Plasmodium even if they are taking drug prophylaxis. We applied this assay to samples collected from controlled malaria infection studies, as well as those collected in field studies in malaria-endemic regions in Uganda and Kenya. The assay was sensitive to vector exposure, malaria infection, and endemicity, demonstrating its potential for use in malaria serosurveillance.Reliably assessing exposure to mosquitoes carrying malaria parasites continues to be a challenge due to the lack of reliable, highly sensitive diagnostics with high-throughput potential. Here, we describe an approach that meets these requirements by simultaneously measuring immune responses to both disease vector and pathogen, using an electro-chemiluminescence-based multiplex assay platform. While using the same logistical steps as a classic ELISA, this platform allows for the multiplexing of up to ten antigens in a single well. This simple, reproducible, quantitative readout reports the magnitude, incidence, and prevalence of malaria infections in residents of malaria-endemic areas. By reporting exposure to both insect vectors and pathogen, the approach also provides insights into the efficacy of drugs and/or other countermeasures deployed against insect vectors aimed at reducing or eliminating arthropod-borne diseases. The high throughput of the assay enables the quick and efficient screening of sera from individuals for exposure to  Anopheles gambiae [Anopheles gambiae, less is known about its sensitivity and specificity in detecting exposure to other Anopheles species or even other mosquito vectors. While high antibody levels to gSG6 are associated with an increased risk of contracting insect-borne diseases [Anopheles-borne pathogen. Here, we address this challenge with a serological analysis approach that simultaneously measures markers of (1) exposure to vector , (2) exposure to pathogen (platform can be adjusted or expanded to cover specific pathogens), (3) infection, and (4) transmissibility in the case of Anopheles-borne Plasmodium falciparum (Pf) malaria parasites.Climate change and the continued destruction of balanced ecosystems contribute to an increased prevalence of insect-borne diseases and require the continued development and monitoring of the efficacy of vector control strategies ,2. While gambiae . This andiseases , the prePf antigen circumsporozoite protein (CSP); the central repeat region (NANP) [Pf, merozoite surface protein (MSP)-1 and apical membrane antigen (AMA)-1 [Pf antigens expressed by sexual blood-stage of Pf , Pfs16 [Pf parasites should be detectable by antibody responses to pre-erythrocytic antigens, such as CSP, regardless of whether an individual is taking chemoprophylaxis.The testing panel used for the present study consisted of the following analytes: two peptides representing the immunogenic regions of the mosquito saliva protein gSG6  to measn (NANP)  and the n (NANP)  of CSP,  (AMA)-1 , to meas), Pfs16  and Pfs2), Pfs16 , to measPf in a controlled clinical environment via mosquito bite. In CHMI, malaria typically progresses through the liver stage (pre-erythrocytic) and into the blood stage, where it is detected and then treated. CHMI subjects will be exposed to vector antigens, as well as Pf antigens, through the asexual blood stage of infection, although this blood stage may be curtailed due to prompt testing and treatment. Second, we use samples collected from subjects following immunization using Pf irradiated whole sporozoite (IMRAS) [We apply the panel to three types of blood samples in order to assess its utility for serosurveillance purposes. First, we validate the panel using blood samples collected from a controlled human malaria infection (CHMI) challenge, wherein subjects are deliberately infected by  (IMRAS) . These sAnopheles mosquito and malaria from samples collected both from controlled clinical studies, as well as in field studies in endemic regions. Successful development of this assay would provide a powerful new tool to complement existing entomological surveillance and gauge the endemicity and transmission of malaria in the field.To date, multiplex serological assays have rarely been used for serosurveillance purposes despite their demonstrated value in serological assessments. In prior studies, we have demonstrated that multiplex serological assessment can be used to assess infectious disease exposure, vaccination, and correlates of immunity ,11,12,13n = 25). Sera from individuals that underwent CHMI were collected under a clinical protocol (WRAIR#2572) (n = 9). Sera from individuals vaccinated with IMRAS were collected under a clinical protocol  (n = 21) [Anopheles stephensi mosquitoes as a means of vaccination [n = 52, medium malaria risk) and Kampala, Uganda ), as previously reported [Malaria-naïve sera were obtained from healthy U.S. donors with no history of traveling to geographic regions with malaria through a blood collection protocol (WRAIR#2567) and a commercial source  and served as negative controls ((n = 21) . The objcination . Malariareported .P. falciparum (3D7 strain) proteins  were produced at Genscript . Peptides derived from the Anopheles gambiae salivary gland protein , AMA-1 (3D7), MSP-1 (Pf clone 3D7), Pfs16 (Pf clone 3D7), Pfs25 (Pf clone 3D7), and bovine serum albumin (BSA) as negative control.Recombinant  1 and 2 ), circumThe described multiplex ECLIA methodology is based on the Mesoscale U-PLEX platform utilizing 10-spot ECLIA plates  and performed as previously described . Brieflyt test or a Wilcoxon signed rank test. We applied a multiple test correction using the Benjamin–Hochberg method; p-values were considered significant if their adjusted p-value was <0.05. Principal component analysis (PCA) was carried out by normalizing and scaling the log-transformed values. Data points were colored by group, and ellipses were generated corresponding to 50% confidence intervals for each group, to identify general trends in the data set. Correlation plots were generated using pairwise Pearson correlation coefficients calculated from the log-transformed data. All statistical analysis was carried out in R using the stats, ggplot2, and corrplot.The MSD assay provides a readout in units of mean luminescence intensity, and all readouts were directly log-transformed prior to analysis, without any normalization or subtraction of background. Univariate analysis comparisons between groups  were made using a Shapiro–Wilk normality test followed by a Student’s Anopheles vector antigens (gSG6-P1 and gSG6-P2), two pre-erythrocytic antigens (CSP.NANP and CSP.Cterm), two asexual blood-stage antigens , and two sexual blood-stage antigens (Pfs16 and Pfs25). An overview of the different panel antigens within the context of the malaria transmission cycle is shown in We selected eight antigens for the panel that include P. falciparum-infected mosquitoes for this sample set, i.e., malaria-naïve individuals exposed once to Plasmodium-infected mosquitoes  is the strongest biomarker of exposure to squitoes A. We obsPlasmodium exposure  were tested to establish antibody profiles of “malaria endemicity” in the malaria MSD panel to determine “malaria exposure” and “endemicity” in geographic regions with distinct malaria transmission rates . Due to p < 0.001, t-test) in Uganda. In contrast, Pfs16 specific antibody responses were not different between the two regions. The degree of malaria endemicity is higher in Kampala/Uganda compared to Kericho/Kenya [We identified significant differences in the serological responses between the two geographic regions except for antibody responses to AMA-1, which was not different  and malaria-immune  vs. malaria-naïve samples (“neg”) . Recent Except for Uganda vs. Kenya, there were statistical differences in the antibody titers specific to the mosquito saliva, demonstrating the utility of these vector antigens as exposure markers.After quantitatively assessing the antibody specificities, correlation matrices were generated that indicate which of the antigen-specificities correlate with each other, thus establishing a serological profile . CompariIn conclusion, serological profiles have the potential to inform of malaria-endemicity; the fact that the saliva marker is strongly associated with exposure will provide invaluable information about the attack rate even in populations that are taking malaria prophylactic drugs.We used principal component analysis (PCA) to map the serological responses to vector and parasite between negative controls, IMRAS subjects, and samples collected from Kenya (low to moderate endemicity) and Uganda (high endemicity). We found that repeat-specific CSP responses were largely orthogonal to responses to the other vector and malaria antigens and formed a single axis that reflects recent malaria exposure and, indirectly, transmission intensity A. The reThese trends can be seen more clearly in a PCA using the three antigens that were found to be significantly different in the CHMI samples: gSG6-P1, reflecting the vector response; CSP.NANP, reflecting sporozoite exposure; and MSP-1, reflecting prior blood-stage infection B. Here, The present study establishes the utility and value of multiplexed, multi-antigen serology for entomological and parasitological surveillance. This tool is invaluable towards accomplishing malaria eradication, which requires the accurate assessment of pathogen prevalence and transmission rates. Conventional entomological surveillance, i.e., monitoring and evaluation methods have notable shortcomings: (1) labor intensive and costly; (2) highly variable, depending on trap type and abiotic factors; (3) vector trap sampling does not correlate with exposure to mosquito bites or disease—especially in the presence of interventions such as insecticide-treated clothing or bed nets; (4) sampling measures like human landing catches are associated with multiple concerns including extensive oversight requirements, intrinsic ethical issues, and operator biases.Serological surveillance, i.e., measuring the presence and magnitude of antibodies specific to arthropod saliva and/or pathogens, has a direct relationship to exposure to vectors and the pathogen on both an individual and community level—indicating an evidence-based potential to serve as a proxy to or even replacement of conventional surveillance and evaluation methods ,20,21. TPlasmodial life-stage specific results. By contrast, because we used samples from experimental models of infection, such as CHMI and IMRAS, that halt infection at specific life stages, we are able to demonstrate stage-specific serological patterns of malaria exposure. This has implications for serosurveillance under chemoprophylaxis, wherein malaria exposure may be specific to certain Plasmodial life-stages.A recent study by Wakeman et al. applied a similar multiplex serology approach to measure antibody responses to vector and malaria antigens, using samples from Kisumu, Kenya, and from United States travelers with documented malaria infection, employing the Luminex platform . Their 1Plasmodium antigens, the main response was against the pre-erythrocytic CSP antigen, while in the CHMI samples there was some elevated response to MSP-1 but not to AMA-1, Pfs16, or Pfs25. This is to be expected in the IMRAS model that lacks the blood stage entirely, but it may be expected in the CHMI model as well, where blood-stage parasitemia is significantly curtailed by early (often pre-symptomatic) detection and rapid treatment.The current study demonstrates that the serological assessment of multiple antigens provides a more in-depth readout for exposure, malaria risk, and transmission rates than analysis of antibody responses to any single antigen. Antibodies to the mosquito saliva protein gSG6 are highly sensitive markers for exposure to arthropod vectors but only an indirect measure of risk for disease transmission. Simultaneously measuring pathogen-specific antibodies increases the accuracy of the assessment. In the CHMI and IMRAS samples, in terms of The endemic samples showed significantly increased responses to all eight antigens compared to negative controls and to all antigens except CSP when compared to IMRAS. When comparing between Kenya (moderate transmission intensity) and Uganda (high transmission intensity), Ugandan samples had significantly higher responses to CSP and MSP-1 and a moderately higher response to AMA-1 but no significant difference in responses to the gametocyte antigens Pfs16 and Pfs25. The reason for this is unclear, and it may be that antibody responses to sporozoite and merozoite antigens are more sensitive to transmission intensity—perhaps because of their abundance and/or immunogenicity—compared to gametocyte antigens. Furthermore, antigen diversity could play a role as well. For example, the modest difference in AMA-1 that distinguishes samples from Kenya vs. Uganda may be due to the high polymorphic nature of the antigen ,25, and Detection of mosquito-saliva specific responses in sera from CHMI participants demonstrated the potential of this readout in detecting exposure to the vector . We did P. falciparum being the dominant parasite species and Anopheles gambiae [An. stephensi [P. vivax and other mosquito species are prevalent by replacing P. falciparum antigens with comparable P. vivax antigens and by utilizing Anopheles species-specific homologs for gSG6. For example, reports from serosurveillance studies using ELISA-based detection of anti-gSG6 antibodies with samples from the South Pacific and in South America reported complete failure [An. dirus, An. maculatus, and An. minimus, only have a 48–87% identity with An. gambiae gSG6-P1 [The current study focused on establishing antibody profiles from samples collected in African malaria-endemic regions with  gambiae ,33,34 antephensi ,36 being failure ,38. More gSG6-P1 , and, thAnopheles mosquitoes, but also to different Plasmodium species such as P. vivax, which is predominant in south and southeast Asia. Fourth, the sample size is limited in this assay and may not capture the full breadth and diversity of serological responses in individuals living in the endemic regions of Kenya and Uganda. Further characterization of this assay using more samples from the broader region is necessary along with stratification of the results by demographic factors such as gender, age, and occupation. Finally, fifth, more development and testing of this assay is needed to ensure that it is robust against batch effects that might result from samples collected and stored under different conditions or for different periods of time.There are several shortcomings to the present study. First, while samples were collected from a longitudinal study conducted in endemic regions of Kenya and Uganda, no data on malaria history was collected from individuals from that study; thus, we could only assess the degree to which the assay was sensitive to malaria endemicity at a group level and not to malaria history at an individual level. Second, more data is needed on negative controls and samples collected from non-endemic regions in order to establish baselines and cutoff criteria for classifying subjects as exposed or quantifying the degree of exposure based on the serological readout. Third, more assessment is needed on the specificity of this assay, not just to different species of The current study demonstrates the feasibility of a multiplex serological assay to determine exposure rates to mosquito vectors, pathogen, and disease. Furthermore, the data demonstrate the value of serosurveillance by establishing antibody profiles to analytes informing on exposure to vector, pathogen, and disease. Lastly, future studies will aim to address field performance of such assay platform with the goal having additional surveillance tools with higher throughput and accuracy available for field and clinical studies.


