Malaria in endemic countries is often asymptomatic during pregnancy, but it has substantial consequences for both the mother and her unborn baby. During pregnancy, anaemia is an important consequence of malaria infection. In Burkina Faso, the intensity of malaria varies according to the season, albeit the prevalence of malaria and anaemia as well as their risk factors, during high and low malaria transmission seasons is underexplored at the household level.Data of 1751 pregnant women from October 2013 to March 2014 and 1931 pregnant women from April 2017 to June 2017 were drawn from two cross-sectional household surveys conducted in 24 health districts of Burkina Faso. Pregnant women were tested for malaria in their household after consenting. Asymptomatic carriage was defined as a positive result from malaria rapid diagnostic tests in the absence of clinical symptoms of malaria. Anaemia was defined as haemoglobin level less than 11 g/dL in the first and third trimester and less than 10.5 g/dL in the second trimester of pregnancy.Prevalence of asymptomatic malaria in pregnancy was estimated at 23.9% (95% CI 20.2–28.0) during the high transmission season (October–November) in 2013. During the low transmission season, it was 12.7% (95% CI 10.9–14.7) between December and March in 2013–2014 and halved  between April and June 2017. Anaemia prevalence was estimated at 59.4% (95% CI 54.8–63.8) during the high transmission season in 2013. During the low transmission season, it was 50.6% (95% CI 47.7–53.4) between December and March 2013–2014 and 65.0% (95% CI 62.8–67.2) between April and June, 2017.This study revealed that the prevalence of malaria asymptomatic carriage and anaemia among pregnant women at the community level remain high throughout the year. Thus, more efforts are needed to increase prevention measures such as IPTp–SP coverage in order to reduce anaemia and contribute to preventing low birth weight and poor pregnancy outcomes. Every year, about 25% of maternal deaths in hyperendemic malaria regions are due to malaria infection in pregnancy (MiP) . In sub-To protect women from asymptomatic MiP and its consequences, the World Health Organization (WHO) recommends combined intervention, including intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp–SP) and iron + folic acid supplementation during antenatal care (ANC) . Albeit,In Burkina Faso, around three-quarters of the population use self-medication or traditional therapy when signs of malaria are suspected . By implThis study, aimed to estimate the prevalence of asymptomatic carriage of malaria parasites and anaemia among pregnant women in their community through a household survey during low and high malaria transmission seasons in Burkina Faso. The study also aimed to identify the potential factors associated with asymptomatic carriage and anaemia among pregnant women surveyed at their household.This study analysed data collected from 24 health districts located in six regions of Burkina Faso . The population consisted of pregnant women who were present in the household during the survey visit and who consented to participate in the study. The main project, entitled “Impact Evaluation for Health Performance-Based Financing in Burkina Faso”, was designed to assess the impact of a performance-based financing strategy on the quality of healthcare delivery . The latData on sociodemographics, health behaviour, and economic factors as well as on the health conditions of pregnant women were collected for each selected household. These data were collected using a computer-assisted personal interview (CAPI) household questionnaire. From these two cross-sectional household surveys, data on 1751 and 1931 pregnant women were drawn in 2013/2014 (from October 2013 to March 2014) and 2017 (From April to June 2017). To provide a snapshot of the prevalence, the timeline of data collection in the field was for a full year of malaria transmission . The first data collection was conducted between October 2013 and March 2014, and the second one occurred from April to June 2017.HRP-II). HRP-II-based diagnostic test accuracy with microscopy as reference test for detection of malaria in asymptomatic pregnant women in Burkina Faso was 96.4% (95% CI 91–99) and 73.9 (95% CI 68–79), respectively, for sensitivity and specificity [During the household-based surveys, blood samples were taken by finger prick to detect the presence of the malaria parasite and to measure haemoglobin level. In both surveys, malaria was confirmed by serology using RDT SD Bioline, which detects histidine-rich protein II . During the field surveys, quality controls were carried out on randomly selected malaria-positive samples (microscopy performed on thick blood smears). Similarly, quality controls were performed on haemoglobin measures using an automated haematology analyzer.Since the definition of anaemia in pregnancy varies according to the trimester of pregnancy to reflect changes in blood volume, in this study the pregnant women was categorized as anemic if the haemoglobin level (Hb) was < 11 g/dL in the first trimester, < 10.5 g/dL in the second trimester and < 11 g/dL in the third trimester .This study assessed two main binary outcomes: asymptomatic carriage of malaria parasites (Yes or No) and anaemia (Yes or No) according to the definition mentioned above . The expOther variables included the mother’s education level (no education and educated), ownership of insecticide-treated nets in the household (No and ≥ 1), the household’s standard of living , and place of residence . To analyze malaria transmission among pregnant women, the study period was cut into two seasons: a high transmission season from July to November and a low transmission season from December to June . It is nThe prevalence of asymptomatic carriage was estimated based on the proportion of pregnant women who tested positive with the malaria RDT. Likewise, the prevalence of anaemia was determined as the proportion of pregnant women with a haemoglobin level less than 11 g/dL in the first and third trimester, and < 10.5 g/dL in the second trimester among those who underwent haematological testing. Descriptive statistics were used to document asymptomatic carriage of malaria parasites, anaemia, and sociodemographic characteristics of pregnant women. Cross-tabulations were performed separately for each time point of the survey.For each main outcome , both univariate and multivariable modified Poisson regressions with generalized estimating equations (GEE) were performed. The selection of variables for multivariable analysis was based on epidemiological interest and based on previous studies that exhibited links between potential risk factors and the study outcomes. The modified Poisson regression with GEE were used to estimate unadjusted and adjusted prevalence ratios (Adj. PRs) with 95% confidence intervals (CIs), and two-tailed p values < 0.05 were considered as statistically significant. The generalized variance-inflation factors were computed to check for multi collinearity between the explanatory variables .All statistical analyses were performed with R statistical software , and regression models were fitted using the “geepack” package.A total of 7844 and 8182 households were included for the 2013/2014 and 2017 surveys, respectively. Of the households included in the main study, 21.8% (1709/7844) and 23.4% (1916/8182) had at least one pregnant woman for the 2013/2014 and 2017 surveys, respectively. The sociodemographic characteristics of the pregnant women for each survey are summarized in Table The prevalence of asymptomatic carriage of malaria parasites and 95% CIs by gravidity and according to malaria transmission season is presented in the Fig. The results of multivariable analyses to identify potential factors associated with asymptomatic carriage are summarized in Tables In 2013/2014, the mean haemoglobin was estimated at 10.5 (SD = 1.6) g/dL and prevalence of anaemia was 35.9% (95% CI 33.6–38.2). In year 2017, the mean haemoglobin was 10.2 (SD = 1.4) and prevalence of anaemia was 46.6% (95% CI 44.3–48.9). The prevalence of anaemia 95% CIs by trimester of pregnancy and according to malaria transmission season is presented in the Fig. In general, as shown in Tables The presence of malaria and anaemia in pregnancy, regardless of the gestational stage, are potentially harmful to both the fetus and mother as well as to the family and community , 37, 38.The study findings show that the prevalence of asymptomatic MiP and anaemia at the household level was high. In this present study, the MiP prevalence was higher among young women, increased significantly during the high transmission season and increased the risk of maternal anaemia. For the survey conducted in 2017, the risk of MiP was higher during the first trimester of pregnancy.. These risk factors have been documented in other previous studies [The prevalence of asymptomatic carriage of malaria parasites found in this household-based study was lower compared with the estimated prevalence reported in previous studies, where it was reported to range from 19 to 51%, among pregnant women attending health facilities as part of their ANC in Burkina Faso –28. Howe studies , 41–43.  studies . From th studies , several studies , 46.The study showed a high prevalence of anaemia among pregnant Burkinabe women living in their communities, though this prevalence seemed to be slightly lower compared with the prevalence among pregnant women attending health facilities as part of their ANC , 28. AccAlthough the findings of this study provide an overview of the extent of asymptomatic malaria and anaemia in pregnant women at the community level, both during the high and the low transmission seasons in Burkina Faso, some potential limitations need to be considered. First, asymptomatic carriage of malaria parasites was defined by using a malaria RDT, which could lead to underestimation  or overestimation  of the true prevalence. False negatives from the malaria RDT may be due to the detection threshold of the test (around 200 parasites/μL) . RegardiThis study showed a high prevalence of both asymptomatic malaria and anaemia during pregnancy, and it indicated the risks increased dramatically during the high transmission season. Thus, infected anaemic (or non-anaemic) women, apart from consequences to their unborn babies (being considered as reservoirs of parasites that may promote mother-to-child transmission), represent an important parasite reservoir contributing to the cycle of malaria transmission in the community. In order to mitigate the harmful effects of asymptomatic carriage of malaria parasites and anaemia for both the mother and her fetus, health programmes aimed at increasing the number of pregnant women coming into contact with health workers should be at least maintained or strengthened. Therefore, more efforts are needed to increase IPTp–SP coverage, at both the health facility and community/household level, to prevent poor pregnancy outcomes , 44. Thi


