P. vivax, the most common causative agent for malaria on the sub-continent. In the study region, Gujarat, severe malaria hospital admissions from vivax malaria outnumber those with P. falciparum infections. Distinct features of SM in P. vivax disease are discussed, such as jaundice, and the observed frequency of severe disease in adults, perhaps indicating a waning of malaria immunity in this population. This paper is an example of “real world” immunology in settings where infections can be complex mixes of multiple parasite species simultaneously, or where a single individual can experience alternating bouts of malaria caused by different species.SM can be the result of infection with lleagues  present This collection of papers reminds us that, along with the need to target mosquito transmission and the necessity of providing rapid access to effective malaria drugs, malaria control strategies must also enlist the defensive capabilities of the human immune system – do not forget the Alamo. This is not an easy task, as illustrated by our dearth of effective vaccines , our lack of diagnostic markers for risk of complicated disease and our currently imperfect understanding of how to minimize harmful pathology in severe malaria. The work described herein is therefore presented as an indication of progress using a number of promising approaches, each of them a reason for optimism that this task may yet be accomplished.


Malaria is a leading cause of morbidity and mortality among children under five years of age, with most cases occurring in Sub-Saharan Africa. Children in this age group in Africa are at greatest risk worldwide for developmental deficits. There are research gaps in quantifying the risks of mild malaria cases, understanding the pathways linking malaria infection and poor child development, and evaluating the impact of malaria on the development of children under five years. We analyzed the association between malaria infection and gross motor, communication, and personal social development in 592 children age 24 months in rural, western Kenya as part of the WASH Benefits environmental enteric dysfunction sub-study. Eighteen percent of children had malaria, 20% were at risk for gross motor delay, 21% were at risk for communication delay, and 23% were at risk for personal social delay. Having a positive malaria test was associated with increased risk for gross motor, communication, and personal social delay while adjusting for child characteristics, household demographics, study cluster, and intervention treatment arm. Mediation analyses suggested that anemia was a significant mediator in the pathway between malaria infection and risk for gross motor, communication, and personal social development delays. The proportion of the total effect of malaria on the risk of developmental delay that is mediated by anemia across the subscales was small (ranging from 9% of the effect on gross motor development to 16% of the effect on communication development mediated by anemia). Overall, malaria may be associated with short-term developmental delays during a vulnerable period of early life. Therefore, preventative malaria measures and immediate treatment are imperative for children’s optimal development, particularly in light of projections of continued high malaria transmission in Kenya and Africa. P. falciparum) has severe cerebral symptoms associated with it [P. falciparum in the country [Malaria is a leading cause of morbidity  and mort with it . Malaria country .Forty-three percent of children under five in low- and middle-income countries (LMICs) are also at risk of not meeting their developmental potential, with children in Africa being at highest risk due to many factors, including extreme poverty . There iP. falciparum infection and cognitive function, Kihara et al. reported that specific parts of the brain may not always be impacted more than others by malaria as the effects can vary and be diffuse [Developmental deficits related to malaria are complex and varied. In a systematic review of the relation between  diffuse . Impairm diffuse . Malaria diffuse ,13, moto diffuse , memory  diffuse ,15,16, e diffuse  in prosp diffuse ,11. The path to cognitive impairment may result from many interacting biological and social risk factors . Risk faThe functions that are impaired may depend on the stage of brain growth at which the infection is acquired and impairment may not manifest until a child is older . Age at Malaria may impact early childhood development through a pathway of anemia/low hemoglobin concentrations. It is well-established that malaria causes anemia, especially in children under five years in Sub-Saharan Africa ,27,28,29Although there is evidence that iron-deficiency anemia can lead to poor child development, there is a research gap in assessing malaria, anemia, and child development. Higher iron concentrations can increase malaria risk among children . HoweverMalaria is widespread throughout Kenya and a leading cause of morbidity and mortality in the country . About 7This study aimed to describe the association between malaria infection and early childhood development. The objectives of the research were to: 1) investigate associations between malaria infection and three child development outcomes, and 2) assess the role of anemia as a potential mediator in the pathway between malaria infection and child development outcomes. The following two connected hypotheses were tested: 1) malaria infection is associated with increased odds of being at risk for gross motor, communication, and personal social developmental delays, and 2) anemia is a significant mediator along the pathway between malaria infection and being at risk for gross motor, communication, and personal social developmental delays.These data were collected during the WASH Benefits trial, which is a cluster-randomized controlled trial assessing the impact of WASH and nutrition interventions individually and combined on child health outcomes ,45,46. THouseholds were visited at enrollment and two follow-up periods, one and two years later. A