Additionally, the levels of this molecule were found to be higher in patients who died from these pathologies compared to those who survived among the two clinical groups  and within the same group . The AUC-ROC for fatality among all the severe cases was 0.77 with a 95%CI of (0.69–0.85). These results suggest that plasma neopterin levels constitute a potential biomarker for predicting fatality among severe falciparum malaria patients.Among the barriers to accessing adequate treatment and high-level monitoring for malaria febrile patients is the lack of effective prognostic markers. Neopterin, which is a marker of monocyte/macrophage activation, was found have increased during severe malaria. In this study, we used quantitative ELISA in order to assess the levels of plasma soluble neopterin in 151 patients from a cohort of Beninese children with severe malaria. We evaluated the prognostic accuracy of this molecule in order to predict the outcome of the disease. Our results show that neopterin levels were not significantly different between patients with different forms of severe malaria, including severe non-cerebral malaria (SNCM) and cerebral malaria (CM). However, the levels of this molecule were found to be higher in patients with severe malarial anemia (SMA) among both CM and SNCM cases ( Similar results were observed in patients experiencing their first malaria infection compared to patients with a history of malaria. These levels were inversely correlated with the number of malaria attacks [The immune activation marker neopterin is produced by activated monocytes or macrophages when stimulated by IFN-γ, which is released by T-helper cell 1 of the cellular immune response. Neopterin has been found to be essential in inflammatory diseases and apoptosis . Neopter attacks ,22. This attacks .Furthermore, Neopterin level and anemia severity were correlated in Zambian children with either severe or cerebral malaria . SimilarHowever, our study sought to investigate the potential of Neopterin as a reliable biomarker for accurately predicting the prognosis of patients who might diefrom severe malaria. Therefore, we assessed the levels of plasma neopterin in children with various clinical manifestations of severe malaria on admission to hospital. Ethical approval for this study was obtained from the institutional ethics committee of the Research Institute of Applied Biomedical Sciences, Cotonou, Benin (authorization no. 006/CER/ISBA/12) and the Comité National d’Ethique pour la Recherche en Santé (CNERS), Cotonou, Benin. Ethical clearance was obtained from No 50, 25 October 2017, IRB00006860.Prior to children inclusion to the study, permission was sought from parents or guardians by the signing of a consent form. Participants were free to opt out of the study at any time point if they felt the need to do so without any coercion.P. falciparum malaria endemic areas with a heterogeneous transmission rate which may exceed thirty infective bites per person each year [This was a cross-sectional study that was conducted in Cotonou and Porto-Novo from June to September 2012; May to July 2013; and then from December 2017 to July 2019. Cotonou and Porto-Novo, two cities located in a subtropical region with two rainy seasons, have been identified as ach year . The samP. falciparum first by using a rapid diagnostic test , then by performing a confirmatory microscopic examinations for P. falciparum mono-infection. The World Health Organization  definition criteria for malaria were used to diagnose and categorize patients with varying degrees of severe P. falciparum malaria [g at 4 °C. Once collected, patients’ personal and clinical data were included in an ad-hoc data form on MS-Excel. The data was anonymized by replacing individual names with unique codes for privacy, thus, preventing bias in data analysis.Children under six years old were recruited after being tested positive to  malaria . Two groNeopterin levels were assessed using a competition ELISA test from IBL International  using 20 µL of undiluted samples. The competition between a peroxidase-conjugated and non-conjugated antigen was evaluated for a fixed number of coated anti-neopterin antibody binding sites. The unbound antigen was removed by washing, and the optical density (OD) was measured after substrate reaction. When the obtained OD values were outside the standard reference range, the dilutions were modified accordingly.Statistical analyses were conducted using GraphPad Prism v9. The median and 95% Confidence Intervals (CIs) of the neopterin levels were compared between the two clinical groups (CM versus SNCM) for the patients who survived versus those who died, and for the patients with severe malarial anemia (SMA) versus the patients without cases of anemia (NSMA) using the Mann–Whitney U test. The validity of neopterin as a biomarker for malaria-related severity, death, and severe anemia was assessed using a receiver operating characteristic (ROC) curve analysis. This is a two-dimensional measure of classification performance where the area under the ROC curve (AUC) accurately measures discrimination, i.e., reflects the power of a quantified parameter in order to distinguish between two clinical groups. The greater the AUC, the better the test.p-value < 0.0001).A total of 151 childrenwere diagnosed with severe malaria. Seventy-six (50.3%) of them had CM while 75 (49.7%) had SNCM. There were no significant differences in age, sex, temperature, parasitemia, or hemoglobin levels, neither for most of the other clinical parameters determined for the patients from either groups . ObviousIn total, 151 plasma samples were available for the neopterin measurements. Seventy-six of such samples were obtained from the CM group and 75 samples were obtained from SNCM patients. Our results showed no significant difference between the medians of the neopterin concentrations for the two groups; the median and 95% CI for the CM group was 43.25 and (41.23–71.63) ng/mL, respectively, and the median and 95% CI for the SNCM group was 42.23 and (39.51–54.1) ng/mL, respectively A.p-value < 0.0001). This result was also confirmed within each clinical group (58.64 (55.63–90.78) ng/mL and 30 (30.18–44.73) ng/mL for the CM and SNCM groups, respectively)  ng/mL, than concentrations in those who survived, with a median of 37.67 and a 95% CI of (36.06–46.7) ng/mL,  of 0.77 (0.69–0.85) ng/mL for fatality C.p-value = 0.022) , with a level of Hb < 5 g/dL, (50.1 (46.29–63.86) ng/mL) compared to those with a level of Hb > 5 g/dL (35.82 (36.16–50.32) ng/mL), (= 0.022) A.Among the 76 children who had CM, 35 had SMA and 41 had Hb levels > 5g/dL. Similarly, among the 75 patients from the SNCM group, 32 of them had SMA and 38 had Hb levels > 5g/dL. The AUC-ROC value shows that neopterin is weak at distinguishing between children with SMA and those without non-severe malarial anemia (NSMA)  ng/mL) B.The challenges in the clinical management of severe malaria partially result from the lack of reliable prognostic markers for differentiating patients who are at risk of death at admission, and for allowing for prompt adequate treatment and special attention to be given from health care providers .It has been shown that neopterin concentrations increase during acute infections, including severe malaria, highlighting the diagnostic value of this molecule as a marker of severity ,27,28,29p-value = 0.6712). Although a rise in mortality was observed at Hb levels below 3 g/dL during admission [Analyzing our results, we observed from dmission ,32, we dP. falciparum malaria are positively correlated with neopterin levels [The neopterin concentrations of those in the CM group did not differ from those in the SNCM group, as shown in A, as botn levels .p-value < 0.0001 among children with CM or SNCM, as well as between the children who died and the children who survived within the same group   C, indica2+ transients in various cell types, thereby affecting cellular redox systems [P. falciparum-infected erythrocytes, causing hyperinflammation, as well as hypotensive reactions [The concomitant release of neopterin, along with the inflammatory mediators of the host defense mechanism upon activation of monocytes/macrophages during malaria, is likely to initiate the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) . Oxidati systems ,36. One  systems . NO has  systems . Additioeactions ,40,41. Neactions , a pro-ieactions . These op-value = 0.02). It is important to note that P. falciparum infections accelerate the development of anemia as the hemolysis of both parasitized and non-parasitized red blood cells leads to a decline in hematocrit [P. falciparum malaria develop ineffective erythropoiesis, which results from inflammation induced by IFN-γ implicated in neopterin production [Our results also indicate increased neopterin concentrations in children with SMA compared to those with Hb levels > 5 g/dL (matocrit ,45,46,47oduction . The prooduction ,51,52,53We have shown, using 251 patients with severe malaria, that neopterin concentrations were high in both the CM and SNCM groups on the first day of admission to the hospital. These concentrations were higher in children with SMA compared to children with Hb levels > 5, and in children who died compared to those who survived. Neopterin may therefore qualify as a good prognostic biomarker of malaria for fatality in patients. Neopterin assessment at admission may also provide clinicians with firsthand information for rapid management of severe malaria patients if it can be developed as a rapid diagnostic test.


