We aimed to investigate the aetiology and outcomes of illnesses in patients presenting to an emergency department after travelling to a malaria-endemic country, in order to raise awareness of both tropical and cosmopolitan diseases.A retrospective chart review was performed for all patients who underwent blood smear testing for malaria at the Emergency Department of the University Hospitals Leuven from 2017 to 2020. Patient characteristics, results of laboratory and radiological examinations, diagnoses, disease course and outcome were collected and analysed.A total of 253 patients were included in the study. The majority of ill travellers returned from Sub-Saharan Africa (68.4%) and Southeast Asia (19.4%). Their diagnoses fell into three major syndrome categories: systemic febrile illness (30.8%), inflammatory syndrome of unknown origin (23.3%) and acute diarrhoea (18.2%). Malaria (15.8%) was the most common specific diagnosis in patients with systemic febrile illness, followed by influenza (5.1%), rickettsiosis (3.2%), dengue (1.6%), enteric fever (0.8%), chikungunya (0.8%) and leptospirosis (0.8%). The presence of hyperbilirubinemia and thrombocytopenia increased the probability of malaria, with a likelihood ratio of 4.01 and 6.03, respectively. Seven patients (2.8%) were treated in the intensive care unit, and none died.Systemic febrile illness, inflammatory syndrome of unknown origin and acute diarrhoea were the three major syndromic categories in returning travellers presenting to our emergency department after a stay in a malaria-endemic country. Malaria was the most common specific diagnosis in patients with systemic febrile illness. None of the patients died.The online version contains supplementary material available at 10.1186/s40794-023-00190-0. Until early 2020, international travel was increasing steadily. In 2018, more than 1.4 billion international trips were undertaken , 4. MalaThe emergency physician faces several challenges: most importantly, life-threatening diseases, such as malaria, must be recognised and treated adequately in a timely manner. Yet there is a knowledge gap in the diagnosis and management of patients with tropical diseases, leading to increased mortality and morbidity . MoreoveThe aim of this study was to investigate the aetiology and outcomes of illnesses in patients presenting to the emergency department (ED) after a stay in a malaria-endemic country, in order to raise awareness of both tropical and cosmopolitan diseases.This retrospective observational study was conducted at the University Hospitals Leuven, Belgium, a tertiary care referral hospital. A computer-assisted search for this chart review was performed to identify all patients who underwent diagnostic blood smear testing for malaria during a three-year period, from 28/02/2017 to 07/03/2020. We chose blood smear testing as a surrogate for illness after a stay in a malaria-endemic, as this test was part of the diagnostic workup for ill travellers returning from these countries [The following information was retrieved from the medical records: demographic data ; travel history ; reason for travel ; prophylaxis and adherence to malaria treatment (non-adherence was defined as not following the prescribed medication regimen); referral source . Duration and onset of symptoms, intake of antimicrobial therapy before presentation, symptoms at presentation (fever (reported or measured in the ED), isolated fever , respiratory symptoms, ear, nose and throat abnormalities, anorexia, diarrhoea, dysuria or urinary frequency, myalgia and arthralgia) were recorded. Relevant physical findings  were collected from patient files. All available laboratory results  were reviewed. Results of all performed radiological examinations were registered. Also records of follow-up care  were reviewed for information on diagnosis, disease evolution and outcome.Travel destinations were grouped in eight global regions corresponding to those defined for the GeoSentinel Surveillance Network: Sub-Saharan Africa, the Middle East and North Africa, Southeast Asia, South-Central Asia, Northeast Asia, Latin