While malaria morbidity and mortality have declined since 2000, viral central nervous system infections appear to be an important, underestimated cause of coma in malaria-endemic Eastern Africa. We aimed to describe the etiology of non-traumatic comas in young children in Benin, as well as their management and early outcomes, and to identify factors associated with death.From March to November 2018, we enrolled all HIV-negative children aged between 2 and 6 years, with a Blantyre Coma Score ≤ 2, in this prospective observational study. Children were screened for malaria severity signs and assessed using a systematic diagnostic protocol, including blood cultures, malaria diagnostics, and cerebrospinal fluid analysis using multiplex PCR. To determine factors associated with death, univariate and multivariate analyses were performed.OR)= 8.6; 95% confidential interval (CI): 2.03–36.1] and lactate > 5 mmol/L . Antibiotic use before admission  and vaccination against yellow fever  protected against mortality.From 3244 admissions, 84 children were included: malaria was diagnosed in 78, eight of whom had a viral or bacterial co-infection. Six children had a non-malarial infection or no identified cause. The mortality rate was 29.8% (25/84), with 20 children dying in the first 24 h. Co-infected children appeared to have a poorer prognosis. Of the 76 children who consulted a healthcare professional before admission, only 5 were prescribed adequate antimalarial oral therapy. Predictors of early death were jaundice or increased bilirubin [odd ratio (Infections were found in all children who died, and cerebral malaria was by far the most common cause of non-traumatic coma. Missed opportunities to receive early effective antimalarial treatment were common. Other central nervous system infections must be considered in their management. Some factors that proved to be protective against early death were unexpected.The online version contains supplementary material available at 10.1186/s40249-022-00956-2. Plasmodium falciparum parasitemia. Both CM and CNS infections are life-threatening conditions that require the administration of adequate medications  and jaundice or its biological equivalent . Antibiotic administration before hospital admission  and vaccination against yellow fever  protected against mortality ]. The remaining three had no evidence of meningitis or BSI and had a favorable outcome. These findings contrast with those from other parts of the world where non-infectious  etiologies of comas are more prevalent [We attempted to fill a gap of knowledge on the etiologies, clinical history, clinical and biological profiles, management and outcome of non-traumatic coma in small children from West Africa, a part of the world where access to investigations is limited most of the times to malaria diagnosis. Our prospective study was performed in teaching hospitals and specifically designed to decipher the etiologies of non-traumatic comas, with the help of an independent panel of experts in order to limit the risk of misdiagnosis, provided that non malarial etiologies could represent a substantial part of the cases. We identified only six children with no evidence of malaria. Three of them had a potentially lethal infection [measles, dengue fever, and revalent , 16.. The first confirmed cases of dengue fever acquired in Benin were reported in 2010; since then, few cases have been reported [Aedes, WNV is transmitted by mosquitoes of the genus Culex which have a nocturnal activity like the anopheles which transmit malaria. The use of mosquito nets thus ensures a prevention of the transmission of these 2 infections. In our study, mortality was higher in children with coinfections, but the small number of children precluded drawing firm conclusions. According to a large study on the role of viral CNS coinfections in CM, viral CNS infections are unlikely to contribute to coma [A study performed in 2013 in Malawi showed that adenovirus infection is the most common cause of viral CNS infection leading to hospitalization or death. Our results do not corroborate this finding. Five types of viruses, including two arboviruses (one dengue virus and one WNV), were identified in eight children. A measles epidemic occurred during the study period, and we found five children with specific measles IgM as indirect diagnosis of measles, three of whom were coinfected with malaria. The infection could have been prevented in the 3 unvaccinated childrenreported . Endemicreported , but we  to coma .P. falciparum malaria, the proportion of BSI was estimated at 6.4% [Overall, three BSIs were identified at admission . Therefore, tracking BSI is essential in the management of non-traumatic coma. We assume that the child with malaria and an aseptic purulent meningitis coinfection died of meningitis. In a systematic review of invasive bacterial coinfection in African children with  at 6.4% , and thi at 6.4% , we beliCM was the most common cause of non-traumatic coma. In this part of Africa, as well as in other areas endemic for malaria, public health policy efforts have focused on implementing strategies for malaria prevention  and severe malaria prevention . Despite this, the remaining substantial burden of severe disease related to malaria should not be neglected. It has been advocated that continuing investment for clinical research on severe malaria is needed . Health A large majority of the children reported one or more visits with a healthcare professional before admission. For most children in whom CM was eventually diagnosed, missed opportunities for early and effective antimalarial treatment were found. Effort is thus needed to enhance access to affordable and efficient drugs for uncomplicated malaria , 25.Our study also provides information on the risk factors of early death in this population, some of them being unexpected. Mortality remained high despite access to standardized care including intravenous artesunate, and broad-spectrum antibiotics, performed by an experienced teaching hospital staff. Most deaths occurred within a few hours of admission. This is likely because this study involved the most severe forms of CM in the majority of children, sepsis, and other life-threatening diseases. However, malaria and/or another infection were found in all children who died. Some of these children could have benefited from intensive care. As in many other low-income countries , there iAmong the biological anomalies encountered in sepsis, regardless of its etiology, Jaundice and/or increased bilirubin, as well as lactate above 5 mmol/L at admission were the strongest predictors of subsequent death. This is not surprising because these are additional severity criteria for coma in severe malaria . FurtherUnexpectedly, a history of antibiotic intake declared before admission and a vaccination against yellow fever were predictors of a better outcome in these children. Antibiotics might have had a protective effect against mortality because some of the children had a bacterial monoinfection or coinfection with malaria. Most deaths occurred shortly after admission, before any emergency antibiotic prescription could have been effective. Antibiotics given before admission may play a role in protecting against a fatal outcome, including in children with CM. It may also be an indicator of a better overall management of these children, independently of the treatment administered.Yellow fever was not among the diseases screened for in this study; therefore, it is not possible to state that no yellow fever co-infection existed. More frequent yellow fever co-infections in the children who died might have worsened their prognosis. Another explanation for the protective effect of the yellow fever vaccine could be a non-specific protective effect of this live vaccine. Non-specific effects of live vaccines have been described for the smallpox, BCG, oral polio, and measles vaccines in experimental and observational studies . In BurkThis study has several limitations. Data on clinical history, based on parents/guardians interview at admission and health booklet review may have not been exhaustive. We raised no brain imaging data and we may have missed for example cerebral tumors or bleeding as a cause of coma. The study was not designed to diagnose measles: there was no repeated serologies, and the diagnosis is less robust than for other etiologies. However, we are confident that the diagnoses of CM and infections by non-malaria pathogens have been quite exhaustive and reliable, due to the high quality of laboratory testing and the use of a panel of experts to ensure the best classification of the patients. Finally, despite the fact that this study ranks among the largest cohorts of severe comas in African children and the only one performed in West Africa, it may have lacked power to extensively identify the factors associated with death outcomes. Although NeuroCM was mainly a case–control study to explore inflammation in comatose vs non-comatose malaria infected children , not speCerebral malaria was the most common cause of non-traumatic coma in the study area and was associated with a high mortality rate. For most children, missed opportunities to receive early and effective antimalarial treatment were detected. A history of antibiotic intake declared before admission and vaccination against yellow fever were protective against death in this population. Efforts against malaria should not overlook the need for health facilities that can provide adequate care to children with life-threatening forms of the disease.Additional file 1: Figure S1. Map of Benin with focus on Cotonou and the 2 study sites. Figure S2. Coma etiologies: graphical presentation.