PfHRP2) specific to Plasmodium falciparum, which is the principal plasmodial species causing malaria in Burkina Faso. However, there is increasing concern about the diagnostic performance of these RDTs in field situations, and so constant monitoring of their accuracy is warranted. (2) Methods: A prospective study was performed in the health district of Nanoro, where 391 febrile children under 5 years with an axillary temperature ≥37.5 °C presenting at participating health facilities were subjected to testing for malaria. The HRP2-based RDT and expert microscopy were used to determine the diagnostic performance of the former. Retrospectively, the correctness of the antimalaria prescriptions was reviewed. (3) Results: Taking expert malaria microscopy as the gold standard, the sensitivity of the employed RDT was 98.5% and the specificity 40.5%, with a moderate agreement between the RDT testing and microscopy. In total, 21.7% of cases received an inappropriate antimalarial treatment based on a retrospective assessment with expert microscopy results. (4) Conclusion: Malaria remains one of the principal causes of febrile illness in Burkina Faso. Testing with HRP2-based RDTs is inaccurate, in particular, due to the low specificity, which results in an over-prescription of antimalarials, with emerging antimalarial drug resistance as an important risk and many children not being treated for potential other causes of fever.(1) Background: Malaria control has strongly benefited from the implementation of rapid diagnostic tests (RDTs). The malaria RDTs used in Burkina Faso, as per the recommendation of the National Malaria Control Program, are based on the detection of histidine-rich protein-2 ( Plasmodium falciparum malaria cases, and associated mortality and morbidity [Recent malaria control efforts have resulted in a significant reduction of orbidity . A globaorbidity . Howeverorbidity .PfHRP2), which is specific to Plasmodium falciparum, or the Plasmodium-specific parasite lactate dehydrogenase (pLDH), either alone or in combination with PfHRP2. Malaria RDTs are easy to implement at the point-of-care, provide results rapidly, and require no specific training and equipment [PfHRP2-based RDTs [The case-detection using malaria rapid diagnostic tests (RDTs) in the test-and-treat strategy advocated by the World Health Organization (WHO) is one of the cornerstones of this success . The majsed RDTs ,4,5,6. Tsed RDTs ,8,9.Plasmodium parasites in Giemsa-stained thick- or thin-blood slides still remains the gold standard for malaria diagnosis [Alternative diagnostics for malaria include microscopy and molecular-biology-based tests. The latter are considered to be the most sensitive, but infrastructural requirements, training, and costs hamper their large-scale implementation in resource-limited settings ,11. Theriagnosis ,12, evenPlasmodium falciparum malaria, and the number of clinically suspected malaria cases was estimated at almost 15 million, with 4000 reported malaria deaths occurring in 2020 [P. falciparum is the predominant malaria species in the country, the National Malaria Control Program (NMCP) has implemented to use of HRP2 RDTs. However, previous studies have demonstrated that its performance in the diagnosis of malaria is reduced [Burkina Faso, amongst the poorest countries in the world, has a high burden of malaria. According to the World Malaria Report 2021, the whole population of the country is at risk of contracting  reduced ,4,5,6. T reduced ,14,15,16PfHRP2 RDT (see below) and managed following NMCP guidelines [The study was performed between September 2018 and August 2019 in the health district of Nanoro, which is located at approximately 85 km from Ouagadougou, the capital city of Burkina Faso. Children under 5 years presenting with a current axillary temperature ≥37.5 °C at one of the participating health facilities  and at the Pediatric ward of “Centre Médical avec antenne chirurgicale  were recruited for the study after obtaining informed consent from a parent or legal guardian. All participating children were tested for malaria using an idelines .The study was approved by the National Ethical Committee “Comité d’Ethique pour la Recherche en Santé” (Deliberation No. 2014-11-130). The study was also approved by the health district authorities and community leaders in different villages before implementation.P.f; Standard Diagnostics, Hagal-Dong, Korea) and for microscopy slides preparation. The result of the malaria RDT testing in the health facilities was recorded on a case record form (CRF) as well as the treatment provided for clinical management.At enrolment, some basic demographic and clinical data were collected from each participant. A small blood sample was collected by finger prick for malaria RDT testing , who were blinded from the results of the RDT testing.The malaria blood slide was fixed with methanol and stained with 3% Giemsa solution (pH 7.2) for identification and quantification of asexual P. falciparum densities were determined by counting the number of asexual parasites per 200 white blood cells and calculated per μL of blood by assuming the number of white blood cells to be at 8000 per μL. Each blood slide was read by two independent expert readers, and in case of discrepancy, a third independent reader was used. Positive microscopy results were recorded as the geometric means of the two readers’ results or the geometric means of the two geometrically closest reading in case of a third reading. These results were expressed as asexual parasites per microliter by using the patient’s white blood cell (WBC) count.To ensure the quality of sample analysis, either in peripheral health centers or in the central laboratory, all diagnostic procedures were conducted in accordance with the standard operating procedures (SOPs) in place at the CRUN. In addition, there was strict adherence to manufacturers’ guidelines with respect to the RDT testing.Plasmodium falciparum, the internal control line confirmed the correct performance of the test. There is no further quality assurance system put in place by the Burkinabe NMCP to subject malaria RDTs to for quality control checks before their use. Furthermore, it is important to mention that all expert microscopists of the CRUN laboratory are frequently subjected to the External Quality Control Assessment (EQA) program with the National Institute for Communicable Diseases  and only qualified readers were allowed to read slides [For malaria rapid diagnostic testing based on the histidine-rich protein-2 antigen specific for d slides .https://www.medcalc.org/calc/diagnostic_test.php . The agreement between the RDT and expert microscopy was determined by calculating the kappa (k) values with 95% confidence intervals using GraphPad software, https://www.graphpad.com/quickcalcs/ (accessed on 30 September 2021).A double-entry system was performed for data in Excel and checked by an independent data manager. The performance of the RDT was evaluated compared to expert microscopy (gold standard) by calculating the sensitivity, the specificity, and the negative and positive predictive value of the RDT using the MedCalc Software Ltd. diagnostic test evaluation calculator, In total, 391 febrile children under the age of 5 were recruited for this study. The recruitment according to the sites was as follows: 115 were included at the Temnaoré health facility, 91 at the Soaw health facility, 82 at the Nazoanga health facility, 68 at the Pediatric ward of the CMA reference hospital, and 35 at the Pella health facility. The basic characteristics of the study population are presented in Taking expert malaria microscopy as the gold standard, the sensitivity of the employed RDT was 98.5% and the specificity was 40.5% . Expert microscopy found 265  cases positive for malaria see . All expartemether–lumefantrine (AL) (both ACTs often combined with paracetamol to suppress fever), were provided to 304  of the children who were malaria-RDT-positive. However, there were 32 malaria RDT-positive children  who did not receive an antimalarial (pyronaridine–artesunate or artemether–lumefantrine) but were treated with antibiotics. Furthermore, 45 RDT-negative children  were not treated with antimalarials, but they were treated with antibiotics . In addition, 10  malaria-RDT-negative children were treated with antimalarials. Overall, there were 349  “right” treatments on the basis of the RDT results  and 42  “wrong” treatments .Antimalarials, mainly artemisinin-based combination therapy (ACT) comprising of either pyronaridine–artesunate (PA) or If we considered the malaria-expert-microscopy-positive results, it was found that 247  of the children who had malaria-expert-microscopy-positive results received the appropriate antimalaria treatment. However, 18  other children in this group did not receive antimalarials. In contrast, 67  children who were malaria-expert-microscopy-negative did receive an antimalaria treatment based on the RDT result but not indicated by microscopy. Accordingly, 59  microscopy-negative-children were not treated with antimalarials.In hindsight , the actual number of right antimalaria treatments provided to children was 306  and the number of wrong treatments was 85 .Our study aimed to assess the diagnostic performance of a malaria-HRP2-based RDT recommended by the National Malaria Control Program (NMCP) of Burkina Faso in remote health facilities using microscopy result as the gold standard. Our study revealed high sensitivity 98.5%) of this RDT used at the peripheral level, but with low specificity 40.5%). This confirms previous observations that the HRP2-based RDTs, the most widely employed diagnostic test for malaria, have a low specificity, particularly in a high-transmission setting ,4,5,6. T0.5%. Thi% of thisSeveral factors may have contributed to the low specificity of the employed RDT. First of all, it is well known that the HRP2 antigen persists for approximately three to nine weeks in the blood after successful treatment ,22,23. TExpert microscopy, as performed in the context of the current study, detected a considerably lower number of malaria cases in contrast to those detected by the malaria RDTs recommended by the NMCP of Burkina Faso. As a result of this, if the results of malaria microscopy would have been followed, a significantly lower number of children would have been treated for malaria, thereby saving costs and avoiding the development of resistance. Microscopy has always proven to be a valuable tool for the diagnosis of malaria, and it comes with a good sensitivity/specificity and allows for quantification and species identification. However, its implementation depends on a well-trained microscopist and appropriately maintained equipment, as this is carried out at the CRUN laboratory . TraininPfHRP2/3 deletion data , researA positive finding of the present study was that the health workers tend to adhere well to the results of the RDT testing, and this is in line with other studies ,31. Yet,Finally, it should be noted that there was also a proportion of febrile children who remained undiagnosed . In many cases, these children were treated with antibiotics irrespective of the actual cause of their fever. Consequently, the management of febrile diseases may be inappropriate and lead to increase morbidity and mortality, which could be avoided. Moreover, this practice will lead to the development of antibiotic resistance, one of the greatest concerns of modern medicine . It is tPlasmodium species, and in many children not being treated for potential other causes of fever.Malaria remains one of the principal causes of febrile illness in Burkina Faso, mostly in rural areas. A large proportion of febrile cases in these areas are tested with HRP2-based RDTs, and this frequently yields inaccurate results, in particular, due to its low specificity, which results in an over-prescription of antimalarials that might increase the risk of emerging resistant