America and the Caribbean, North America and Australia, New Zealand and Oceania . When diDiagnoses were made in accordance with internationally recognised case definitions . All diaCategorical variables are reported as totals and frequencies, and continuous variables are expressed as medians with interquartile ranges (IQR). We used 2 × 2 tables to calculate sensitivity, specificity, positive and negative likelihood ratios of signs, symptoms and laboratory findings. The confidence intervals (CI) for sensitivity, specificity and accuracy were 'exact' Clopper-Pearson confidence intervals.The CI of the likelihood ratios were calculated using the log method . Data wen = 39), repeated blood smears from the same patient (n = 38), absence of travel to malaria-endemic country within the past year (n = 26), incomplete patient records (n = 4), blood smear taken outside the ED (n = 1) or being asymptomatic (n = 1).A total of 362 blood smears from the returning travellers in our ED were performed in the predefined period, and 109 blood smears were excluded from further analysis because of failure to meet the age criterion . Detected bacteria included Salmonella Paratyphi A (n = 1), Salmonella Paratyphi B (n = 1), Salmonella group B (n = 1), Salmonella group D (n = 1), Escherichia coli (n = 3), Streptococcus agalactiae (n = 1) and Streptococcus pneumoniae (n = 1). The other three positive cultures were contaminated with coagulase negative staphylococci (n = 1), Acinetobacter Iwoffii (n = 1) and Corynebacterium spp. (n = 1). Urine cultures turned positive in 5.2% (10/192). Stool cultures were positive in 13 out of 38 tests: isolated pathogens consisted of Salmonella spp. (6/13), Shigella spp. (5/13) and Campylobacter spp. (2/13). All 54 patients that were tested for HIV were negative. Only 12 (6.9%) of the 175 chest X-rays performed were abnormal, showing an infiltrate (n = 10), pleural effusion (n = 1) or pulmonary nodule (n = 1). Abdominal ultrasound was performed in 138 patients (54.5%) and showed abnormalities in 23.9% of the examinations, including splenomegaly (n = 10), hepatosplenomegaly (n = 6), hepatomegaly (n = 3), lymphadenopathy (n = 3), colitis (n = 7), signs of enteritis (n = 3), acute appendicitis (n = 1) and sedimentation at the bladder base (n = 1). In 1 patient, 2 abnormalities were detected .Blood cultures were obtained from 231 out of 253 patients and were positive in 5.2% (n = 2), subarachnoid haemorrhage (n = 1) and lymphoproliferative disorder (n = 1).Table n = 33) or non-adherent (n = 7) with the prophylaxis (early discontinuation). In our study 5/40  had a negative blood smear. In four of these patients the rapid antigen detection test  turned positive (presence of histidine-rich protein II detected). One patient needed a PCR test ) for diagnosis. Plasmodium falciparum was found in 75% of the malaria cases. Four patients  with falciparum malaria met the WHO criteria for severe malaria . The majority of patients diagnosed with malaria returned from Sub-Saharan Africa (95.0%) and their most frequently reported symptoms were fever (97.5%), headache (75.0%), myalgia (55.0%), anorexia (37.5%), arthralgia (25.0%), abdominal pain (20.0%) and diarrhoea (17.5%). In three patients with a malaria diagnosis , a co-infection was detected (1 rickettsiosis (positive IgG antibodies to Rickettsia conorii), 1 acute hepatitis E and 1 influenza B).Malaria was detected in 40 patients (15.8%), all of them not taking malaria prophylaxis  and 6.03 (95% CI: 4.08 – 8.90), respectively (Table The majority of the 253 patients (55.7%) were treated in ambulatory care setting; 44.3% required admission to the hospital, and 2.8% were admitted to the intensive care unit. The median length of hospital stay was 4 days (IQR 3–6). None of the patients died.This study focusses on characteristics and outcomes of ill returning travellers presenting to the emergency department after a stay in a malaria-endemic country.n = 40) was the most common specific diagnosis in the patients with systemic febrile illness, followed by influenza (n = 13) and rickettsiosis (n = 8). Plasmodium falciparum, the