While rapid diagnostic tests are an alternative diagnostic tests for microscopy in the diagnosis of malaria in rural settings, their performance has been inconsistent. Performance of rapid diagnostic tests might be affected by manufacturing process, transportation and storage, parasitemia level, and skill of personnel who perform the tests. Therefore, periodic evaluation of the local field performance of rapid diagnostic tests is mandatory in order to make early corrections in case of decreased performance. Plasmodium infection by CareStart™ rapid diagnostic test kit and thin and thick blood film microscopy. Data were analyzed using statistical software for social sciences version 20 and MedCalc software version 19.3. Sensitivity, specificity, positive and negative predictive values, and kappa value were calculated to evaluate the performance of rapid diagnostic tests against microscopy.  A facility-based cross-sectional study was conducted from January to May 2020 among 257 malaria-suspected patients attending selected health centers in Bahir Dar Zuria district. Capillary blood was collected from each participant and tested for Plasmodium infection by at least one of the diagnostic methods. Rapid diagnostic tests revealed 3 false positive and 3 false negative results. The sensitivity and specificity of CareStart Malaria Pf/Pv Combo test were 93.2% and 98.6%, respectively (kappa = 0.918).  Among 257 study participants, 47 (18.3%) were tested positive for  CareStart™ rapid diagnostic test has comparable performance with microscopy for malaria diagnosis. We recommend continued use of CareStart Malaria Pf/Pv Combo test at health posts in Ethiopia where microscopy is not available. Plasmodium [Plasmodium (P) falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [Plasmodium species, P. falciparum and P. vivax are the two epidemiologically important species in Ethiopia comprising 60% and 40% of infections, respectively. Both species are prevalent in all malaria endemic areas with their relative frequency varying in time and place within a given geographical range [Malaria is a major global health problem which is caused by obligate intracellular protozoan parasites of the genus asmodium . There aknowlesi . Malariaknowlesi . In Ethiknowlesi , 4. Amonal range . Hence, Plasmodium species. Most RDTs are based on the detection of either histidine-rich protein 2 (HRP2), a protein synthesized by P. falciparum and lactate dehydrogenase (LDH) or aldolase, enzymes produced by all human Plasmodium species [The World Health Organization (WHO) recommends that antimalarial treatment should be initiated after laboratory confirmation of suspected cases –7. Altho species , 10.Rapid diagnostic tests have a significant impact on the reduction of malaria burden in the world . They arMalaria-suspected patients should be confirmed by laboratory diagnosis and treated with efficacious antimalaria drugs within 24 hours of the onset of symptoms. Early diagnosis and immediate treatment is one of the strategies in malaria control and prevention . Diagnoskebeles. In the district, there are 9 health centers and 36 health posts. The district receives an average annual rainfall of about 1035 mm. The minimum and maximum temperature lies at 10°C and 32°C, respectively [A facility-based cross-sectional study was conducted in Yinesa, Andasa, and Robit health centers in Bahir Dar Zuria district, northwest Ethiopia, from January to May 2020. The Bahir Dar Zuria district is one of the 14 districts of West Gojjam Zone, which is located at a distance of 560 km from capital city of the country, Addis Ababa, and the district is situated surrounding Bahir Dar city, capital of Amhara National Regional State. The altitude of the district ranges from 1700 to 2300 meters above sea level [ectively .Sample size for assessment of CareStart RDT performance was determined using the Buderer formula for diagnostic test studies . (1)Studn = sample size, z = 95%statistic for level of confidence (Z = 1.96), SN (sensitivity) = 95.4% from previous study from Sheraro, northwest Ethiopia [P  = 13.4% from previous study at Sheraro, northwest Ethiopia [d = margin of error tolerated (d = 0.07), and n = ((1.96)2 × 0.954(1 − 0.954))/(0.07)2 (0.134) = 257.where Ethiopia , P  was 2. Of the first two participants, one patient was randomly selected by lottery method, and then, every 2nd patient was selected to participate in the study.Therefore, a total of 257 malaria-suspected patients were included in the study to evaluate the test performance of CareStart™ RDTs against microscopy. Three health facilities, Andasa health center, Yinesa health center, and Robit health center, were selected using simple random sampling technique among nine health centers. On average, 214, 198, and 142 febrile patients visited the laboratory at Yinesa, Andasa, and Robit health center laboratories, respectively, per two months from January to March in 2019. Therefore, by proportional allocation, 100, 92, and 65 patients were allocated in Yinesa, Andasa, and Robit health centers, respectively. Then, systematic random sampling was used to select study participants, and the sampling interval  provided with the kit, followed by adding 2 drops (60 μl) of reagent buffer on the buffer well. Results were read 20 mins after addition of the sample and buffer [Sociodemographic and related data were collected through face to face interview before blood sample collection by trained laboratory technicians using semistructured questionnaires prepared in English and translated to Amharic. Each febrile patient was sampled with capillary blood and used for protocol , 12. RDTd buffer .Data were checked for completeness and entered and analyzed in Statistical Package for Social Science software version 20. Descriptive statistics was manipulated to explain the study participants and to show the malaria prevalence by CareStart™ RDT and microscopy. Performance of RDTs was measured by calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) against microscopy result using MedCalc software version 19.3. Kappa value was also calculated to show the strength of test agreement between “RDTs and microscopy.” Kappa value was interpreted as follows: values = 0 as indicating no agreement, 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreement .The research was carried out after ethical approval was obtained from the Institutional Review Committee of College of Medicine and Health Sciences, Bahir Dar University. Additionally, supportive letters were obtained from Amhara Public Health Institute, West Gojjam Zone health department, and Bahir Dar Zuria district health office, and permission was obtained from each health center authority. Information obtained at any course of the study was kept confidential. Informed verbal consent was obtained from each participant and assent from guardians for children under 18 years old. Moreover, no personal and health facility identifier was included as participants were given a unique code. Malaria-suspected patients who were positive for malaria or other haemoparasites by any of the tests were linked to the respective health center for appropriate treatment.Among a total of 257 febrile patients participated in the present study, 151 (58.8%) and 106 (41.2%) were males and females, respectively. The participants' age ranged from 1 to 82 years with mean age of 21.0 (±17.1 SD) years. All the study participants were rural inhabitants. Regarding to their educational status, 194 (75.5%) were unable to read and write and the rest 63 (24.5%) were able to read and write or have attended formal education .Plasmodium infection by at least one of the diagnostic methods (RDT or microscopy). Each test separately detected Plasmodium infections in 44 (17.1%) participants. Blood film microscopy results revealed that 30 (11.7%), 11 (4.3%), and 3 (1.2%) participants were positive for P. vivax, P. falciparum, and mixed infections, respectively. CareStartTM RDT detected P. vivax, P. falciparum, and mixed infections in 32 (12.5%), 10 (3.9%), and 2 (0.8%) participants, respectively  of the infections had parasite density greater than 2000 parasites/μl. There were two cases that had a parasitic load less than 200 parasites/μl, which were negative by RDTs.Among 257 participants, 47 (18.3%) were tested positive for ectively . The meaP. falciparum and P. vivax, respectively, by microscopy. On the reverse, 3 samples were false positive by RDT as microscopy results were negative  [Comparison of RDTs' performance against blood film microscopy in the present study revealed sensitivity, specificity, PPV, and NPV of 93.2%, 98.6%, 93.2%, and 98.6%, respectively. This result showed that the sensitivity of RDT in detecting  (95.0%) , Mayani  (95.0%) , Serbo,  (95.0%) , Kenya ( (95.0%) , and Chi (95.0%) . On the  (95.0%)  and Fele (95.0%) . Such va (95.0%) , lot-to- (95.0%) , parasit (95.0%) , observe (95.0%) , mutatio (95.0%) . The RDTP. falciparum infections indicated the low level of sensitivity (81.8%) which was comparable with previous report in Jimma, southwest Ethiopia (85.6%) [P. vivax (93.3%) was in consistent with the study conducted in Kola Diba, northwest Ethiopia (90.9%) [Plasmodium infections [μl while all positive samples by both RDT and microscopy had a parasitemia level of >4000/μl. Moreover, identification of mixed infection by microscopy is also prone to reader errors and needs microscopy experts. Hence, large-scale studies recruiting reasonable number of patient samples with mixed infection and including more sensitive molecular methods are recommended to give definitive conclusion on the performance of RDTs in detecting mixed infections.The attempt to evaluate the performance of the CareStart Malaria Pf/Pv Combo test to diagnose  (85.6%) . However (85.6%) , Mayani  (85.6%) , in Kola (85.6%) , Serbo,  (85.6%) , and Chi (85.6%) . The sen (90.9%) , Serbo,  (90.9%) , and Chi (90.9%) . Likewis (90.9%) . On the  (90.9%) . This difections , 31, 33.Plasmodium, P. falciparum, and P. vivax, respectively, revealed that RDTs could be used for malaria diagnosis at health post and other public health facility levels in the absence of microscopy [P. falciparum and P. vivax  was comparable with previous findings at Felegeselam in northwest Ethiopia (97.8% and 99.9%) [Plasmodium species was comparable with study done at Felegeselam in northwest Ethiopia (97.7%) [P. falciparum-specific HRP2 in patients who had been treated [Based on the WHO limit of detection (specificity > 90%), specificity of 98.6%, 99.6%, and 98.2% for overall croscopy . The sped 99.9%) , Serbo, d 99.9%) , and Chid 99.9%) . The spe (97.7%) , Kola Di (97.7%) , Mayani  (97.7%) , and Chi (97.7%) . Likewis (97.7%)  and west (97.7%) . Such va treated .The RDTs had high NPV (98.6%) and PPV (93.2%) in the present study, which was relatively consistent with a study conducted at Felegeselam Health Center in northwest Ethiopia (99.7% and 97.8%) , Kola DiCareStart™ RDT had very good sensitivity and specificity for malaria diagnosis with an excellent agreement with the reference blood film microscopy. We recommend continued use of CareStart Malaria Pf/Pv Combo test at health posts in Ethiopia where microscopy is not available. However, the test performance in detecting light infections should be evaluated against a more sensitive molecular technique.Different factors affect performance of rapid diagnostic testsPrevious study results do not help to infer for the present situation