All PDTs were performed using red light at around 635 nm wavelength. Safety and tolerability were documented along with pharmacokinetics. In both MUsTs, ALA plasma concentrations were transiently increased to a maximum concentration at about 2.5 to 3.3 times above endogenous baseline with time to maximum concentration at ≈3 hours after dosing. Plasma levels subsequently returned to baseline within 10 hours after dosing. Overall baseline‐adjusted mean area under the baseline‐adjusted plasma concentration‐time curve from time zero to the last sampling time point at which the concentration was at or above the lower limit of quantification ranged from 142.8 to 146.2, indicating that a similar, minor fraction of topical ALA is systemically absorbed under both dosing regimens. Systemic PpIX exposure after administration of either dose of BF‐200 ALA was equally minimal. Application site skin reactions were treatment area size‐related, albeit transient and consistent with the known safety profile of BF‐200 ALA.The nanoemulsion‐based 10% aminolevulinic acid (ALA) hydrochloride gel BF‐200 ALA optimizes epidermal penetration of its active ingredient and is approved for topical photodynamic therapy (PDT) for the treatment of actinic keratosis in the United States and Europe. To characterize systemic absorption from dermal application during PDT, ALA and its key active metabolite protoporphyrin IX (PpIX) were analyzed in 2 maximal usage pharmacokinetic trials (MUsT) in patients severely affected with actinic keratosis. The primary objective of both MUsTs was to assess baseline‐adjusted plasma concentration–time curves for ALA and PpIX after a single PDT treatment applying either 2 g (1 tube) of BF‐200 ALA on the face (MUsT‐1) or applying 6 g (3 tubes) of BF‐200 ALA on the face/scalp or body periphery (MUsT‐2), to 20 or 60 cm Actinic keratoses (AKs) are atypical proliferations of epidermal keratinocytes that are regarded as precancerous and typically develop because of chronic exposure to ultraviolet radiation.18 In these pivotal clinical trials, complete clearance of patients 12 weeks after the first PDT with BF‐200 ALA was shown to be up to 61.8% and up to 90.9% after a maximum of 2 PDTs.2 for BF‐200 ALA,Photodynamic therapy (PDT) using topically applied prodrugs of the endogenous, photosensitizing heme precursor protoporphyrin IX (PpIX), such as 5‐aminolevulinic acid (ALA), displays high selectivity and efficacy and is therefore recommended in international guidelines for field‐directed treatment of AK.The 2 MUsTs were conducted between 2013 and 2020 in accordance with the Declaration of Helsinki, guidelines of Good Clinical Practice, and other relevant regulatory guidelines. Both studies were approved by competent institutional review boards/independent ethics committees before implementation. For MUsT‐1, the ethics committee was of the Medical Association North Rhine, in Duesseldorf, Germany, and for MUsT‐2, it was the central institutional review board Advarra, in Columbia, Maryland. All participants provided written informed consent before study enrollment.2 on the face or forehead. Treatment fields were allowed to be discontinuous but must have been within 2 illumination areas of the PDT lamp (6 cm × 16 cm each). A fixed‐sequence design was chosen, in which all patients received PDT with placebo (period 1) followed by PDT with BF‐200 ALA (period 2) with an interim washout period of 7 days. Both placebo and BF‐200 ALA were applied at a thickness of 1 mm in a quantity of 2 g to an area of 20 cm2 on the face or forehead. For BF‐200 ALA, this resulted in a total drug dose of 156 mg. Treatments fields were allowed to be discontinuous but must have been within 2 illumination areas of the PDT lamp (6 cm × 16 cm each). The study medication was incubated for 3 hours under an occlusive, light‐blocking dressing. Thereafter, the occlusion and remaining gel were removed, and a PDT illumination was conducted, delivering a total light dose of 37 J/cm2 at a peak wavelength of 635 nm with 1 or, when needed, 2 PDT lamps simultaneously. On both treatment days, a total of 15 blood samples were taken per patient. Their time points in relation to BF‐200 ALA application were: 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, and 10 hours. A graphical overview of the treatment and sampling procedures in MUsT‐1 is given in Figure The first MUsT  was a single center, nonrandomized, open‐label, placebo‐controlled, fixed‐sequence study to evaluate the pharmacokinetics of ALA and PpIX in the plasma of patients with AK following topical application of 2 g of BF‐200 ALA (containing 78 mg/g ALA) under maximal use conditions when using PDT. It was conducted in 1clinical phase I unit at CRS Clinical Research Service, Moenchengladbach, Germany. In this study, eligible patients were aged 18 to 85 years and presented with at least 10 lesions of AK of mild to moderate intensity (Olsen grade 1 and 2)2 in total. Treatments fields were allowed to be discontinuous but must have been within 2 illumination areas of the PDT lamp (6 cm × 16 cm each). This study was designed open‐label as the use of a placebo arm was considered unnecessary based on the previous observation from MUsT‐1 that plasma concentrations of ALA and PpIX are not subject to circadian rhythm. The PDT procedure  was performed similar to MUsT‐1 with the exceptions that 3 tubes of BF‐200 ALA  were applied per patient in an extended skin area and the treatment was performed on the face/scalp or in the periphery (neck/trunk/extremities) with 1 or, when needed, 2 PDT lamps simultaneously. The number of blood samples taken for pharmacokinetics as well as the time points of blood sampling were determined on the basis of the results of the previous MUsT‐1, taking into account expected time to reach maximum concentration (tmax) and apparent terminal half‐life (t1/2), thus avoiding unnecessary blood sampling and shortening the time over which blood samples were collected. On treatment days, a total of 14 blood samples were taken per patient. Their time points in relation to BF‐200 ALA application were –0.5, 0 (predose), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, and 10 hours. An additional predose sample was taken at the screening visit, such that 3 predose samples per patient could be analyzed to establish individual baseline plasma levels of ALA and PpIX. A graphical overview of the treatment and sampling procedures in MUsT‐2 is given in Figure The second MUsT  was a single‐center, nonrandomized, open‐label phase I study to evaluate the pharmacokinetics of ALA and PpIX in the plasma of patients with AK after topical application of 3 tubes of BF‐200 ALA 10% gel for PDT under maximal use conditions. It was conducted in 1 clinical phase I unit at DermResearch, Inc., Austin, Texas. In this study, eligible patients were 18 to 85 years of age, and presented with at least 12 clinically confirmed mild to severe AK lesions  methods. The methods were developed and validated according to current international standards.13C2, 15N HCl as an internal standard. Calibration standards were prepared by spiking buffer pH 6 as replacement matrix with ALA at 8 concentration levels ranging from 1.000 ng/mL through 100.000 ng/mL. Before analysis, ALA was derivatized using a method described below (see MUsT‐2). For PpIX, working solutions were prepared by spiking methanol with PpIX in methanol/dimethylformamide (1/1). Midalzolam‐D4 was used as an internal standard, and calibration standards were prepared by spiking blank phosphate buffered saline in bovine serum albumin, fraction V, as artificial matrix with PpIX in methanol at 8 concentration levels ranging from 1.000 ng/mL through 100.000 ng/mL. For ALA, QC samples were prepared by spiking buffer pH 6 with 3.000 ng/mL (low‐QC), and by spiking blank plasma diluted by a factor of 4 with 33.654 ng/mL (mid‐QC) and 78.654 ng/mL (high‐QC), respectively. Additionally, bio‐QC, which represents the endogenous concentration, was prepared by diluting blank plasma by a factor of 4. The sum of the mean diluted endogenous background concentrations plus the respective spiked concentrations were used as theoretical concentrations for the mid‐ and high‐QC. The mean diluted endogenous background is used as theoretical concentration for the bio‐QC. The mean endogenous background concentration was 14.619 ng/mL. Since plasma is diluted by a factor of 4 with buffer pH 6, the diluted concentration is then calculated to be 3.654 ng/mL. For PpIX, as endogenous levels in blank plasma could not be determined with sufficient certainty, spiked concentrations were regarded as theoretical concentrations. QC samples were prepared by spiking blank bovine serum albumin as artificial matrix with PpIX in methanol. QC samples were prepared at 3 concentration levels: 3.000 ng/mL (low‐QC), 23.000 ng/mL (mid‐QC), and 80.000 ng/mL (high‐QC). Analysis was performed as a single determination. Samples were ordered according to their expected concentration to avoid carryover.In MUsT‐1, for the determination of ALA and PpIX in plasma, a total of 360 blood samples were shipped to CRS laboratory (30 samples per period from each of the 12 patients). All collected samples were analyzed and had valid results. The lower limits of quantification (LLOQ) of ALA and PpIX in plasma were 1.000 ng/mL. ALA was determined by an internally standardized LC‐MS/MS method using atmospheric pressure chemical ionization in the positive ion mode. Buffer at pH 6 was used as artificial matrix. Analyte PpIX was determined by an internally standardized LC‐MS/MS method using electrospray ionization in the positive ion mode. Bovine serum albumin (fraction V) was used as artificial matrix. Before detection in multiple reaction monitoring on a mass spectrometer, analyte and internal standard were separated on an analytical narrow bore column. An overview of instrument settings and equipment is given in Table 13C2, 15N HCl as an internal standard. ALA was extracted from samples (50 μL) of diluted human plasma (containing lithium heparin as an anticoagulant) or buffer pH 6 by protein precipitation using methanol (150 μL) and then 100 μL of the supernatant was transferred to a new well and evaporated to dryness under nitrogen. The analyte was derivatized by adding 50 μL 1‐butanol:HCl (2:1 v/v), heating at 65°C for 30 minutes, and then evaporating until dry. The derivatization product 5‐aminolevulinic acid butyl ester was reconstituted in 100 μL reconstitution solvent  for quantitative determination by LC‐MS/MS with multiple reaction monitoring and atmospheric pressure chemical ionization in the positive ion mode. An overview of instrument settings and equipment is given in Table A total of 960 plasma samples were shipped to ACM Bioanalytical Services for the determination of ALA (480 samples) and PpIX (480 samples). All collected samples were analyzed and had valid results, with 1 single exception: as 1 of the samples for PpIX analysis had not been adequately protected from light during transport, it had to be excluded from final analysis. Buffer  was used as an artificial matrix for the calibration standards, LLOQ, and low‐QC. Calibration standards were prepared at 0.00 (blank), 1.00, 2.50, 5.00, 12.5, 25.0, 50.0, 80.0, and 100 ng/mL, from the ALA reference material. LLOQ and low‐QC samples were prepared at 1.00 and 3.00 ng/mL, respectively. Mid‐ and high‐QC samples were prepared by spiking ALA into plasma to give concentration increases of 120 and 300 ng/mL, respectively. Since plasma samples were diluted 4 times before extraction, the diluted mid‐ and high‐QC measured concentrations were 30.0 and 75.0 ng/mL plus the diluted plasma endogenous content determined from the bio‐QC. The quantification of ALA in human plasma was achieved via external calibration using 5‐ALA‐Plasma devoid of endogenous PpIX and was used to prepare calibration standards using PpIX reference material at 0.00 (blank), 1.00, 2.50, 5.00, 12.5, 25.0, 50.0, 80.0, and 100 ng/mL. QC samples containing PpIX in depleted plasma were prepared at 1.00, 3.00, 30.0, and 75.0 ng/mL. The quantification of PpIX in human plasma was achieved via external calibration using PpIX D6 as an internal standard. PpIX was extracted from samples (100 μL) of plasma by protein precipitation. The analyte was extracted using methanol:acetonitrile  in 0.1% formic acid. After centrifugation, the supernatant was transferred to a second 96‐well plate for quantitative determination using LC‐MS/MS with multiple reaction monitoring and electrospray ionization in the positive ion mode. An overview of instrument settings and equipment is given in Table All samples for a given patient were analyzed together in a single batch except when samples had to be reanalyzed. A