cause of severe  malaria, was identified in 75.0% of the patients with malaria. Compared to the study of Siikamaki et al., who performed a similar retrospective study in the ED looking at febrile adults after travel to malaria-endemic areas, our data show a higher percentage of Plasmodium falciparum malaria (11.9% vs 3.5%) [Malaria (osis n = . Plasmodvs 3.5%) . Althougosis n = . Plasmodenza n =  and rickEarly diagnosis and treatment of malaria is of utmost importance, since delay in diagnosis is associated with fatal outcome . When anSalmonella Enteritidis and E. coli)) [It is important to stress that malaria may be acquired together with other pathogens that are endemic in the visited areas , and that symptoms can overlap. Therefore, additional diagnoses should always be considered, even when a diagnosis of malaria has already been made: a tropical co-infection may be present, may require specific therapy and may cause significant morbidity when left untreated . In our . coli)) .Although recognition and early treatment of life-threatening tropical diseases by the emergency physician is crucial, in our study only 22.5% of the patients had a ‘tropical’ diagnosis  was almost exclusively seen after return from Sub-Saharan Africa (7 of 8 patients), especially South-Africa (6/8), based on epidemiological data presumably 6 cases of African tick-bite fever. Enteric fever was only found in Asia in our study . Dengue was diagnosed in 4 patients (1.6%), all after travels to Asia or Latin America. These data are in accordance with the GeoSentinel surveillance data, and emphasize the importance of the region of return when determining the prior probability for a specific disease [When we looked at the prevalence of tropical disease according to geographical region of exposure, rickettsiosis  and/or positive IgG antibody titer to  disease .For a large proportion of the travellers presenting at the ED (23.3%), the aetiology of illness remained unclear, which is consistent with other reports in and outside the ED (no specific diagnosis in respectively 25.1 and 24.4% of patients) .Strongyloides hyperinfection syndromes [Mortality in returning travellers has been reported to range from 0.2 to 0.5% in other studies , 5, 7, ayndromes –21. In oSince this is a single-centred study performed in a tertiary care hospital, there are restrictions on generalisability to other non-tertiary centres. We used the blood smear test as a surrogate for illness in the returning travellers after a stay in a malaria-endemic country. Theoretically we may have missed some patients that erroneously did not have a blood smear, but we believe this is unlikely since training on imported diseases is organised on a yearly basis in our centre and the threshold to request a blood smear is low. We were not able to collect reliable data on pre-travel vaccination status and so we did not include it on our study. Furthermore inpatients presenting with diarrhoea; parasitic testing was not routinely performed. The indication was evaluated case by case. In our study, there were no diagnoses of parasitic diarrhoea (e.g. giardiasis or amebiasis) and this could be an underestimation. Finally, this study discusses the patient population before the onset of the Covid-19 pandemic. For now, it remains unclear if the pandemic will have a permanent influence on international travel and cause a shift in the epidemiology of diseases in returning travellers.Our study confirms the broad differential diagnoses of illnesses in returning travellers presenting at the ED after a stay in a malaria-endemic country, and thus the need for continued vigilance in the ED physician. Both tropical and non-tropical, infectious and non-infectious causes should be taken into account. Whether they are tropical or non-tropical, potentially life-threatening diseases should be excluded or confirmed first. A thorough clinical examination, directed laboratory tests and knowledge about the prevalence of tropical disease according to the geographical region of exposure can help narrow down the possibilities.Additional file 1. Final diagnoses.