Reactive and proactive case detection measures are widely implemented by national malaria elimination programs globally. Ethiopia decided to include Reactive Case Detection (RCD) and targeted Mass Drug Administration (tMDA) approaches as part of their elimination strategy along with rigorous evaluation. The purpose of this study is to compare the impact of RCD and tMDA on malaria elimination over the 2-year study period, by looking at the annual parasite incidence before and after the intervention.The study will be conducted in the East Hararghe zone of Ethiopia. Malaria transmission in the area is low to moderate. This study will deploy a community-based, three-arm, cluster-randomized control trial implemented over 2 years. Forty-eight clusters (16 clusters per arm) will be selected based on the annual number of confirmed malaria cases seen in the cluster. All clusters will receive the current standard of care in terms of malaria elimination interventions provided by the national malaria control program. In addition, following the identification of malaria parasite infection, individuals who reside within a 100-m radius of the index case will receive a diagnosis for malaria and treatment if positive in the RCD arm or presumptive treatment in the tMDA arm. The primary effectiveness endpoint will be measured at baseline and endline for each intervention arm and compared to the control arm using a difference in difference approach.This randomized controlled trial will provide evidence of the impact of the proposed intervention approaches for malaria elimination.NCT04241705. Registration date: January 27, 2020.ClinicalTrials.gov  The order of the items has been modified to group similar items  was 9 million higher than the previous year [Plasmodium falciparum  and Plasmodium vivax , co-exist with Plasmodium malariae and Plasmodium ovale infections (< 1%) [Ethiopia has a unique malaria eco-epidemiology based on altitude. The predominant malaria species, s (< 1%) . Malarias (< 1%) . Similars (< 1%) .Encouraged by the observed reductions, the Ministry of Health (MOH) of Ethiopia set an ambitious goal to be malaria-free by 2030. To achieve this goal, it has taken on a stepwise strategy to eliminate malaria at the district level first, starting in 239 low transmission districts [P. falciparum cases, to use PQ (0.25 mg/kg) for 14 days in addition to chloroquine (CQ) for P. vivax infections in all transmission settings as recommended by WHO [P. falciparum gametocytes, which is responsible for onward transmission, as well the P. vivax hypnozoites responsible for future relapsing episodes.To optimize case management in the context of malaria elimination, the national treatment guideline was amended to include single low-dose primaquine (PQ) 0.25 mg/kg) in addition to artemether-lumefantrine (AL) for all passively detected  mg/kg inIn many countries, the utilization of PQ is hindered by the widespread prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is common in malaria-endemic areas . PQ admiIn addition to optimizing case management, case investigation and response strategies (active surveillance) such as reactive case detection (RCD) and targeted mass drug administration (tMDA) of passively detected cases are being considered as part of the elimination strategy. Cautious of the limited evidence that tMDA or RCD works in elimination settings, there is a need to optimize both the operational implementation of such strategies and the information systems used so as to allow for testing the effect of these strategies on malaria outcomes in elimination settings.P. vivax, and mixed infection whereby the treatment of P. falciparum results in P. vivax relapse, should be considered when developing elimination strategies [P. vivax and gametocidal treatment for P. falciparum of all patients with malaria in regions of co-endemicity could therefore have important implications for ensuring the removal of parasites.In settings like Ethiopia, having multiple parasite species circulating will certainly have implications for malaria elimination. There is already global evidence that treatment failure, recurrence of rategies –11. The Under low transmission settings, acquired immunity and low parasite clonal complexity may allow most infected hosts to remain asymptomatic with undetected, low-density parasitemia , 13. As The highest probability of finding additional cases surrounding the index case was noted to be in the immediate household and within a 100- to 200-m radius of the index case , 25. BasThe primary objective of this study is to measure the effect of RCD and tMDA on malaria annual parasite incidence (API). Secondary objectives include measuring the prevalence of malaria, as well as the measurement of intervention coverage, acceptability of interventions, antimalarial treatment adherence, serious adverse events, cost and cost-effectiveness of the RCD and tMDA strategies relative to a control arm.This study will deploy a three-arm, cluster-randomized control trial implemented over 2 years. Kebeles  will be the unit of randomization (referred to as clusters hereafter). A cluster has one Health Post (HP) linked to the nearest Health Center (HC) providing health services in the catchment area. Forty-eight clusters (16 clusters per arm) will be selected based on the annual number of confirmed malaria cases seen in the cluster. Those clusters which reported confirmed malaria cases between 1 and 50 will be eligible for the study , 4 town administrations, and 587 kebeles (the lowest administrative unit) out of which the majority, 541, are classified as rural. The estimated population is 3,737,469, of which approximately 48% are female . This poP. falciparum cases, 2066 (14%) were P. vivax cases, and treated according to the national treatment guidelines. In addition,1669 (9.9%) clinical cases were treated without parasitological confirmation, which makes the average API 4.5/1000 population. In addition to increased treatment, the zone has also rolled out other prevention interventions such as LLINs, IRS, and a relatively strong surveillance system.Malaria transmission in the study area is low to moderate. According to the MOH updated malaria risk stratification in 2017, 5 woredas were stratified as malaria-free, 13 woredas are categorized as low (API < 5), and 6 woredas are classified as having moderate transmission (API 5 to 100/1000 population)  children less than 6 months of age or < 5 kg; (2) known allergy or history of adverse reaction or chronic/congenital disease contra indicated to any of the intervention drugs; (3) individuals with severe malnutrition or signs of severe disease, with evidence of any organ failure or hemoglobin (Hgb) level < 8gm/dl; and (4) household members already covered by the intervention drugs less than or equal to one month before the intervention. In addition, the following individuals will be excluded from receiving primaquine: (1) individuals with G6PD level ≤4 U/Hgb, (2) pregnant women, and (3) lactating women breastfeeding infants less than 6 months of age or with unknown G6PD status.All intervention activities will be conducted by trained health care and study team members. Those study staffs participating in intervention activities will be trained in informed consent procedures, administration of pharmaceuticals, and data collection involving both survey data and blood specimens.Informed consent will be obtained from all study participants in all arms of the study. The informed consent process and documentation will be administered from trained study team members at the time of data collection at the household or health facility where the subject was identified.As part of the consent process, the informed consent form will also describe the potential use of the collected data for secondary analyses. Any request for secondary analyses needs approval from the institutional review board (IRB) of the requesting institute. Data will be aggregated and no personal identifying information will be distributed as part of any secondary analyses.This study will have three arms: a control arm, RCD arm, and tMDA arm. Kebeles will be randomized to one of these arms. As all kebeles in the study area are comparable on key characteristics, it is likely that randomization will produce similar comparators, with potential confounders distributed evenly throughout control and intervention areas.P. falciparum cases; CQ plus 14 days of PQ (0.25 mg/kg daily) for P. vivax cases; and AL plus 14 days of PQ (0.25 mg/kg daily) for mixed infections as well as follow-up at a health facility on days 3, 7, and 13 for those receiving 14 days of PQ to assess adverse events and adherence as per the national treatment guidelines.Kebeles in the control arm will receive the current standard of care in terms of malaria elimination interventions provided by the national malaria elimination program, which are referred to as “optimized malaria control interventions” defined as strengthened surveillance and commodities management systems, scale-up of vector control and case management services, and social and behavior change communication to seek prompt treatment and use LLINs. Case management will follow national treatment guidelines. This includes passive detection of malaria cases and treatment with AL plus single low dose PQ (0.25 mg/kg once) for Kebeles in the RCD arm will get optimized malaria control interventions as described above. In addition, following identification of parasite infection via microscopy or RDT among passively-detected index cases at the HP or HC, individuals who reside within a 100-m radius of the index case will receive diagnosis for malaria using a conventional RDT. Positive individuals will receive treatment and follow-up as per the national treatment guidelines. Fourteen days of PQ will only be administered to those found to have G6PD levels ≥4 U/Hgb on the standard G6PD test . Additional procedures will include the collection of a dried blood spot (DBS) for molecular confirmation and secondary analyses.Kebeles randomized to the tMDA arm will get the optimized malaria control interventions as described above. In addition, following the identification of a parasite infection via microscopy or RDT among positive index cases at the HP or HC, individuals who reside within a 100-m radius of the index case will receive presumptive treatment with AL plus 14 days of PQ (0.25 mg/kg daily); 14 days of PQ will only be administered to those found to have G6PD level ≥4 U/Hgb.Dark urine with Hillmen  urine coHypersensitivity reaction necessitating the discontinuation of the study drug(s), as indicated aboveSymptomatic anemia or hemoglobin level < 5 g/dlA patient who developed a serious adverse event will be referred to the nearby hospital and the study will facilitate the transportation. If any of the following symptoms occur, the patient will be withdrawn from the study and will be managed at the HC or referred to a nearby hospital for better care:In both RCD and tMDA arms, one family member will be recruited as a treatment supporter and will be instructed to directly observe pill(s) being swallowed at all times.Subjects who do not attend the clinic for follow-up will be traced by the study team member who will counsel the subject on the importance of follow-up care. Any subject in the intervention arms who develop a serious adverse event (SAE) will be referred to a hospital and the study will facilitate transportation.None, beyond the standard care of patients in the Ethiopian malaria treatment guideline. Study participants will exit the study after the completion of the intervention. There will be no further follow-up.The effect of RCD or tMDA will be defined as a change in malaria API among kebele residents measured by microscopy or RDT tests at HCs or HPs. The malaria API will be defined as all