batch, at a minimum, consisted of duplicate sets of calibration standards and duplicate low‐, med‐, and high‐QC samples. Analysis batches that contained study samples that were diluted into range also contained a QC sample that was similarly diluted.In both MUsTs, collection of adverse events (AEs) started at signing informed consent until the end of the study. AEs were discriminated as pretreatment adverse events and treatment‐emergent adverse events (TEAEs). Both application site pain and application site reactions were collected from the outset of treatment. In MUsT‐1, this assessment encompassed local and overall tolerability, including pain, graded by the investigator on a 4‐point scale . In this study, application site pain and skin reactions in the treatment area triggered by the PDT were documented but not considered as AEs. Pain was documented after the illumination. A pretreatment with 1 g acetaminophen 1 hour before illumination was considered at the discretion of the study physician. Application site skin reactions were collected on days 1 and 7 after treatment and allocated to the predefined terms: burning, dryness, edema, erosion, erythema, and itching. In MUsT‐2, patients assessed the experienced pain during PDT using an 11‐point numeric rating scale ranging from 0  to 10 (worst possible pain). This score reflected the patient's respective maximum pain during treatment. In this study, skin reactions occurring in the treatment field(s) after starting PDT treatment were subclassified into the more specific categories application site discomfort and application site skin reactions and were documented on the treatment day and on days 7 and 28 after treatment. The assessment on day 7 was via phone call and captured the patient‐reported events only. Application site discomfort encompassed events such as “burning,” “pain,” “itching,” “stinging,” and others. Intensity was assigned to 1 of 4 categories ranging from “none” to “severe” based on the patient's report. Application site skin reactions, like “erythema,” “edema,” “induration,” “vesicles,” and others were assessed by the investigator on the treatment day and at the subsequent visit. Intensity assessment followed the discomfort intensity grading. AEs were tabulated by the Medical Dictionary for Regulatory Activities preferred term and system organ class. In addition to this, routine laboratory testing from blood and urine was performed before and after treatment. The safety end points were analyzed descriptively and in an exploratory way.Both ALA and PpIX are endogenous molecules, naturally occurring in human plasma. Hence, bioanalytical data were corrected by each patient's individual baseline and the primary end point analyses focused on baseline‐corrected concentration‐vs‐time profiles and baseline‐corrected PK parameters. Baseline was established from one predose plasma sample (MUsT‐1) or the mean of 3 predose plasma samples . Patient‐specific baseline values were used for baseline adjustment in each study. Values less than the LLOQ and negative values after baseline‐adjustment were set to 0 and disregarded for the calculation of geometric statistics and on the logarithmic scale. In MUsT‐1, data were analyzed by period (placebo and BF‐200 ALA). In MUsT‐2, data were analyzed overall and according to strata .max), tmax, (area under the baseline‐adjusted plasma concentration‐time curve from time zero to the last sampling time point at which the concentration was at or above LLOQ (AUC0‐t), area under the baseline‐adjusted plasma concentration‐time curve, data extrapolated to infinity by calculating AUC = AUC0‐t + Ct/Kel, terminal rate constant, assuming first order elimination kinetics (Kel), and t1/2 . Considerations on reliability of calculated parameters are presented in the supplement.The pharmacokinetic set per analyte included all patients who had at least one evaluable predose and postdose pharmacokinetic sample for the respective analyte. A sample was regarded as evaluable if the plasma concentration was above 0 (MUsT‐1) or at or above LLOQ (MUsT‐2). All pharmacokinetic parameters were derived from plasma concentrations of ALA and PpIX using noncompartmental methods. The following parameters were determined: observed maximum baseline‐adjusted plasma concentration , and median as well as minimum and maximum. For descriptive statistics of pharmacokinetic parameters and plasma concentrations, the geometric mean, geometric standard deviation, and geometric coefficient of variation were additionally included. For tDescriptive statistical analyses were conducted using SAS version 9.2 or higher . The noncompartmental pharmacokinetic analysis of the data were accomplished by using Phoenix WinNonlin version 6.0 or higher  (MUsT‐2) and by using SAS version 9.2 or higher (MUsT‐1). Prism version 9.1.0  was used for graphical representation of data.The calibration standards and quality controls of ALA and PpIX sample analysis runs displayed sufficient interbatch precision (<15%) and interbatch accuracy (<±5%) to confirm methodical quality see  and S4. 2. Eight of 12 patients received simultaneous illumination with 2 PDT lamps; 4 received illumination with only 1 lamp.Altogether, 12 patients were included in the treatment phase of the study and completed the entire course of the study receiving both treatments (placebo [period 1] and BF‐200 ALA [period 2]). Analysis sets are highlighted in Table max exposure to ALA were increased after application of the BF‐200 ALA compared to the placebo gel  compared to period 1 (placebo gel). A reasonable explanation for this outlier value could not be identified. The outlier value was supposed to be within normal interindividual variability of plasma concentrations.Concentrations of PpIX were generally low in all patients. Four patients showed concentrations below the LLOQ at all sampling time points  is aggregated over these 2 time points. An overview of application site skin reactions and discomfort is given in Table The calibration standards and quality controls of ALA and PpIX sample analysis runs displayed sufficient interbatch precision (<15%) and interbatch accuracy (<±5%) to confirm methodical quality see  and S4. 2. The number and size of the lesions within treatment field(s) and the severity were comparable for both strata, thus allowing the comparison of pharmacokinetic and safety data. A summary of treatment field and AK target lesion assessments is presented overall and by strata in Table In total, 46 patients were enrolled, and a total of 32 patients were included in the treatment phase of the study. Of these, 16 patients were treated on the face or scalp and 16 on the body periphery. For a tabular view of patient disposition, see Table With one exception, all patients showed ALA concentrations above the LLOQ at all sampling time points. Hence, ALA was systemically absorbed; that is, a measurable increase of ALA concentrations was observed after application of BF‐200 ALA  was clearly higher than the normal physiological range in plasma of healthy adults. This is reported in the literature to be below 10 ng/mL.0‐10h) of ALA (x axis) to the baseline‐adjusted AUC0‐10h of PpIX (y axis)  Figure . Based oThe overall incidence of patients with treatment‐emergent application site conditions was 100% for patients treated on the face/scalp and for patients treated in the periphery. In both strata, the most commonly reported TEAEs were those of the application site. Application site erythema and application site pain were the most common individual events in both strata  or 6 g (MUsT‐2) of BF‐200 ALA 10% gel in conjunction with red‐light PDT under maximal use conditions in patients with AK.2 located on the face or forehead , no systemic elevation of PpIX levels was observed. A slight and transient elevation of systemic ALA levels was detected, which was well below the daily rate of ALA synthesis (200‐354 mg).In MUsT‐1, in which 1 tube of BF‐200 ALA was applied on treatment fields of 20 cm2.MUsT‐2 was designed to assess pharmacokinetics and safety of PDT treatment in 2 strata  to account for a possible influence of varying epidermal thickness between different body regionsThe pharmacokinetic results of MUsT‐2 demonstrated a transient increase of systemic total and maximum exposure to ALA, which was slightly higher in the stratum face/scalp than in the stratum periphery. Possible explanations might be a generally higher skin temperature of face and scalp in comparison to peripheral skin,max determined for ALA was about 3.3 times the overall geometric mean of the baseline concentrations, indicating that only a minor fraction of the topical dose is systemically absorbed: From a total topical dose of 468 mg, the overall geometric mean baseline‐adjusted AUC indicates that the predominant portion of the topical ALA dose remains locally in the skin and that relative topical bioavailability is very low. ALA was eliminated quickly from plasma returning to approximate baseline levels within 10 hours. The results obtained in MUsT‐2 are similar to the results of MUsT‐1, although a 3‐fold higher dose was administered to a 3‐fold larger area. The overall baseline‐adjusted geometric mean AUC was even higher in MUsT‐1 compared to the overall geometric mean AUC in MUsT‐2. This difference may be related to the different treatment areas, as facial skin is characterized by less epidermal thickness compared to other body parts, particularly scalp and hands.The overall geometric mean of CTaking into consideration the available data from the scientific literature, the observed elevation in the plasma concentration of ALA in both studies is not expected to yield any noticeable clinical effect and thus regarded as uncritical. PpIX should be regarded as the key factor for systemic safety. Mild systemic adverse effects such as nausea and vomiting have been reported in patients with PpIX plasma concentrations of 0.56 to 1.59 μg/mL following oral dosing with 30 to 60 mg/kg ALA.All patients in both studies had at least 1 application site skin reaction or discomfort after PDT with BF‐200 ALA. In MUsT‐2, the overall incidence of related treatment‐emergent application site conditions was comparable for both strata, with certain exceptions as highlighted in the results section. In both studies, application site pain, erythema, and edema were the most frequently recorded events in connection to BF‐200 ALA Tables . This al2.Evaluation of pharmacokinetics indicates that the systemic exposure profile after treatment with 3 tubes of BF‐200 ALA is not significantly different from the systemic exposure profile after treatment with 1 tube of BF‐200 ALA. Based on the obtained safety as well as pharmacokinetics data, no safety concern due to high PpIX exposure is apparent upon application of 2 to 6 g of BF‐200 ALA for PDT of extensive AK in treatment fields located on the face/scalp or periphery. Evidently, systemic metabolism of ALA to PpIX is not increased under maximal use conditions. In summary, the studies demonstrate that field‐directed treatment of AK with a dose of up to 6 g BF‐200 ALA is safe on surface areas of up to 60 cmB.N., K.O., B.S., C.Z., and H.L. are or were employed by the sponsoring company, and B.N., K.O., B.S., and H.L. hold either stock or stock options of Biofrontera AG. The remaining authors declare no conflicts of interest.B.S. and B.N. provided clinical pharmacology expertise, designed the studies, and analyzed the data. J.D. and T. Papusha provided medical expertise, advised on study design, and collected the data. O.C. and T. Philippi provided bioanalytical expertise, developed and validated the assays, and collected the data. S.H. performed the pharmacokinetics and statistical analysis of MUsT‐2. C.Z. contributed to study design of MUsT‐2 and provided safety expertise and editorial assistance. B.N., K.O., and O.C. wrote the manuscript. H.L. provided pharmacological guidance, advised on study design, and revised the manuscript for important intellectual content. All authors critically revised and approved the final manuscript.These clinical studies were sponsored and funded by Biofrontera Bioscience GmbH.http://www.clinicaltrials.gov/ct2/show/NCT04319159Data supporting the results of this publication are publicly available in the ClinicalTrials.gov database under SUPPLEMENTARY INFORMATIONClick here for additional data file.