Plasmodium falciparum infections and exposure, P. falciparum apical membrane antigen 1 (PfAMA1) antibody (Ab) response, in the Central Highlands of Madagascar (CHL).Targeted research on residual malaria transmission is important to improve strategies in settings pursuing elimination, where transmission reductions prove challenging. This study aimed to detect and characterize spatial heterogeneity and factors associated with fokontany (villages) within 7 health districts of the CHL. Rapid diagnostic tests (RDTs) and a bead-based immunoassay including PfAMA1 antigen biomarker were used to estimate malaria prevalence and seroprevalence, respectively. Local Moran’s I index was used to detect spatial “hotspots”. Remotely sensed environmental data—temperature, vegetation indices, land covers, and elevation—were used in multivariable mixed-effects logistic regression models to characterize factors associated with malaria infection and cumulative exposure.From May to July 2014, a cross-sectional school-based survey was carried out in 182 Among 6,293 school-children ages 2–14 years surveyed, RDT prevalence was low at 0.8% (95% CI 0.6–1.1%), while PfAMA1 Ab seroprevalence was 7.0% (95% CI 6.4–7.7%). Hotspots of PfAMA1 Ab seroprevalence were observed in two districts (Ankazobe and Mandoto). Seroprevalence increased for children living > 5 km from a health centre (adjusted odds ratio (OR) = 1.6, 95% CI 1.2–2.2), and for those experiencing a fever episode in the previous 2 weeks , but decreased at higher elevation . A clear age pattern was observed whereby children 9–10 years old had an OR of 1.8 (95% CI 1.2–2.4), children 11–12 years an OR of 3.7 (95% CI 2.8–5.0), and children 13–14 years an OR of 5.7 (95% CI 4.0–8.0) for seropositivity, compared with younger children (2–8 years).The use of serology in this study provided a better understanding of malaria hotspots and associated factors, revealing a pattern of higher transmission linked to geographical barriers in health care access. The integration of antibody-assays into existing surveillance activities could improve exposure assessment, and may help to monitor the effectiveness of malaria control efforts and adapt elimination interventions.The online version contains supplementary material available at 10.1186/s12936-022-04260-0. Malaria remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. While many countries in the African region have the potential to eliminate malaria in the medium or long term—malaria case incidence reduced from 363 to 225 cases per 1000 populations at risk in 2000 and 2020, respectively—Madagascar has still been aiming to improve case management for at least 95% of diagnosed cases and to ensure the permanent availability of diagnostic-and-treatment tools for 95% of health facilities since 2013 . The NatPlasmodium falciparum are influenced by age and exposure to infection  were performed with finger prick blood to detect malaria infections in all selected children. Children with a positive RDT were treated with artesunate-amodiaquine with age-appropriate doses, as recommended by national guidelines. Additionally, capillary blood was collected for all children in microvette tubes  for later serological analyses. Collected samples were transported to Institut Pasteur de Madagascar’s immunology laboratory in Antananarivo and stored at − 20 °C until used .P. falciparum antigens: three soluble recombinant proteins , and bovine serum albumin-conjugated peptides (PfCSP and PfGLURP) from P. falciparum, using procedures previously described ), and every 10% (one unit) increase in school-level RDT prevalence was associated with a doubling in the odds of PfAMA1 Ab response aggregated at fokontany-level . Out of all the environmental and climatic variables, only elevation was significantly associated with P. falciparum exposure, with a 30% decrease in the odds for every 100 m (one unit) increase in elevation  (Table For on Table . After e95% CI 1.–2.2), anP. falciparum exposure risk, or the considered district-and-commune level random effects.No evidence of residual spatial autocorrelation was found in the final multivariable model for PfAMA1 Ab response at the fokontany-level; and no deviance to normal distribution was observed to its residuals  model at the fokontany-level  the previous month, the percentage of land cover such as woodlands, and the elevation of vel Fig. . In addiP. falciparum infection, approximately 7.0% of children had been exposed to P. falciparum according to the Ab response despite a very low prevalence of malaria infection using RDTs (0.8%). Furthermore, hotspots of PfAMA1 Ab seroprevalence were observed in multiple districts in the area, many of which were not observed via malaria RDTs. Exposure to the malarial parasite, as indicated by seroprevalence, increased with a child’s age  although it was not representative of this age group because the study was restricted to school-aged children; and in rural Madagascar, children generally start school at age 5. A sub-analysis of factors associated with exposure of ≤ 5 years children  suggests the absence of the barrier of distance to health facilities  were significantly more exposed to