passively confirmed malaria cases over a period of 12 months among residents of the selected kebele divided by the estimated population in the selected kebele multiplied by 1000. The primary effectiveness endpoint will be measured at baseline, and at endline for each intervention arm and compared to the control arm using a difference in difference approach.Prevalence of malaria infection, measured cross-sectionally at baseline and 24 months later at endline using a household survey in all three arms: prevalence will be estimated using RDT and molecular testing.Proportion of households reporting intervention coverageProportion of household respondents reporting acceptability of intervention in RCD and tMDA arms, measured cross-sectionally at endline using a household survey in all three armsProportion of household respondents reporting adherence to antimalarialsThe total costs of RCD and tMDA, measured using program recordsCost-effectiveness of intervention (RCD and tMDA) relative to control arm standard of care measured using program records and financial reportsThe proportion of imported versus locally acquired incident cases, measured cross-sectionally at baseline and at endline using a household survey with travel questions in all three arms, in addition to measurement at health facilities during visitsMalaria burden as measured by antigen, serology, and molecular testingThe proportion of adverse events and serious adverse events identified in the RCD and tMDA armThe study duration is 24 months. Subjects residing in selected study arms will receive a baseline household survey at time zero, and a second after 24 months. Routine health facility visits will take place as usual throughout the study period.t test of the event-rate difference was used with a significance level of 0.05.Assuming that the RCD intervention would decrease API by at least 40% and the tMDA intervention by more than that, based on the previous year’s data, we assume a baseline API of 7.8/1000 population and expect to decrease to 4.7/1000 population in the intervention arms. Sampling 16 clusters in each group or arm with an average of 500 person-years per cluster achieves 80% power to detect a difference of − 0.03100 between the treatment event rate 0.047 and the control event rate 0.078. The between-cluster coefficient of variation in the control group and in the intervention group was assumed to be 0.5. A two-sided Total population of a kebele is estimated to be 5000 people; data from the HP was used to determine the burden to inform sample size calculation. The cross-sectional survey population will include all residents in 20 randomly selected households in 48 study clusters (16 per arm). A total of 960 households will be needed to detect a reduction in sero-prevalence from 17 to 8% between baseline and endline between arms .Random selection will be used to recruit respondents for the household survey and prevalence testing. Routine health facility visits will be used to recruit index cases for the intervention strategies, and for the collection of incidence data.N/AN/AIntervention and standard of care activities in all arms will be implemented by the national malaria elimination program and study team members.In the control arm, the surveillance officer based at the district level will monitor the collection of malaria data by health staff at HPs/HCs using standard reporting form(s), which includes a follow-up and adherence assessment for primaquine treated cases. In addition, the officer will document any other interventions not routinely conducted in the cluster. In short, the surveillance officer will ensure that the standard of care is executed in all the control kebeles by closely working with the woreda focal persons and other responsible government institutes.P. falciparum, CQ+ 14 days of PQ for P. vivax, or AL + 14 days of PQ for mixed infections. Women between 12 and 49 years of age with an unknown pregnancy status will be tested for pregnancy as per national guidelines, upon obtaining verbal consent. For women ages 12–17 years, verbal consent from parents and verbal assent from the mature minor will be obtained for a pregnancy test. Primaquine will not be administered to pregnant women or lactating women who are breastfeeding infants less than 6 months of age. Women in the first trimester of pregnancy will be treated with quinine tablets for P. falciparum, while those in the second and third trimesters will receive AL for P. falciparum or mixed case. CQ will be given for P. vivax cases in all trimesters. Age-eligible women refusing the pregnancy test will only receive appropriate first-line blood-stage treatment without PQ as per the national treatment guideline. Infants < 6 months of age will be treated with standard treatment according to national guidelines, which does not include PQ.In the study clusters, individuals with a history of fever or axillary temperature ≥ 37.5 °C and who seek care at a HC or HP will be tested with microscopy or RDT, respectively, per national guidelines . IndividP. falciparum, CQ plus14 days of PQ for P. vivax, or AL plus 14 days of PQ for mixed infections. G6PD testing will be done in P. vivax and mixed infections using the SD Biosensor STANDARD G6PD test. The result will be provided to the participant and 14 days of primaquine administered only to those confirmed to have a G6PD value ≥4 U/Hgb. A designated family member will serve as a treatment supporter and directly observe the medicines being taken every day. Women of childbearing age with an unknown pregnancy status will be tested for pregnancy and provided treatment as per the national treatment guidelines. Infants < 6 months of age will be excluded from the study.In the RCD arm, the surveillance assistant will notify the district malaria focal person and the study surveillance officer within one day of a positive diagnosis at the HP or HC. Case investigation will be done within three days using the case investigation form at their respective facilities. Within 7 days, the study team in charge of RCD, surveillance officer, surveillance assistant, and health extension workers (HEWs) will visit the patient at her/his home. Then, all consenting index household members and members of households within a 100-m radius will be tested for malaria using RDT. In addition, three dried blood spots will be collected on Whatman 903 filter paper for subsequent laboratory-based malaria testing. Individuals found positive by RDT will be treated with AL plus a single dose of PQ for In the tMDA arm, case reporting and investigation will be done as stated in the RCD arm. The tMDA team will visit the diagnosed index case, at his/her home within 7 days. All consenting and eligible household members and members of households within a 100-m radius will be treated without RDT testing for malaria with AL plus 14 days of PQ after receiving G6PD testing and only those with G6PD value ≥4 U/Hgb will be given PQ. The G6PD test results will be provided to the participant. Febrile household members will be tested with RDT and will be treated regardless of the test result. If the RDT is negative, they will be advised to seek further care at the nearest health facility. Women of childbearing age with an unknown pregnancy status will be tested for pregnancy with consent. Women in the first trimester of pregnancy will be treated with quinine while those in the second and third trimesters will receive AL as per the national guidelines. A designated family member will serve as a treatment supporter and directly observe the medicines being taken every day. Age-eligible women refusing the pregnancy test will be excluded from the study. Infants < 6 months of age will be excluded from the study.P. falciparum and administered only a single low dose PQ will be asked to return for follow-up only on day 3 to assess for adherence and adverse events. During the follow-up, the surveillance assistant will administer a brief questionnaire regarding adherence to the treatment regimen and ask about any side effects experienced. He/she will also conduct a pill count and check for any leftover medications or empty drug packaging. In addition, if a participant fails to come to HP/HC for follow-up on scheduled dates, the surveillance assistant will follow up with a phone call and/or a home visit. During the home visit, there will be a questionnaire to determine the reason for missing the follow-up visit.To ensure participants’ adherence to their medication, the community will be educated on how to take their medications, the side effects of the treatment, and the importance of adherence. Participants in the control arm, will follow the national guidelines which requires follow-up on days 3, 7, and 13 using the national follow-up form if taking 14 days of PQ. Those receiving other regimens will only return to the health facility if presenting with symptoms as per the national guidelines. A more detailed adverse events assessment will be conducted at all follow-up visits. Similarly, for participants in the RCD and tMDA arms participants taking 14 days of PQ will be instructed to come to the health post on days three, seven, and thirteen after the initial treatment to assess adherence to and adverse events of treatment. In the RCD arm, those testing positive for According to the Ethiopian national malaria treatment guideline, laboratory confirmation of malaria is done using RDT at the HP level and microscopy at HC’s and above. G6PD test is conducted by SD Biosensor standard G6PD test. To ensure quality laboratory diagnosis, the study team at the initiation of the study will provide appropriate training to the laboratory personnel at the HC, and the HEWs from the intervention kebeles. The study team will make regular supervision visits to the study sites and perform quality assessments using the procedure recommended by the national malaria diagnostic quality assurance scheme. The project will also make sure all laboratory consumables will be quality checked prior to importation and utilization at the sites. An internal quality check will also be instituted on a subsample of specimens. The following tests will be performed to strengthen the interpretation of the findings:P. falciparum parasites will be quantified using standard curves generated from a serial dilution of NF54 ring-stage parasites [P. vivax parasite quantification will be done using plasmid constructs to infer copy numbers as described before. Blood samples in RNA protect buffers will be used for extraction of RNA using the RNeasy Mini Kit (QIAGEN) for gametocyte quantification, gametocyte commitment and maturation assays, sex ratio estimation [DNA will be extracted from 6 mm diameter punches that will be treated with 20 μl Proteinase K (QIAGEN) in a total volume of 200 μl containing Tissue Lysis Buffer (QIAGEN) at 56 °C overnight in a water-bath, followed by extraction using commercial DNA extraction kit (QIAGEN). Real-time quantitative (qPCR) for parasite detection will be performed by targeting the 18S small rRNA gene for Pf and Pv using primer and probe sequences as described , 30. P. arasites . P. vivatimation , asexualtimation –34.Sero-prevalence will be determined using LUMINEX-based multiplex assays as described before . Age-speP. falciparum, by the presence of HRP2 antigen. Any samples found to be positive for any antigen will have total DNA extracted from DBS samples for malaria-specific PCR reactions. As the multiplex laboratory antigen test is more sensitive than field RDTs, estimates can be generated for how well field RDTs performed in detecting malaria antigens.Testing for pan-Plasmodium antigens aldolase and LDH