Plasmodium falciparum malaria episodes in sub-Saharan Africa while others remain free of disease most of the time. In contrast, malaria risk heterogeneity remains little studied in regions where P. vivax is the dominant species. Are repeatedly infected people in vivax malaria settings such as the Amazon just unlucky? Here, we briefly review evidence that human genetic polymorphism and acquired immunity after repeated exposure to parasites can modulate the risk of P. vivax infection and disease in predictable ways. One-fifth of the hosts account for 80% or more of the community-wide vivax malaria burden and contribute disproportionally to onward transmission, representing a priority target of more intensive interventions to achieve malaria elimination. Importantly, high-risk individuals eventually develop clinical immunity, even in areas with very low or residual malaria transmission, and may constitute a large but silent parasite reservoir.Extensive research has examined why some people have frequent  Plasmodium vivax is the dominant malaria-causing species, we searched the PubMed and SciELO databases for publications in English, Spanish, Portuguese, or French that appeared until Aug 1, 2021. We used the search terms (“risk heterogeneity” OR “superspreading” OR “Pareto” OR “overdispersed distribution” OR “genetic factors”) AND . We also searched reference lists of identified studies.Extensive research has examined why some people have repeated malaria episodes in Sub-Saharan Africa while others remain free of disease most of the time. To identify studies that describe malaria risk heterogeneity and their causes and consequences, especially in endemic settings where The burden of malaria is heterogeneously distributed in communities and tends to conform the 20/80 rule or Pareto principle: 20% of the individuals experience approximately 80% of the malaria episodes. Some malaria-exposed people have repeated malaria episodes, while most individuals remain free of disease over extended periods of time . Fig 1 sThe zero-inflated negative binomial (ZINB) fitted to incidence data combines the negative binomial distribution and the logistic model. It allows to simultaneously estimate the proportion of at-risk people, i.e., those who contribute cases following a negative distribution , and the proportion of people who are not at risk, the “structural zeroes” component described by the logistic model . Not-at-Why is malaria risk so heterogeneous and why are some people little susceptible or “protected”? To begin with, people differ in the frequency at which they are bitten by infected vectors . Some inP. falciparum sporozoite inoculations in typical rural African communities  and j = 2 for high risk [HR]) to describe the transmission dynamics of P. vivax malaria [pj  of the population and is associated with a risk factor xj>0 . We assume that the overall average risk is equal to 1 (x1p1+x2p2 = 1), since the factors xj are modifiers of individual responses to a force of infection that was allowed to vary. This setting configures a risk distribution with variance v = p1(x1−1)2+p2(x2−1)2.The individual risk of vivax malaria varies widely at the community level as a result of sociodemographic, genetic, and behavioral factors. The paradoxical finding of asymptomatic infections in low-endemicity settings might be explained by the presence of a minority of high-risk people who are repeatedly exposed to blood-stage parasites and eventually become immune, while low-risk individuals are seldom infected and remain susceptible to infection and disease during their lifetime. To estimate the proportion of high-risk individuals living in Brazil’s main malaria hotspot, we fitted compartmental susceptible-infected-susceptible (SIS)-type transmission models simultaneously to (i) age-stratified vivax malaria incidence densities and (ii) the frequency distribution of ver time . Instead malaria . Each ria)-dependent force of infection λ(a) [xj, according to the risk group j, a partial immunity weight σ(i) = eα∙i−  and the recovery rates γ and γ′ of infected symptomatic and asymptomatic individuals, respectively. For each risk group j, we obtained the dynamics of individuals according to their epidemiological status: susceptible , this group is estimated to contribute 86% of the overall vivax malaria burden. The model also suggests that as many as 25 symptomatic P. vivax infections per individual, on average, are required to reduce the probability of developing clinical malaria risk by half [Assuming equilibrium, in our modeling approach   modified by half . Some peP. falciparum transmission [P. vivax transmission elsewhere in the tropics [In addition to acquired immunity, differences in parasite virulence might also account for the high proportion of asymptomatic infections at times of low transmission . Virulensmission  as well  tropics .P. vivax transmission [P. vivax infections are more likely to cause adverse clinical outcomes—e.g., anemia among PQ-ineligible pregnant women and infants [Repeatedly infected people—including relapse-prone individuals who cannot take or metabolize PQ—are commonly found even in communities with relatively low smission , with im infants –76.P. vivax infection and transmission in the population—to be targeted to more intensive and effective measures.Risk heterogeneity has also clear implications for malaria control and elimination. The 20/80 rule implies that control programs selectively targeted at high-risk people are more likely to be effective ,13. IndePlasmodium vivax malaria in communities follows the 20/80 rule or Pareto principle: 20% of the people account for 80% or more of all infections.The burden of P. vivax disease and transmission potential by rendering some people more likely to have relapses.Factors that preclude the use of primaquine to eliminate hypnozoites  or reduce its efficacy  may specifically increase the burden of Inherited hemolytic anemias, caused by defects in hemoglobin synthesis, RBC membrane structure, or RBC enzymes, are classically regarded as “malaria resistance” factors but do not necessarily lower the risk of vivax malaria.P. vivax infection  or affect the efficacy of antirelapse treatment  have no such effects on falciparum malaria risk; these are “vivax-specific” malaria resistance factors.Human polymorphisms known to reduce the susceptibility to P. vivax and the impact of control interventions, especially those selectively targeted at high-risk individuals.Mathematical modeling must consider individual risk variation to describe the transmission dynamics of Plasmodium vivax malaria: the problem of G6PD deficiency and cytochrome P-450 2D6 polymorphisms. Malar J. 2018;17:42. doi: 10.1186/s12936-018-2190-zBaird JK, Battle KE, Howes RE. Primaquine ineligibility in anti-relapse therapy of Plasmodium vivax malaria in a heterogeneous host population: a case study in the Amazon Basin. PLoS Comput Biol. 2020;16:e1007377. doi: 10.1371/journal.pcbi.1007377Corder RM, Ferreira MU, Gomes MGM. Modelling the epidemiology of residual Plasmodium vivax transmission in the Amazon: pooled analysis of individual participant data from population-based cross-sectional surveys. Lancet Reg Health Am. 2022;9:100169. doi: 10.1016/j.lana.2021.100169Ferreira MU, Corder RM, Johansen IC, Kattenberg JH, Moreno M, Rosas-Aguirre A, et al. Relative contribution of low-density and asymptomatic infections to 10.1097/MOH.0000000000000039Taylor SM, Fairhurst RM. Malaria parasites and red cell variants: when a house is not a home. Curr Opin Hematol. 2014;21:193–200. doi: 10.1073/pnas.94.1.338Woolhouse ME, Dye C, Etard JF, Smith T, Charlwood JD, Garnett GP, et al. Heterogeneities in the transmission of infectious agents: implications for the design of control programs. Proc Natl Acad Sci U S A. 1997;94:338–42. doi:


Many of the atypical antipsychotics induce metabolic side effects, limiting their use in clinical practice. Alpha-lipoic acid (ALA) was proposed as a new approach in schizophrenia to improve metabolic effects of atypical antipsychotics. The aim of the study is to evaluate the effect of ALA on metabolic and clinical parameters among schizophrenic subjects.15 schizophrenic subjects, in stable atypical antipsychotic monotherapy were included in the study. ALA was administrated at the oral daily dose of 600 mg/d in addition to antipsychotic therapy. Metabolic, clinical, and psychopathological parameters were measured at typical antipsychotics. e initial screening, and after 12 weeks.p = 0.012), blood glucose (p = 0.005), AST (p = 0.021), γGT (p = 0.035), CPK (p = 0.005) and prolactinaemia (p = 0.026). In contrast, there was a significant increase in HbA1c (p = 0.026). No effects on body weight and blood lipid levels  emerged.ALA produced a statistically significant reduction in QTc (ALA treatment appeared to be effective for reducing diabetes risk, liver functionality parameters, hyperprolactinaemia and QTC interval. ALA appears to be safe as adjunctive components in schizophrenia. •Atypical antipsychotics induce the frequent occurrence of metabolic side effects.•Prevalence rates of Metabolic Syndrome in patients with schizophrenia are high.•No specific therapies for metabolic effects of SGAs in schizophrenia are still available.•ALA was suggested as a potential therapeutic agent in various chronic diseases.•ALA appeared to be effective to improve metabolic values in schizophrenia. HoweverManagement and reduction of metabolic side effects remain a major concern when considering atypical antipsychotics for the treatment of schizophrenia. While there is a general consensus regarding routine monitoring of metabolic syndrome indices in patients receiving atypical antipsychotics, adherence to the guidelines is not common  and no sAlpha lipoid acid (ALA) is a natural substance commonly found in dietary components such as vegetables and meats, which is an essential cofactor of mitochondrial respiratory enzymes and is crucial for normal functioning of oxidative metabolism . ALA plaThe therapeutic use of ALA in schizophrenia has recently been investigated in human populations. A case series by Based on this background, ALA may be a potentially interesting therapeutic agent to improve the metabolic effects of atypical antipsychotics. The purpose of this study was to assess: (1) the efficacy of ALA on metabolic factors and (2) its safety and potential therapeutic effects in a sample of schizophrenic patients in stable therapy with atypical antipsychotics.22.1This was a 12-week, open-label, uncontrolled study, which aimed to evaluate the efficacy of adjunctive ALA in a sample of schizophrenia patients receiving atypical antipsychotic therapy. ALA was administrated in capsules at a fixed oral daily dose of 600 mg for the entire duration of the study in addition to the atypical antipsychotic therapy. The maximum dose of 600 mg per day was established according to 2.2The accidental sampling method was used to recruit the participants. Patients who met DSM-5 criteria for schizophrenia, aged between 18 and 60 years old in stable atypical antipsychotic monotherapy  for least 3 months  were conAll the patients