lth care , could rlth care . Communilth care , since tP. falciparum cumulative exposure, PfAMA1 Ab response at the fokontany-level was higher at lower elevations, but there was little additional effect of other environmental and climatic factors examined. Elevation is widely used in malaria mapping as an established proxy of malaria transmission due to its association with precipitation and temperature which, along with vegetation cover, are generally found to be important predictors of malaria incidence and transmission, given their role on Anopheles spp survival, development, breeding, and biting rates [. In this setting, elevation was significantly and negatively correlated with temperature and grasslands or cereals land covers, but positively with vegetation cover proxies (NDVI and EVI)  and vector ecology [This study had several limitations ecology , 66. Sec ecology .. Nevertheless, sub-optimal testing and treatment of malaria infections in CHL and surrounding Fringes areas could undermine elimination efforts by NMCP, at the moment when Madagascar should adapt interventions to face the current challenge of plateauing or increasing malaria cases [In this setting, serological markers (PfAMA1 Ab) enabled to highlight hotspots of malaria seroprevalence in multiple districts in the highlands of Madagascar (CHL and Fringe areas)—many of which were not observed via malaria RDTs—and associated factors, revealing a pattern of higher transmission linked to geographical barriers in health care access. Targeting these residual pockets could reduce malaria transmission at the community level , 68. Nevia cases , 49. Seria cases , and helia cases .The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.Additional file 1: Method S1. Two-component gaussian mixture model; Method S2. Environmental and remotely sensed data processing; Method S3. Mixed-effects binomial logistic regression model frameworks and selection; Table S1. List of environmental and climatic data; Table S2. Univariable and multivariable P. falciparum exposure (PfAMA1 Ab response) model of individual-, household- and fokontany-level covariates, using mixed-effect logistic regression at district and commune level; Figure S1. Cut-off value for PfAMA1 Ab seropositivity (dashed-red line) by using two finite Gaussian mixture models and the serological data  described in Steinhardt et al. [;Figure S2.Fokontany neighbour definition by maximum distance, using Great Circle distance around 17 km between two contiguous fokontany; Figure S3. Malaria infection prevalence by RDT versus PfAMA1 Ab seroprevalence detected high clusters fokontany across districts. High clusters fokontany are the combination of “High-High” or “Low–High” clusters; that represent fokontany with expected values  matching with the weighted mean of each fokontany’s neighbours, or those with abnormally high expected but low expected values, respectively; Figure S4. Weighted global Moran’I statistics versus simulated random distribution of logit scale of (A) malaria infection prevalence by RDT (I = 0.24), and (B) PfAMA1 Ab seroprevalence (I = 0.59); Figure S5. Moran Scatterplots of clusters and hotspots of (A) malaria infection prevalence by RDT, and (B) PfAMA1 Ab seroprevalence; Figure S6. Pearson’s correlation scatterplot and peer’s significance of P. falciparum infection prevalence by RDT, PfAMA1 Ab seroprevalence and quantitative environmental and climatic covariates. Levels of significance are marked with (***) for p < 0.001, (**) for p < 0.01, and (*) for p < 0.05. Lagged values of temperature and vegetation at 2 and 3 months were less correlated to malaria infection prevalence by RDT and PfAMA1 Ab seroprevalence than one-month lag at fokontany-level; Figure S7. Pearson’s correlation scatterplot and peers of P. falciparum infection prevalence by RDT, PfAMA1 Ab seroprevalence and quantitative environmental and climatic covariates associations. There was high correlation between multiple pairs of covariates such as {NDVI, EVI, forests, woodlands, or grasslands/cereals}, {LST day, LST night, elevation, woodlands or grasslands/cereals} and {grasslands/cereals, wet/croplands/mosaics}; Table S5. Univariable and multivariable P. falciparum infection model at individual-, household- and fokontany-level covariates using mixed-effect logistic regression at district and commune level; Figure S8.P. falciparum exposure (PfAMA1 Ab response) model residuals diagnostics of normality at fokontany-level. No significant deviance to normal distribution was observed in PfAMA1 Ab seroprevalence model residuals using Kolmogorov–Smirnov normality test (p = 0.3); Figure S9. Spatial autocorrelation of residuals diagnostics using Moran’s I index of non-spatial P. falciparum exposure (PfAMA1 Ab response) model at fokontany-level; Table S6. Sub-model for P. falciparum exposure (PfAMA1 Ab response) model of individual- and fokontany-level covariates, using binomial logistic regression.t et al. ;Figure Additional file 2: Table S3. Malaria infection prevalence by RDT measures of local indicators of spatial association.Additional file 3: Table S4. PfAMA1 Ab seroprevalence measures of local indicators of spatial association.