will determine active infection with malaria parasite and if the infecting species is Additional molecular work to assess parasite relatedness, genetic variants of G6PD, and drug metabolism, e.g., cytochrome p450 2D6 as it relates to malaria as well as antigen and antibody testing for diseases of public health importance may be conducted. No other human genetic testing including whole genome sequencing or HIV testing will be undertaken.It was not possible to blind both the participants and health professionals. This is an open-label trial.Not applicable. There is no blinding as part of the interventions in this study.This study will deploy both secondary and primary data collection methods. The secondary data will be used to collect community-level data captured by the routine health systems, while primary data collection will be used to collect household-level information using household surveys.Five years of retrospective malaria case data will be obtained from the routine health management information system (HMIS), population census data, and meteorological data will be obtained from districts and the National Meteorological Agency. These data will be used to describe the study settings using descriptive statistics, mean values for rainfall and temperature, and API .There will be baseline and endline survey. The survey will be conducted by trained data collectors and supervisors using a pretested questionnaire. A census of each cluster will be conducted electronically using tablets. Twenty households from each cluster will be selected randomly for the household survey. For the selected households, information collected will include socio-demographic characteristics, knowledge about malaria transmission and prevention, ownership, and utilization of LLIN, coverage of IRS, and risk factors for malaria infection and access to other malaria control interventions. In addition, all members of the selected households will be tested for malaria to estimate the malaria prevalence in the kebele and those turned positive will be treated according to the national treatment guideline. DBS samples will be collected for further testing.In the control arm data related to important indicators for the study will be collected using a standardize form by the woreda surveillance officer. The information will be collected from the standard HP/HC registration books and reporting forms. In the RCD and tMDA arm, data will be collected using a standardized form prepared for the study. The information will be collected from the standard HP/HC registration books and from the study participants.Study participants will remain in the study until they finish the intervention treatment. Participants are encouraged to visit the study sites for follow-ups and reminded via phone call or home visit.Data collection will be performed using a combination of manual data entry and electronic forms. Data collected in electronic format will be sent in real-time to a central server. Daily feedback will be given to the field team by the data managers. Data collected using manual data entry from all arms will be managed, double entered, cleaned, and verified at the central level. At the end of the study, the cleaned dataset will be shared with the investigators for analysis.All information regarding the participants will remain confidential to the extent allowed by law. Unique identifiers will only be used for data entry and will not be linked to any personal identifiers other than household GPS coordinates. GPS coordinates will be offset by a distance, to allow for standardized spatial analyses, but not the exact location of the household.All case report forms will be kept in a secured location at HPs and transferred to the Addis Continental Institute of Public Health office. Electronic data will be password protected with access limited to authorized study staff. Unique identifiers will be used for the computer-based data entry and blood samples and will not be linked to personal identifiers. Publications will contain only aggregated data; no identifying information will be included.The study will collect DBS to measure malaria antigen, antibody, and molecular  prevalence. A finger prick blood sample (~ 0.3 ml) will be collected from consenting study participants. No human genetic studies will be performed on stored samples. The samples obtained from the study participants will be de-identified when stored for 5 years and will be destroyed thereafter.Association of different factors such as travel history, education status, occupation, and sex, with the outcomes of interest, will be analyzed using univariate and bivariate tests; all analyses will factor in survey design, weights, and clustering as needed.Baseline survey and routine surveillance data will be used to determine the prevalence and API for each arm, respectively, at the start of the study. Data from surveys and routine surveillance will also be used to measure the rate of change in the intervention versus control group, at baseline versus endline time points. Interrupted time series regression models will be used to measure the relative percentage change in API, assuming a 95% confidence interval. The proportion of individuals who adhere to the treatment will be determined by self-report, counting pills, and calculating the number of individuals completing the full, partial, or no dose of medication in the intervention and control groups. The proportion of malaria infections that would be missed by screening will be estimated by counting the number missed by the diagnostic method, compared to a more sensitive diagnostic tool such as antigen-based multiplex PCR. The sensitivity and specificity of RDT(s) against more sensitive diagnostic tool(s) will also be calculated. A difference in difference approach with logistic regression will be used to assess the impact on malaria parasite prevalence between arms using both baseline and endline survey data.Costs will be calculated using an ingredients approach that involves enumerating both the quantity of specific inputs  and the time spent during the intervention. Existing infrastructure and recurrent salary inputs that would be present in the absence of the intervention will not be included in the cost analysis. Although the emphasis of the cost analysis is on determining the cost of RCD and tMDA alone, the cost-effectiveness will then be measured through malaria cases averted as measured through the difference in API in all study kebelesThere will be no planned interim analyses.At this time, there is no planned additional subgroup or adjusted analyses.The study will use several strategies to ensure that participants adhere to the assigned intervention, including tracking lost to follow-up participants. However, we recognize that some non-adherence is unavoidable. Therefore, the result of the study will be analyzed per protocol. The number of participants with missing information will be clearly stated and the missing data will be mentioned.The manuscript is the full protocol. Anyone interested in the consent forms, de-identified participant-level data, or any future statistical code can contact the authors.At each study sites the HEWs and surveillance assistants are responsible for the trial day-to-day activities and ensure the trial is in accordance with the study protocol. The zonal level study coordinator will follow the day-to-day activities and motivate the site study team to recruit all eligible participants, assist with all trial-related questions. A weekly report will be given to the central study coordinator. A monthly site supervision will be conducted to ensure the sites are collecting high-quality data and DBS, and to follow the trial is in accordance with the good clinical practice guidelines. A group of community representatives will be identified after the consultative meeting to serve as a community advisory and engagement committee. Community engagement training will be provided for the committee members and HEWs. Community engagement activities will include house-to-house communication through community advisory and engagement committees. The information provided will focus on why the specific community is in that arm  and, why, when, and how RCD or tMDA will be carried out. All investigators will meet at a regular interval to monitor the progress and review the relevant information.An independent data and safety monitoring board (DSMB), composed of a multidisciplinary group of experts will be established. The DSMB will assess trial progress quarterly and report the ongoing scientific and ethical integrity of the study to investigators and responsible agencies. The DSMB will ensure that the study is conducted and the data are handled in accordance with the provisions of the research protocol.All SAEs and unanticipated problems, regardless of whether there is sufficient evidence to establish causality with the study drug, will be reported to the PI. The PI then will report to all relevant IRBs within 48 hours of the event occurring. Representatives from the study team, including at least one member from each participating institution, will review the event to establish definite causality, probable causality, possible causality or no causality with the study drug or procedures for administering the drug. In the event of a drug-related SAE, Armauer Hansen Research Institute (AHRI) will convene a meeting within a week of the event with the representatives from the study team, the MOH, and Addis Continental Institute of Public Health (ACIPH), to review the case and take any remediating action deemed necessary.The research team will have a regular meetings to ensure enrollment, recruitment, and follow-ups are in accordance with the study protocol. The study is subjected to submit annual report and renewal of the license from the institutional and national ethical review boards.Any amendments will be submitted to the institutional and national ethical review boards prior to implementation. The protocol amendments will be communicated to relevant authorities. epidemiology and biostatisticsThe trial results will be disseminated to all concerning bodies by organizing a dissemination workshop. In addition, the results will be disseminated through academic conferences and published in peer-reviewed journals.This study is a three-arm, cluster-randomized, open-label, controlled intervention trial conducted over 2 years, which has aim of evaluating the impact of tMDA and RCD within 100 m of the index case compared to standard malaria control intervention in reducing malaria incidence in low transmission settings of Ethiopia. A team of researchers from MoH, AHRI, ACIPH, Tulane University, PMI, and other relevant partners from in-country and abroad are involved in this evaluation. The outcome of this study will contribute to the impact and further targeting of Ethiopian MoH malaria elimination interventions strategies.The baseline survey was conducted in January 2020 as scheduled. Although our timeline was to initiate the interventions, immediately after the baseline assessment, we could not make that happen because of the COVID-19 crisis in the country and the limitation of movement. Therefore, the intervention started in November 2020. We acknowledge the delay to start the intervention as planned shortened the planned intervention time.The study is ongoing. The baseline survey was conducted form January to February 2020. The intervention started in November 2020. There was a delay due to the COVID-19 pandemic. Participant recruitment is ongoing and expected to end in January 2022. Endline survey is scheduled for January to February 2022.


Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates.Knowledge of the  Unfortunately, progress has plateaued, and billions of people live in areas of endemic transmission with illness impeding social and economic well-being. Naturally acquired immunity to malaria builds after repeated exposure and prevents illness4; however, it does not prevent re-infection5. A pre-erythrocytic vaccine that attenuates or prevents liver-stage infection, blood stage parasitemia, and gametocyte transmission would be a critical tool for malaria elimination.Despite control efforts, malaria remains an infectious disease of formidable impact. Since 2000, global malaria mortality has decreased by about half, to 405,000 in 20189. Moreover, the pre-erythrocytic phase of Plasmodium parasite development represents a bottleneck during which infection is confined to the liver without symptoms or transmission and is thus an ideal target for vaccine-induced immunity. Therefore, identification of new pre-erythrocytic vaccine targets is a priority that would aid vaccine development. To accomplish this, we previously developed a rationale to classify antigens recognized by the human immune system within days after controlled human malaria infection (CHMI) and before blood stage parasitemia is detected by PCR11. The pre-erythrocytic antigens discovered with this approach are distinct from blood stage antigens recognized by naturally exposed individuals experiencing blood stage parasitemia. Residents of malaria endemic regions develop naturally acquired immunity that includes elevated antibody levels against blood stage parasites that interfere with blood-stage parasite replication and reduce parasitemia levels enough to prevent individuals from experiencing debilitating symptoms12. However, parasitemia in asymptomatically infected individuals with naturally acquired immunity is sufficient to sustain transmission13.Blood stage parasites that can be propagated in vitro have been extensively studied, and the blood stage proteome is well characterized; however, the antigens comprising the pre-erythrocytic proteome in the human liver have been more difficult to classifyPlasmodium falciparum, the deadliest species, occur at various sites in the human host, and are accessible to antibodies detectable in the peripheral circulation. Sporozoites, the parasite stage that invades hepatocytes, are predominantly coated with circumsporozoite protein (CSP) and other proteins. After hepatocyte invasion, merozoites are released from liver schizonts into the peripheral circulation where merozoite surface proteins elicit antibody responses, including antibodies directed against erythrocyte invasion machinery. Merozoites then invade and mature inside red blood cells, where variant surface antigens including PfEMP1 are expressed by the parasite on the surface of infected red blood cells, facilitating endothelial adhesion and sequestration in the brain14 and placenta17 while also eliciting antibody responses20. A minority of blood stage parasites further differentiate into gametocytes that are taken up by the mosquito to complete the lifecycle; antibodies to these gametocytes are also taken up by the mosquito and result in transmission reducing immunity, and possibly transmission enhancing immunity21. In all cases, the prevailing assumption is that after IgM is initially expressed by B cells, isotype switching results in an IgG predominant antibody response with observed and purported functions that promote protection from illness22.Several life cycle stages of P. falciparum. Secretory IgA is a homodimer typically elicited by antigens presented to mucosal sites where known functions include inhibition of virus infectivity, bacterial toxin neutralization, and prevention of bacterial adhesion to host cells23. Nonetheless, IgA could hypothetically be induced in response to pre-erythrocytic antigen traversal through liver mucosal surfaces, a proposed explanation for the presence of mucosal associated invariant T (MAIT) cells enriched for CD161 in non-human primate livers following immunization with whole organism sporozoite vaccine24. Alternatively, infected red blood cells at the intestinal-vascular barrier may have immune effects such as induction of T-cells with gut-homing properties25 or perhaps IgA induction. These hypotheses support the scant previous observations of IgA antibody induction following malaria infection or vaccination27.By contrast, IgA, which is found in the serum and in mucosal secretions, is not thought to be induced by Here we report an expanded pre-erythrocytic immunoproteome that includes IgA reactive antigens in acutely infected CHMI volunteers who were administered infectious sporozoites either by mosquito bite or by intravenous (IV) direct inoculation. We compared the immunoproteomes from these two routes of administration to the immunoproteome resulting from infection in children living in Mali who had previous malaria exposure.P. falciparum (Pf) polypeptides representing 214 unique Pf3D7 genes were probed with sera from three cohorts  CHMI30, blood samples were collected 1, 29, and 57 days post-infection; samples from the 22 volunteers who were parasitemic after challenge were used for the current study. In the third study, 47 Malian children ages 1–6 years of age who were control participants in a vaccine study31 were monitored longitudinally during the malaria transmission season beginning in May 2007 for naturally acquired infection, and pre- and post-infection blood samples were selected for the current study. Heat maps and bar graphs showing the IgG and IgA antibody responses after CHMI challenge and natural exposure are shown in Fig. Protein microarrays displaying a total of 251 The IV and bite CHMI induced IgG antibody responses against a collection of antigens that were not reactive in naturally exposed children  and “low IgA-correlated” (n = 37) with the adult CHMI groups . The breadth scores for the IV CHMI day 29 and 57 timepoints were similar . For IgA, breadth scores increased after infection in the CHMI cohorts and in the subset of Malian children whose responses correlated with the adults who underwent CHMI, but not for the Malian children whose responses had low correlation with the CHMI adults     increased for participants in both CHMI studies (pre-infection compared to day 29) and after malaria illness in Malian children Fig. . The earlts Fig. . Overall2 = 0.13, p = 0.012, Fig. 2 = 0.33, p = 0.000024, Fig. IgG breadth scores were increased in post- versus pre-infection samples Fig. . IgA breP. falciparum proteins than those recognized by IgG responses in semi-immune Kenyan adults11. For the pediatric samples, both the increase in IgG responses with increasing age and the decrease in IgG responses with increasing days after infection for the post-infection blood draw demonstrate expected kinetics of IgG antibody responses in children33.This study compared antibody responses in children who had previous malaria exposures to antibody responses in adult volunteers who had a first malaria exposure by CHMI via two administration routes (mosquito bite and IV). We show that the serologic profile after natural infection in Malian children is different from the response to CHMI in malaria-naïve adults. Malian children demonstrated reduced IgA antibody prevalence and variability in the antigen specificity and breadth of IgG antibodies measured. These findings align with previously published results; healthy adult volunteers who underwent CHMI while simultaneously receiving chloroquine developed IgG responses that were specific to different P. falciparum strain, or factors that lead to inhibition of antibody production34. Notably, when comparing the CHMI responses to responses in Malian children, we observed that CHMI elicited IgA to a greater breadth of antigens and with a higher intensity of seroreactivity than was observed in Malian children following natural infection. The production of IgA post-infection in malaria naïve individuals is an intriguing observation that has several possible explanations, which will be explored in planned future experiments, including experiments that test the extent to which IgA antibodies inhibit invasion of hepatocytes and/or erythrocytes.Lower IgA and IgG responses were observed in the IV CHMI study compared to the bite CHMI study  in Tanzanian adults35 and non-human primate hepatocytes24, mucosal-associated invariant T (MAIT) cells were observed following immunization. MAIT cells, a component of the innate immune response, are enriched at mucosal sites and have been observed to stimulate IgA, IgG, and IgM production in an ex vivo model36. Another study demonstrated that altered signal transduction and inflammation in host hepatocytes during the pre-erythrocytic phase of malaria infection results in upregulation of a human transmembrane mucin, MUC1337. The presence of MAIT cells and upregulation of a mucin both suggest a role of mucosal immunity in the response to P. falciparum infection, perhaps induced when pre-erythrocytic antigens traverse through the hepatic mucosal surface and elicit mucosal immune responses, including IgA. Alternatively, during malaria blood stage infection, γδ T cells migrate through colonic mucosa and subsequently induce an IgA response39. On the other hand, studies conducted in germ-free mice with no mucosal microbiota stimulus and in mice with defective intestinal homing signals demonstrate serum IgA production separate from mucosal IgA production42.One possible explanation for the observation of IgA following malaria infection is that mucosal immunity may be a significant but until recently under-recognized component of the immune response to Babesia, a red blood cell pathogen that causes erythrocyte deformation and lysis, splenic enlargement, increase in number of splenocytes, and transient increases in IgA and IgG antibody titers within weeks43. In humans, malaria is associated with splenomegaly that may be provoked by the spleen’s main function of recycling aged and damaged red blood cells, including those that contain parasites44. Despite parasite-mediated changes in splenic architecture and resulting immunomodulation, the spleen mounts an immune response that includes production of antibodies from splenic plasma cells45. In humans, newly class-switched IgA circle transcripts were as abundant in the spleen as in gut mucosal sites and lymphoid follicles, indicating that the spleen is likely a major source of the predominating monomeric IgA1 in serum46. In mice, a recent review concludes that both peritoneal “innate-like” B1 B cells and splenic marginal zone B cells can contribute to IgA production42. Thus, our data suggest that a transient rise in newly generated IgA positive plasmablasts and plasma cells, possibly induced in the spleen, liver, gastrointestinal tract, and/or peritoneum of infected patients, may have given rise to the transient IgA titers seen in our study population.Our data are consistent with a recent report of experimental infection in naive mice with 47. Bone marrow infection blunts the ability to mount B cell responses including diminution of B memory cells48 that could ultimately attenuate IgA