provided written informed consent after a full explanation of the experimental design, according to the Declaration of Helsinki. Patients were recruited from June 2021, and the follow-up was completed in December 2021.2.3Patients attended two visits: initial screening (day 0), and final visit (week 12). Standard laboratory methods were used to determine, as primary outcome measures, fasting levels of glucose, glycated haemoglobin (Hb1c), total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and as secondary outcome measures, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (γGT), creatine phosphokinase (CPK), uric acid, creatinine, azotaemia, and prolactinaemia. Moreover, a physical examination was performed to measure systolic and diastolic blood pressure, heart rate, body weight, and body mass index (BMI). Electrocardiography (ECG) tracing was performed to measure QT interval and QTc. Finally, psychopathologic symptoms were assessed using the Positive and Negative Schizophrenic Symptoms Scale (PANSS) , which w2.4Prior to the start of the study, a power and sample size estimation was conducted (G∗Power 3.1.9.2.), under the assumption of an effect size of 0.8, a significant level of 0.05 with a power of 0.80, a minimal sample size of 12 was determined. Considering dropouts to normally be around 20%, a final sample size of 15 was selected for the study.Due to the small sample size, no randomization/allocation in subgroups was performed, and the analysis was carried out by nonparametric tests. Given the absence of dropouts, no last observation carried forward analysis was performed. Continuous data were expressed as mean (±S.D.), and the within-group differences between baseline and final tests were analysed by the Wilcoxon rank-sum test for dependent samples. As for the magnitude of the treatment effect, effect size was calculated based on Cohen’s d statistic and values lower than 0.50, ranging from 0.50 to 0.79, and 0.80 or greater, were considered small, moderate, and large, respectively . Results3Baseline characteristics, duration of illness, drug type and dose range in final sample are detailed in p = 0.012), blood glucose (Glucose T0 vs T1 (mean ± SD) = 105.4 ± 14.05 vs 88.8 ± 12.56, p = 0.005), AST (AST T0 vs T1 (mean ± SD) = 23.2 ± 7.16 vs 17.8 ± 3.36, p = 0.021), γGT (γGT T0 vs T1 (mean ± SD) = 18.2 ± 7.06 vs 15.6 ± 6.13, p = 0.035), CPK (CPK T0 vs T1 (mean ± SD) = 203.4 ± 177.13 vs 84.2 ± 50.35, p = 0.005) and prolactinaemia (Prolactinaemia T0 vs T1 (mean ± SD) = 389.76 ± 405.65 vs 190.16 ± 196.75, p = 0.026). In contrast, there was a significant increase in HbA1c (HbA1c T0 vs T1 (mean ± SD) = 5.2 ± 0.53 vs 5.34 ± 0.62, p = 0.026) and azotaemia values (Azotaemia T0 vs T1 (mean ± SD) = 25.6 ± 4.14 vs 30.8 ± 4.91, p = 0.021), both of which remained within the normal range. No significant differences in other clinical  and metabolic parameters  were observed. Effect sizes were large in Glucose (d = 1.26), AST (d = 0.9), CPK (d = 0.9) and Azotaemia (d = 1.1), moderate in QTc (d = 0.7) and Prolactinaemia (d = 0.6), and small (d < 0.50) in other clinical and metabolic parameters explored.No significant changes were observed in the subscales and PANSS total score , suggest4Schizophrenia patients show a high incidence of MetS with prevalence rates much higher than that of the general population and healthy controls . AlthougUndoubtedly, the metabolic effects of atypical antipsychotics represent a serious medical problem that needs to be addressed with specific therapeutic approaches to improve quality of life and life expectancy of patients with mental disorders. Pharmacological treatments have been proposed to reduce antipsychotic-induced metabolic abnormalities, but standard treatments have shown limited efficacy and pose safety concerns, especially in patients with a long disease history . As adjuThe main finding of the current work is that patients treated for 12 weeks with ALA at 600 mg/d experienced highly significant improvement of blood glycaemia, while glycated haemoglobin, although showing a tendency to worsen, is maintained within normal values. This result indicates the effect of ALA in reducing the risk of MetS since hyperglycaemia is the most ascertained mechanism in the pathogenesis of diabetes. In addition, this result is consistent with the previous findings showing a dose-dependent decline in glycaemic parameters in diabetic patients treated with ALA at different daily doses .No direct effects on body weight and blood lipid levels  were observed in our sample. This result is in accordance with other previous studies that found no effective weight reduction and no significant differences in cholesterol levels, triglyceride levels, and high-density lipoprotein cholesterol levels in schizophrenia patients who had taken ALA . HoweverIn the present study, ALA significantly reduced prolactinaemia after 12 weeks of treatment. Antipsychotic-induced hyperprolactinaemia provokes suppression of the hypothalamic–pituitary–gonadal axis with consequences such as sexual dysfunction, adverse effects on bone mineral density, and osteoporosis . Common An unexpected and interesting finding was the significant reduction in QTc interval in patients after ALA administration. No studies explored the relationship between the role of ALA and ECG parameters. Psychiatric drugs such as antipsychotics, seem to result in QT prolongation, and that is why it is considered a significant limiting factor when prescribing medications. . Our res5In conclusion, our findings indicated that adjunctive ALA treatment at the oral dose of 600 mg/d administered for 12 weeks resulted in a statistically significant reduction of relevant metabolic parameters. The primary result from this study reflects the benefits of ALA regarding blood glucose and confirms that oral administration of ALA can help improve glycaemic status in schizophrenia patients and encourages its use as an adjuvant therapy in atypical antipsychotics-induced diabetes. The unexpected reductions in QT interval and in hyperprolactinaemia are novel findings to be considered for further study, since these two parameters represent vital issues for psychiatrists in clinical practice. Finally, administration of ALA at a dose of 600 mg/d was well tolerated during the 3 months of treatment, given the observed absence of adverse events, ALA appears to also be neutral regarding psychopathological symptoms, suggesting its safe use as an adjunctive in psychiatric therapies.The findings of the present study need to be interpreted with caution because of the presence of several limitations such as the low dosage used, the small sample size, the lack of a comparative treatment, and the uneven distribution between sexes. Additionally, the length of the study was relatively short and thus it was not possible to assess the long-term effects of ALA supplementation. Furthermore, no safe conclusions can be drawn about patient compliance, as therapeutic drug monitoring has not been performed despite its importance in optimizing a treatment. Further clinical trials with adequately powered and well-designed methodology  are required to better evaluate the effectiveness of the ALA on atypical antipsychotic-induced metabolic side effects.Fiammetta Iannuzzo: Writing – original draft, managed the literature searches and wrote the first drafts of the manuscript. All Authors contributed to and have approved the final manuscript. Gianpaolo Antonio Basile: Writing – original draft, managed the literature searches and wrote the first drafts of the manuscript. All Authors contributed to and have approved the final manuscript. Domenica Campolo: Data curation, Formal analysis, organized recruitment, collected the data and undertook the statistical analysis. All Authors contributed to and have approved the final manuscript. Giovanni Genovese: Data curation, Formal analysis, organized recruitment, collected the data and undertook the statistical analysis. All Authors contributed to and have approved the final manuscript. Gianluca Pandolfo: Data curation, Formal analysis, organized recruitment, collected the data and undertook the statistical analysis. All Authors contributed to and have approved the final manuscript. Loretta Giunta: Supervision, Methodology, Formal analysis, supervised the Method procedures, analysis and interpretation of the results, and revised the manuscript. All Authors contributed to and have approved the final manuscript. Domenica Ruggeri: Supervision, Methodology, Formal analysis, supervised the Method procedures, analysis and interpretation of the results, and revised the manuscript. All Authors contributed to and have approved the final manuscript. Antonio Bruno: Writing – original draft, Formal analysis, designed the study, wrote the protocol, and supervised the various drafts and the final version of the manuscript. All Authors contributed to and have approved the final manuscript. Antonino Di Benedetto: Writing – original draft, Formal analysis, designed the study, wrote the protocol, and supervised the various drafts and the final version of the manuscript. All Authors contributed to and have approved the final manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


However, the frequency of severe AKI (stage 2 or 3 AKI) was comparable . Circulating markers of both immune and endothelial activation were associated with severe AKI. Children who had malaria and AKI had increased mortality , while there was no difference in mortality among children with NMFI . AKI is a common complication in children hospitalized with acute infections. Immune and endothelial activation appear to play central roles in the pathogenesis of AKI.Acute kidney injury (AKI) is a life-threatening complication. Malaria and sepsis are leading causes of AKI in low-and-middle-income countries, but its etiology and pathogenesis are poorly understood. A prospective observational cohort study was conducted to evaluate pathways of immune and endothelial activation in children hospitalized with an acute febrile illness in Uganda. The relationship between clinical outcome and AKI, defined using the Kidney Disease: Improving Global Outcomes criteria, was investigated. The study included 967 participants  with 687 (71.0%) positive for malaria by rapid diagnostic test and 280 (29.1%) children had a non-malarial febrile illness (NMFI). The frequency of AKI was higher in children with NMFI compared to malaria (AKI, 55.0% vs. 46.7%,  Globally, 85% of acute kidney injury (AKI) cases occur in low-and-middle-income countries (LMIC) . In sub-Plasmodium falciparum is the primary cause of severe malaria and is characterized by the cytoadherence of parasitized red blood cells to the endothelium [In the context of infection, the host response is an important determinant of disease severity and survival ,21. Endoothelium ,29. Paraothelium . Severe othelium ,30. HoweIn the present study, we characterize pathways of immune and endothelial activation in hospitalized children < 5 years of age with an acute febrile illness. We hypothesize that children with severe malaria-associated AKI experience greater endothelial