The combined application of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are commonly used malaria interventions that target indoor Anopheles vectors. Recent studies on the effects of house screening (HS) and LLINs have demonstrated a reduction in indoor vector densities and malaria when the interventions are combined. In addition, complementary interventions are needed to curb co-occurring pest populations which pose menace to agricultural crop productivity and food security. However, interventions that impact malaria mainly centre on public health strategies, overlooking subtle but important component of agricultural measures. Addressing the coexisting risks of malaria and crop pests could contribute to improved livelihood of communities.A four-armed household, cluster-randomized, controlled study will be conducted to assess the combined impact of HS, LLINs and push-pull agricultural technology (PPT) against clinical malaria in children in Ethiopia. The unit of randomization will be the household, which includes a house and its occupants. A total of 838 households will be enrolled in this study. In this trial 246 households will receive LLINs and HS, 250 will receive LLINs, HS and PPT, 175 households will receive LLINs and PPT. The remaining 167 houses which receive LLINs only will be used as control. One child aged ≤14 years will be enrolled per household in each treatment and followed for clinical malaria using active case detection to estimate malaria incidence for two malaria transmission seasons.Episodes of clinical malaria, density of indoor biting malaria vectors, sporozoite infection rate, improved crop infestation rate, crop yield gain, livestock productivity and cost effectiveness analysis will be the end points of this study. Socio-economic, social demographic, cost-effectiveness analysis will be conducted using qualitative and participatory methods to explore the acceptability of HS and PPT. Documenting the combined impact of LLINs, HS and PPT on the prevalence of clinical malaria and crop pest damage will be the first of its kind.https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=11101.Pan African Clinical Trials Registry, PACTR202006878245287. 24/06/2020. The online version contains supplementary material available at 10.1186/s12889-022-12919-1. Malaria continues to be a major health threat in Africa where 93 and 94% of the global cases and deaths, respectively, are reported . Long-laHouse screening (HS), a promising supplementary vector control tool, is a valuable non-chemical strategy for preventing indoor biting by vectors, by acting as a physical barrier against entry into human dwellings –8. HS coIn addition to introducing HS for malaria vector control, we are interested to know the impact of providing farmers with agricultural technologies on productivity. This is because agriculture and health has always been interconnected in many ways. Increasing agricultural productivity improves the overall livelihoods of communities through improved health, nutrition, and income generation through sale of crops and livestock . A studyA combination of interventions is commonly evaluated to improve malaria outcomes. For instance, the joint implementation of LLINs and IRS were found to significantly reduce malaria incidence , 20, altThe benefits of HS and PPT have been separately documented by several studies , 22–27. The general objective of this intervention is to determine whether the combined intervention of long-lasting insecticidal nets, house screening and push-pull technology provide better protection against malaria and improves agricultural productivity in the intervention households compared to control households who received LLINs alone in Jabi Tehnan area, Northwest Ethiopia.i.To determine whether adding house screening of windows and doors of houses and implementing PPT reduces the rate of malaria parasite infection, parasite density, and anemia in children (aged between 5 to 14) compared to situations where only LLINs are used.ii.To assess whether the proposed interventions reduce human mosquito-interaction ) inside houses compared with LLINs alone.iii.To determine the incremental costs, benefits, and cost-effectiveness of adding house screening and PPT to usage of LLINs.iv.To assess the economic, social, and environmental feasibility of the combined intervention of house screening, LLINs and PPT.