sub-study assessing environmental enteric dysfunction, malaria infection, and hemoglobin concentrations from biological samples enrolled 2,304 households from four arms of the WASH Benefits trial  ,44,47. TCaregivers brought study children to a central location for sub-study data collection between August 2015 and April 2016. Blood samples for malaria and anemia diagnoses were taken by trained phlebotomists and health surveys were completed. Caregivers provided written informed consent. Consent for blood collection and a sufficient blood sample was obtained for 699 children and therefore malaria and hemoglobin measurements only existed for this subset of children. An average of two to three months later, caregivers brought study children to a central location for main trial data collection, including a child development assessment. Local enumerators conducted most surveys in Kiswahili, the regional language. Surveys and data collection tools were developed from validated measures  and locaP. falciparum, P. vivax, P. ovale, and P. malariae were conducted using rapid diagnostic kits . The kits can detect parasite concentrations up to 28 days after infection. All children with a positive test result were defined as having malaria in these analyses. Axillary temperatures were taken for all children and if the malaria parasite test was positive and the child’s temperature was 37.5 degrees Celsius or higher, he/she was referred to the nearest clinic for malaria treatment. Hemoglobin concentrations were measured using a Hemocue (Hb 301) machine . Anemia was defined as having a hemoglobin concentration below 11.0 g/dL [Venous blood samples were drawn from sub-study participant children in a central location within villages for biomarker analyses and drops of blood were used to assess malaria parasites and hemoglobin concentrations. Rapid diagnostic tests (RDTs) for 1.0 g/dL . A medicHousehold demographics, including asset ownership and maternal education, were assessed using standardized questionnaires administered at enrollment. Socioeconomic status was defined with an asset index using enrollment survey results for all sub-study participants. The asset index was created using the first principal component from a principal components analysis, which has been shown to be an appropriate proxy for household wealth and is correlated with consumption expenditures ,52,53. IThree domains of child development were assessed: gross motor, communication, and personal social. The corresponding subscales of the Extended Ages and Stages Questionnaire (EASQ) were used to measure each developmental outcome among all WASH Benefits trial participants during the last data collection round (at approximately age 24 months). The EASQ is a modified version of the Ages and Stages Questionnaire (ASQ), which is widely used for the developmental screening of children under five years of age ,55,56 anAll statistical analyses were conducted in Stata 14 . Three sets of logistic multivariate analyses were undertaken to test the hypotheses corresponding to each child development outcome . Two regression models were built for each child development outcome to assess the pathways between malaria infection (binary) and being at risk for gross motor, communication, and personal social delays (binary). The first model included malaria infection (binary) and the child development outcomes (three separate binary outcomes). Anemia status  was added to the second model. The interaction between malaria status and participation in the nutrition intervention was also analyzed in multivariate models to assess whether being in a nutrition treatment arm differentially affected malaria infection. All models were adjusted for child characteristics , household demographics , study cluster, and whether the household received the nutrition intervention (binary). Analyses controlled for arms that received nutrition interventions because they were likely confounders. Mediation analyses were then undertaken to assess whether anemia was a mediator along the pathway between malaria infection and being at risk for gross motor, communication, and personal social delays. The binary_mediation method in Stata ,60,61 waChildren with missing malaria data or child development assessments were removed from analyses. Nine percent of children had missing data from one of these measurements and thus were not included in the final analyses. The final sample size was therefore 592 children. Differences between those who provided consent for blood collection and had a sufficient blood sample, and those who did not were assessed due to the low response rate and no significant differences or systematic attrition were found . https://clinicaltrials.gov/ct2/show/NCT01704105). Ethical approval was obtained from the University of California, Berkeley Center for the Protection of Human Subjects and the Kenya Medical Research Institute Ethical Review Committee. Adult participants provided written consent for themselves and their children prior to enrollment.The WASH Benefits trial is registered as a clinical trial with the U.S. National Institutes of Health , communication (21%), and personal social (23%) delays in this population (had Z-scores in the lowest quartile based on control group domain Z-scores) were similar across domains.Maternal education in this population was low: 53% of mothers did not complete primary school . The meaHaving a positive malaria test was significantly associated with increased odds of a child being at risk for gross motor , communication , and personal social  delays  adjusting for child characteristics, household demographics, study cluster, and participation in the nutrition treatment arm Model 1, . The oddAnemia was a significant mediator in the pathway between malaria infection and risk of gross motor, communication, and personal social development delays  . Across A positive malaria blood test was associated with increased odds of being at risk of gross motor, communication, and personal social delays  while controlling for a range of individual- and household-level variables. The