and immune activation compared to children with NMFI-associated AKI.The analysis included 967 hospitalized children enrolled between February 2012 and August 2013 . The meap = 0.020). Malaria was associated with 38% reduced odds of AKI compared to children with a NMFI with a odds ratio (OR) of 0.62 (95% CI 0.46 to 0.83) after adjusting for child age, sex, and disease severity (p = 0.001). The prevalence of severe AKI was comparable in children with malaria vs. NMFI at 12.1% and 10.5%, respectively (p = 0.45). Differences in participant characteristics based on malaria status in children with or without severe AKI are presented in p < 0.0001) and NMFI (OR 5.06 (95% CI 1.71 to 14.97), p = 0.003). Delayed capillary refill time, a clinical sign of shock, was associated with severe AKI in malaria (OR 1.91 (95% CI 1.04 to 3.51), p = 0.04) but did not reach statistical significance among children with NMFI (OR 1.79 (95% CI 0.68 to 4.72), p = 0.24).The prevalence of AKI at admission was 49.1%. AKI was observed in 46.7% of children with malaria and 55.0% in children with a non-malaria febrile illness (NMFI) (p < 0.001). Furthermore, among children with malaria, a positive cystatin C test was associated with 3.59-fold increased odds of severe AKI (95% CI 2.12 to 6.09) compared to an 11.46-fold increase in the odds of severe AKI (95% CI 5.02 to 26.14) in children with a NMFI.We quantified levels of cystatin C as an alternate functional biomarker of AKI in children at enrollment. AKI represents an abrupt loss of kidney function due to changes in kidney filtration (assessed using filtration markers such as creatinine or cystatin C) or urine output. Cystatin C levels had moderate ability to discriminate between children with or without AKI in malaria with an area under the receiver operator characteristic curve (AUC) of 0.71 (95% CI 0.64 to 0.77) and good discriminatory ability in children with NMFI with an AUC of 0.82 (95% CI 0.72 to 0.91). Using a pre-established cystatin C cut-off of >0.8 mg/L , 19.4% oTo study the pathophysiology of severe AKI in children based on the etiology of infection, we quantified biomarkers involved in the host’s response to infection . Immune p < 0.05 for all). In children with both malaria and NMFI, there was evidence of endothelial activation (increased Angpt-2 and sFlt-1), as well as immune activation  in children with severe AKI  and AKI status (no severe AKI vs. severe AKI) . When evp < 0.0001 for both), and were higher in fatal cases (p < 0.0001 for both), providing evidence of convergent validity of the scales. Higher IAI and EAI were associated with severe AKI in both malaria and NMFI , while there was no difference in mortality among children with NMFI . A similar relationship was seen in children with severe AKI where children who had malaria and severe AKI had increased mortality , while the difference in mortality among children with NMFI was not significant . In models adjusting for age, sex, and disease severity, the presence of severe AKI showed a stronger relationship with mortality in children with malaria compared to children with NFMI  2.73 (95% CI 0.94 to 7.90), p = 0.06; NMFI, RR 1.04 (95% CI 0.34 to 3.18), p = 0.94). Similarly, when positive cystatin C was used as the diagnostic marker for AKI, there was an association with increased risk of mortality among children with malaria (RR 2.97 (95% CI 1.29 to 6.87), p = 0.01) but not among children with NMFI (RR 1.83 (95% CI 0.77 to 4.30), p = 0.17) after adjusting for age and sex and severity of illness.Overall, 2.9% of study participants died in-hospital. We evaluated the relationship between AKI, severe AKI, and mortality among children based on infection etiology. Children who had malaria and AKI had increased mortality  and endothelial  activation associated with AKI in children with both malaria and NMFI. In this population, AKI was associated with more pronounced endothelial activation and a higher risk of mortality in children with malaria, but not among children with NMFI.P. falciparum, an intraerythrocytic parasite, and results in the release of free heme, a known nephrotoxin [The mechanisms of AKI in febrile Ugandan children are likely multifactorial. Jaundice may be a clinical sign of hemolysis and was associated with AKI in both malaria and NMFI patients in this study. Hemolysis is common with hrotoxin . Delayedhrotoxin . ChildreP. falciparum cytoadherence [ANGPT2 gene associated with a functional decrease in Angpt-2 production is associated with reduced risk of developing a sub-phenotype of AKI defined based on sTNFR1, Angpt-2, and Angpt-1 [ANGPT2 gene may also modify the risk of AKI severity and recovery in the context of severe malaria.Endothelial activation appears to be a common pathway leading to AKI in both malaria and NMFI but may be more accentuated in malaria-associated AKI. Increased endothelial activation in the context of malaria-associated AKI may reflect direct injury to the glomerular endothelium due to dherence . Howeverdherence ,34,35,36dherence ,37. A po Angpt-1 . ChangesIn the present study, severe AKI was also characterized by immune activation with specific elevations in sTNFR1, sTREM-1, CHI3L1, and IL-8. A higher immune activation index (IAI) was associated with severe AKI in both malaria and NMFI. These findings are consistent with markers of immune activation as biomarkers of AKI and disease severity , to riskTo evaluate immune activation and endothelial activation as concepts, we used confirmatory factor analysis to derive indices of immune and endothelial activation based on panels of correlated biomarkers with similar biological functions . The IAIThe strengths of this study include the large sample size which enabled investigation of the relationships between AKI severity, immune and endothelial activation, and mortality in children hospitalized with an acute febrile illness due to malaria and NMFI. Limitations of this study include the lack of information on the etiology of febrile illness in children without malaria and a lack of data on co-infections. As most children tested positive for malaria, the study was underpowered to detect smaller differences in AKI-related mortality in children with NMFI. Despite these limitations, we show distinct differences in markers of life-threatening host response to infection in children with malaria-associated AKI compared to children with AKI due to other causes and demonstrate that children with malaria are particularly susceptible to AKI-related mortality. Together, these data suggest that pathways of immune and endothelial activation underlie the pathophysiology of AKI in children with both severe malaria-associated AKI and AKI related to NMFI. However, endothelial activation may be more pronounced in the context of severe malaria than NMFI-associated AKI. Further, endothelial activation may explain– in part– worse clinical outcomes in children with severe malaria and AKI. Additional studies are needed to delineate differences in AKI etiology and pathophysiology using urine biomarkers to differentiate between functional and structural kidney injury. Finally, studies are needed to evaluate pathways of adaptive vs. maladaptive repair following AKI in children with malaria vs. NMFI to identify opportunities to improve kidney recovery among children with severe malaria-associated AKI.P. falciparum histidine-rich protein 2 (HRP2) and pan-malaria lactate dehydrogenase (pLDH)  [This study was nested within a previously described prospective cohort study of children hospitalized with an acute febrile illness at Jinja Regional Referral Hospital in Uganda ,47. Chil, India) .Within the parent study, we conducted a nested sub-study to evaluate differences in the host response associated with AKI in children with malaria and a non-malarial febrile illness (NMFI). As a secondary outcome, we examined the association between AKI and in-hospital mortality in children based on malaria status. A sample size calculation indicated that we would need a minimum of 715 patients to detect a difference in mortality between patients with and without AKI at admission, assuming a baseline mortality rate of 4% , a preva2 as described [AKI was defined using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria based on a 1.5-fold increase in serum creatinine from estimated baseline and staged as follows: stage 1, 1.5–1.9-fold increase in creatinine over baseline; stage 2, 2.0–2.9-fold increase over baseline; stage 3 ≥ 3.0-fold increase over baseline. Baseline creatinine was estimated using a height-independent approach, assuming a GFR of 120 mL/min per 1.73 mescribed . AKI wasescribed . Creatinescribed .® Performance Assay (R&D Systems) in EDTA anticoagulated plasma stored at −80 °C until testing [Two panels of biomarkers were designed to interrogate host pathways of: (1) immune activation; and (2) endothelial activation. Selected molecules have been previously validated as biologically plausible and clinically informative markers host response to infection. Immune activation was assessed using circulating (plasma) levels of C-X-C motif chemokine Ligand 10 (CXCL10)/interferon γ-induced protein 10 kDa (IP-10) ,26, chit testing .t-test or Wilcoxon rank-sum test. Categorical data were analyzed using Pearson’s Chi-square test. To evaluate the relationship between clinical signs and severe AKI, logistic regression was used. To evaluate biomarker signatures across groups, biomarker concentrations were standardized to have a mean of 0 and a standard deviation of 1. The mean standardized concentration is presented by AKI and malaria status and differences analyzed using linear regression was used to evaluate differences in biomarker levels based on the presence of severe AKI and malaria. The reference category was children without malaria or severe AKI and the p values from malaria, severe AKI and the interaction term were used to evaluate significance. To adjust for multiple comparisons, the Benjamini–Hochberg procedure was used, adjusting for 36 comparisons. The relationships between biomarkers were evaluated using the non-parametric Spearman correlation. Confirmatory factor analysis was used to derive a single latent construct of immune activation  and endothelial activation . Differences in the indices were evaluated in children based on malaria and severe AKI status using a one-way ANOVA with Sidak’s multiple comparison test. To evaluate the relationship between AKI and a positive cystatin C test and mortality, Poisson regression was used with robust variance estimates and models adjusted for participant age, sex, and disease severity (assessed using LODS).Data were analyzed using STATAv14.0 and GraphPad Prism v7.0. Continuous data were analyzed using Student’s AKI in children hospitalized with severe malaria is associated with greater endothelial activation and higher mortality. With the International Society of Nephrology goal of eliminating preventable deaths due to AKI by 2025 (0 by 25 initiative), and efforts to reduce and eliminate malaria, increased collaborative research efforts directed towards the study of malaria-associated AKI are urgently needed.