production. In a mouse model, blood stage infection attenuated the development of antibodies to circumsporozoite protein, a highly immunogenic pre-erythrocytic antigen49. The method of IgA and IgG detection used in this microarray study precludes quantitative comparison of IgA to IgG, namely, each isotype was detected with a different fluorophore, and the fluorescence intensity rather than the amount of antibody was the readout. Thus, immune dysregulation in the setting of chronic infection may be more readily detectable as attenuated IgA levels rather than attenuated IgG levels.A possible explanation for the IgA responses observed in only a subset of malaria-exposed children could include immune dysregulation in children. Chronic exposure to malaria leads to attenuation of the antibody response50. Likewise for malaria infections, the first parasite exposure may elicit an IgA response; on subsequent exposure an IgG dominant response may develop. For both dengue and malaria, controlled human infection models allow for the study of first infections in a naïve host. Even the youngest Malian children likely experienced a first exposure as infants while being protected by maternal antibodies; hence “first infections” may not have been observed in this Malian pediatric population and may explain the lower IgA response34. Previously, P. falciparum specific IgA was found in sera from malaria naïve participants undergoing bite-CHMI but not in semi-immune Kenyan adults51. IgA may not have been present in adult Kenyan serum because IgA may have a short half-life or because IgA is only elicited after first or early P. falciparum infections. Concomitantly, IgA levels have been shown to be higher in people who experienced fewer than five malaria infections26.Differential IgA responses in naïve adults vs. malaria-exposed children could alternatively or additionally be explained by timing-dependent changes in class switch recombination. In a human dengue virus challenge model, class-switched IgA plasmablasts following primary but not subsequent dengue infection were unexpectedly observedPost-infection IgA responses for a subset of the Malian children correlated with those of the adult CHMI participants, notably the children with more antibody responses above the detectable level Fig. . By cont52. In the IV CHMI study, volunteers received either NF54 or 7G8, a Brazilian strain that was intentionally chosen because of its genetic distance from NF54. Naturally acquired malaria infections are from diverse parasites rather than specific strains. Therefore, it is possible that the detected antibody responses were lower in people who were infected with strains other than NF54. However, for the IV CHMI study, when serological responses to NF54 (n = 5) and 7G8 (n = 17) were compared, breadth score and seroreactivity did not differ between the two groups, albeit the sample size was small. Therefore, it is possible that strain specific humoral immune responses are difficult to see on a whole protein microarray because conserved immunodominant epitopes elicit high antibody binding53. Peptide microarrays may allow better discernment of strain-specific antibody responses.While all samples were probed on an array with proteins from the 3D7 strain, an important difference between the studies is the infecting parasite strain. The bite CHMI study used NF54, the parent strain of the 3D7 clone that is nearly genetically identicalP. falciparum sporozoites (PfSPZ) or five infectious mosquito bites are standard for CHMI; however, these doses may be higher than what is experienced in any single natural infection. Moreover, the bite CHMI used aseptically reared mosquitos that have increased salivary gland PfSPZ counts as compared to conventionally reared mosquitoes, which may result in elevated challenge sporozoite doses. However, for the original aseptic mosquito bite CHMI study, although a trend was present for dose response when comparing number of bites to parasite density at time of malaria diagnosis28, in the current study we did not observe a dose response when comparing number of bites to IgA or IgG responses, suggesting that the sporozoite dose may not explain the increased antibody responses in CHMI compared to natural infection.Another important difference between malaria infection via CHMI vs. natural infection is the sporozoite dose. A dose of 3200 This study is significant because it compared the antibody response profile to malaria infections induced by mosquito bite vs. administration of aseptic, purified, cryopreserved PfSPZ administered intravenously by direct venous inoculation , showing similarities and differences. Intravenous administration of PfSPZ Challenge is a practical way to conduct CHMI studies worldwide. Mosquito bite challenge, which requires insectaries for propagating infectious mosquitoes, is only available in a handful of institutions worldwide. Knowledge gained from the thorough analysis of these data will be valuable to interpret CHMI study results. This capacity will be important to malaria vaccine researchers and the greater vaccine research community.The relevance of this work to malaria vaccine research and development is the expanded list of pre-erythrocytic antigens recognized by the immune system early post-infection and are candidates for a multi-antigen pre-erythrocytic vaccine. A vaccine against these target antigens would potentially induce sterile protective immunity and prevent malaria transmission.Serum samples from three previously conducted clinical trials were studied Table :n = 32; volunteers who had previously agreed to future use of their samples) aged 18–40 years received 1, 3, or 5 bites from aseptically reared Anopheles stephensi mosquitoes infected with the PfNF54 strain using Sanaria® PfSPZ Challenge Mosquitoes 29. P. falciparum parasitemia was detected by thick blood smear and treated with chloroquine. Serum samples were collected before challenge and two- and four- weeks following challenge.Malaria-naïve adults (n = 30) 18–45 years of age were randomly assigned to one of five treatment groups, four dose groups of Sanaria® PfSPZ Challenge (7G8) or one dose group of PfSPZ Challenge (NF54), all administered intravenously (IV) by direct venous inoculation 30. PfSPZ Challenge (7G8 and NF54) were composed of aseptic, purified, cryopreserved P. falciparum sporozoites (PfSPZ). Serum samples were collected before challenge, 29 and 56 days after challenge. P. falciparum parasitemia was detected by ultra-sensitive PCR in 22 participants and was treated with atovaquone/proguanil. Data from the 22 malaria infected participants were included in the final analysis.Malaria-naïve adults (n = 47) 1–6 years of age were randomized to receive an adjuvanted malaria subunit vaccine (AMA1/AS02A) or rabies vaccine as a control 31. Only volunteers who received the control vaccine were included in the current study. From six scheduled blood draws throughout the first six months of the study, paired serum samples were selected for which a single intervening malaria illness occurred 6–41 days after the first blood draw and 14–42 days before the second blood draw. Malaria diagnosis was via thick blood smear, and all children received standard animalarial treatment when they presented with symptoms.Malian children  reactions and fabricated as previously described54. Seven control spots containing IVTT reaction substrates without DNA template were included on each array (“no-DNA” controls). Affinity-purified goat anti-human IgA and goat anti-human IgG conjugated to Qdot 585 and Qdot 800 , were provided by Grace Bio-Labs, Inc., . Serum samples were probed, arrays were imaged, and signals were quantified for IgA and IgG as previously described51. For each array, the mean of the fluorescence intensity (FI) of the no-DNA controls was subtracted from the FI of each of the other spots to create seroreactivity values for each sample-antigen pair. To minimize batch effect, all arrays were probed within two days. Samples from the three studies were randomized across all arrays with samples from the same individual but different timepoints probed on the same slides. Samples probed on different days and from different batches of slides were correlated, showing that slide printing and probing were reproducible. Samples from one participant were excluded from analysis because duplicate correlation was low.Protein microarrays displaying a total of 251 n = 251), at all timepoints and all participants in the group. A cutoff point for each group was defined as the mean of the negative antibody signal distribution plus three standard deviations (SD) as previously described10. Antigens were defined as serorecognized by a sample if the seroreactivity was above the mixture model cutoff point. This approach allows for clustering observations (antibody responses) that are assumed to arise from a mixture of finite distributions (positive and negative antibody responses) against P. falciparum antigens on the array. Mixture models were created on a per-group basis with the presumption that pre-existing exposure was similar for each group. Seroprevalence was defined by study group for each antigen as the proportion of all samples with FI above the serorecognition threshold. Antigens with a seroprevalence of less than 10% amongst the post-infection samples in all three study groups were excluded from further analyses. The number of responders for each antigen per cohort was compared using a two-proportion z test with Yates continuity correction. All tests used α = 0.05 with application of the false discovery rate based on the Benjamini-Hochberg procedure. Boxplots are presented with median, interquartile ranges, and confidence interval defined as +/− 1.58*IQR/sqrt(n) indicated. All reported p values are two-sided. Analysis was conducted in R and visualized using ‘ggplot2’56. Venn diagrams for Supplementary Fig. http://bioinformatics.psb.ugent.be/webtools/Venn/.For timepoint comparisons, a baseline group that was comprised of Day 1 FI from both CHMI studies was compared to post-infection timepoints. A protein was considered differentially seroreactive for a study group when the median FI of the baseline group and the post-infection timepoint were significantly different by Wilcoxon signed-rank test. Proteins were considered differentially seroreactive between study groups when the median FI for the two study groups were significantly different by Wilcoxon rank sum test. For each study group, a two-component mixture model was constructed using seroreactivity for all proteins n = 51, at alProtocols were approved by institutional review boards of the University of Maryland, Baltimore  and the Faculty of Medicine, Pharmacy and Dentistry, Bamako, Mali . Written informed consent was obtained from the participants of the Aseptic Mosquito Bite CHMI study, the IV CHMI study, and the unexposed controls. Written informed consent was obtained from the parents or guardians for the Malian pediatric vaccine study. All methods were performed in accordance with the relevant guidelines and regulations.Further information on research design is available in the Supplementary InformationSupplementary Data 1Reporting Summary