odds of personal social delays were greater than the odds of delays in the other two domains. Across subscales, the addition of the potential mediator (anemia) to models led to smaller odds of delays. Formal mediation analyses demonstrated that anemia was a mediator in the pathway between malaria infection and risk of developmental delay in all three domains. However, the proportion of the total association mediated by anemia was small.The prevalence of malaria (18%) in this population was lower than expected given that the prevalence of malaria in the region is 42% using RDTs . This coThis study has several strengths. The use of blood tests to indicate the presence of malaria parasites provides a robust measure of infection. Including all cases of malaria rather than only severe or cerebral incidents is beneficial for understanding associations between mild forms of malaria that are more widespread and child development. There may be a lag between infection and the manifestation of measurable effects on development, and the measurements in this study were longitudinal; however, the amount of time between the malaria blood test and the child development assessments varied. Incorporating multiple domains of child development yields a more comprehensive understanding of the implications of malaria infection. The use of a control group to calculate standardized child development scores enabled analyses to accurately assess whether a child is at risk of delay in this population. Building models by systematically adding a hypothesized mediator to analyses allowed for a thorough assessment of the potential pathways of impact. Further mediation analyses strengthened findings.There are limitations to these analyses as the pathways between malaria and child development are complex. While there were no systematic differences found between those who provided consent for blood collection and those who did not, the low response rate could have led to selection bias. Resistance to venous blood collection was the primary reason for the low response rate, which was likely due to the high burden placed on respondents related to the collection of other biological and environmental samples, long questionnaires, and the child development assessment. The relatively small sample size could limit the generalizability and precision of the findings. Moreover, the rapid diagnostic tests detect parasites up to approximately one month after infection and do not quantify parasite load. Thus, it is difficult to determine when a child was infected and the severity of malaria, and therefore the extent to which infection could affect child development outcomes. Only one malaria test was performed and repeated infections, which may be common in this endemic area, could have larger effects on child development. Although there was time between the malaria diagnosis and child development assessment, there was only one measurement of each and therefore causal implications cannot be drawn. Further, children in the sample may have had malaria multiple times before the malaria test and child development assessment since they live in an endemic area. This could have made it more challenging to detect effects, thereby making these findings conservative. The single malaria test result is not indicative of general exposure to malaria, hence hindering interpretation of the association with the child development outcomes. Despite there being a lag between malaria and child development measurements, malaria and anemia measurements were taken at the same time, further limiting the conclusions about the pathways of impact. Finally, the mediation analysis did not yield direct and indirect effects of anemia in the complex pathway between malaria infection and the child development outcomes, simplifying the implications of the mediation findings. Overall, this study’s findings highlight how malaria may be associated with child development outcomes. The focus on children under five years adds to the literature as exposure to risk factors like malaria during this vulnerable time period is critical for development, yet understudied. Significant associations between malaria and gross motor, communication, and personal social domains in this young population emphasize the importance of preventing and treating malaria early in life. Patterns of malaria are likely to shift throughout Africa with climate change as certain geographies may be more impacted. Climate change coupled with human population density projections for the continent suggest that malaria transmission will remain high in the coming years ,64, addiMore longitudinal studies with repeated measures of malaria and child development outcomes can inform causal relations in addition to the timing of effects. The assessment of additional domains of development  and long-term versus short-term effects can provide a comprehensive understanding of the impacts of malaria. The role of less severe and repeated cases of malaria can also support the targeting of interventions to the largest number of children who are most susceptible to risks for developmental delay. Longitudinal research assessing other mediators is important for better understanding the nuances of mechanisms of impact and when programs can be most effective. This study provides evidence of how malaria infection may be associated with short-term child development outcomes during a critical developmental period. These findings suggest that anemia, in this case caused by infection rather than lack of iron in the diet, may play a significant role in the pathway. The high prevalence of malaria and large percentage of children at risk for developmental delays in LMICs motivates the need for policies and programs to address the complexities of malaria infection. Preventative malaria measures and immediate treatment are imperative, particularly in light of projections of continued high malaria transmission in Africa. Overall, combating malaria may enable children to reach their full developmental potential, which can have lifelong and intergenerational benefits.


However, platelets function as a source for plasma S1P in malaria needs to be examined experimentally. RBC being the preferential site for parasite seclusion, and having the ability of trans-cellular S1P transportation to EC upon tight cell-cell contact, might play critical role in differential S1P distribution and parasite growth. In the present review, we have summarized the significance of both the S1P pools in the context of malaria, and how the RBC content of S1P can be channelized in better ways for its possible implication in therapeutic opportunities to control malaria.Sphingosine 1-Phosphate (S1P) is a bioactive lipid intermediate in the sphingolipid metabolism, which exist in two pools, intracellular and extracellular, and each pool has a different function. The circulating extracellular pool, specifically the plasma S1P is shown to be important in regulating various physiological processes related to malaria pathogenesis in recent years. Although blood cells (red blood cells and platelets), vascular endothelial cells and hepatocytes are considered as the important sources of plasma S1P, their extent of contribution is still debated. The red blood cells (RBCs) and platelets serve as a major repository of intracellular S1P due to lack, or low activity of S1P degrading enzymes, however, contribution of platelets toward maintaining plasma S1P is shown negligible under normal condition. Substantial evidences suggest platelets loss during  Chemotherapeutics is the mainstay of malaria control in absence of high efficacy approved vaccine and ineffective vector control measures , or from degradation of membrane sphingolipids (such as sphingomyelin and glycosphingolipids) in lysosomes or in the outer leaf-let of cellular membranes   showed opposite results  from endothelial cells in a non-ATP dependent manner ]. These S1PRs vary in their distribution and expression in response to various stimuli , S1PR4−5 have much narrower patterns of expression compared to S1PR1−3 receptors, with ubiquitous expression of S1PR1 in the brain, kidney, lung, spleen and cardiovascular system , degradation  and signaling receptors (S1pr1−3) leading to plasma level of S1P concentration and clinical outcome in malaria. Screening of functional genetic variations in these genes may be useful for better understanding of S1P biology in malaria.Although intracellular receptor for S1P is not well-characterized, it is shown to regulate important intracellular function like ubiquitination, histone deacetylation and gene expression, and respiration /acute respiratory distress syndrome (ARDS) patients  initially, it down regulates S1PR1 function through irreversible induction of S1PR1 internalization and degradation  for execution of immune-protective response against malaria. Some transient pharmacological inhibition studies of S1PR1 in brain endothelial cell also highlight enhanced delivery and accumulation of therapeutics drugs in to the CNS  and RBCs (anemia), the two major sources for plasma S1P to be frequent and are responsible for clinical severity with fatal outcome  were shown essential for platelet formation from megakaryocytes and their shedding in to the circulation  terrify the condition of reduced plasma S1P, there is no study conducted till date addressing whether erythrocytic content of S1P is maintained despite decreased plasma S1P level during severe malaria. Previous study documents RBC content of S1P in non-malaria anemic patients to be similar compared to healthy individual  and Siponimod (antagonist to S1PR1 and S1PR5) are FDA–approved drugs , urinal detection of S1P could serve as a prognostic indicator and needs investigation in this line. Hitherto, substantial evidences suggest plasma S1P is crucial for differential manifestation of malaria. Therefore, selective inhibition of S1P synthesis in RBC, its degradation or abatement though supplementation of adequate acceptor molecules leading to augmented plasma level S1P may impair RBC growth of parasite and protect from severe malaria. Although the involvement of S1P in malaria pathogenesis has been studied only recently; more studies are warranted to answer several of these un-resolved questions before its implication as therapeutics.In summary, S1P seems to play a double-edged sword in malaria pathogenesis. While intracellular content of S1P in infected RBCs is essential for parasite growth and development, its low level in plasma is associated with cerebral malaria in mouse model and human studies. However, how reduced S1P concentration in plasma affects other forms of sub-clinical severe malaria is yet to be materialized. Although diminished S1P concentration in plasma is likely to induce S1P synthesis in RBC, it is yet to be confirmed in parasite infected and/or non-infected RBCs through experimental studies in malaria. Further, whether S1P is involved in inducing rosette formation during GD: conceived the original idea, prepared all figures, and the first draft of manuscript. SS: interpreted the manuscript drafting for important intellectual content. All authors discussed and contributed to the final 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.