(Kebeles), which is the lowest administrative unit of the country, and three town administrations. More than 90% of the people in the district live in rural areas practicing mixed farming.This study will take place between September 2020 and December 2022 in the Jabi-Tehnan district of Amhara regional state, Northwest Ethiopia. The capital town is Finote-Selam, which is about 387 km from the National capital, Addis Ababa, and 176 km Southwest of the regional capital, Bahir Dar. The population of the district was 211,516 in 2017 with an average annual growth rate of 2.8% . The dis(Kebeles) of the district, malaria is a major health problem threat resulting in total mortality of 4345, and average Plasmodium annual parasite rate (API) of 11% . A cross sectional active malaria prevalence survey from randomly selected kebeles of the district in 2013 showed the disease prevalence of 2.8% [The altitude of the district ranges from 900 to 2300 m above sea level. Much of the area lies in the higher altitude range, closer to 2300 m. Agro-ecologically, 88% of the district is classified as mid land and the remaining 12% as low land. The topography of the district is dominated by areas of flat plain. According to Asmare & Gure, 2019 , the top of 2.8% .The source population of this study will be the total population of the Jabi Tehnan district, Amhara Regional State, Northwest Ethiopia.The study population will be all households that are found in the rural areas of Jabi-Tehnan district having at least one child (under 14 age) in their family.(Kebeles) in the district showed a 2.8% malaria prevalence and used as a benchmark for this trial. Accordingly, 838 households, will be selected from 30 villages (Kebeles), using 80% power at the 5% significance level.Studies conducted in Ethiopia  and The According to WHO guideline for malaria entomology , a repredoi.org/10.1257/rct.5642-1.0).We will use the baseline data from the 3010 households collected in the district for the socioeconomic analysis. The sample is randomly selected from a fresh list of households in the rural and semi-urban part of the district. We will follow these households and collect additional information two times . The data collection procedure for the social data were discussed in detail in Abro et al. (2020)  will be based in the study district and form a recruitment committee. The committee will be composed of the head of local administration, representative from village HEWs, the head of local health center and the principal investigator (AA). The team will make house-to-house visits and explain the objective of the study to the candidate household heads. The list of candidate households will be separately generated from the roster containing the list of household heads of the district by the data manager (ZA). In instances where the candidate household refuses to participate, reserve candidate households will be invited to participate.A longitudinal study of four-armed household clustered randomized control trial will be conducted to estimate the incremental benefit of combining house screening, long-lasting insecticidal nets, and push-pull technology.The study design is summarized in a flow chart as shown in Fig. In this study, a total of 167, 246, 250 and 175 houses will be included in four different treatments, treatment 1 , treatment 2 (LLINs and HS), treatment 3  and treatment 4 (LLINs and PPT), respectively. The differences in sample size across arms is due to differences in number of households within each sub-village. To full fill the total sample size, we did the random selection based on population size of each sub-village. Census of children under 14, and above 14 years of age will be made in all 838 households selected. One child will be randomly selected from each house for annual malaria parasite survey. In addition to the annual parasite screening survey, monthly follow up will be implemented in all households to monitor clinical malaria by health extension workers. If a child moves or quits the study, we will replace the child from the same house. In instances where the whole family leaves the area, we will document them as lost to follow up.Plasmodium species infection, parasite density, and the prevalence of anaemia. Collections will be made from both indoor and outdoor settings.The selected children will be checked up every month for clinical symptoms of malaria infection, splenomegaly, and development of anaemia. Blood test will be conducted if the child develops clinical symptoms. The child will also be surveyed at the beginning and end of each transmission season to estimate the prevalence of sub-kebele) level. For this study, we divided the 30 villages (Kebeles) into 66 sub-villages/sub-kebeles. In each sub-kebele, we will obtain a fresh list of farmers, which are organized into one-to-five groups.sub-kebele will be randomly assigned into the four treatment arms.As described in the Section of study population all the households in the district are eligible to the treatment. The unit of randomization is the sub-village . Therefore, the study area will be first clustered into kebeles and then each kebele will be clustered in to sub-kebeles. The randomization process will be conducted using the STATA’s sampsi package and using sub-kebele as a unit of randomization. Efforts will be made to ensure that each kebele receives balanced number of treatments following the standard protocol developed by Pinder et al. (2016) [sub-kebeles will take out the kebele effect and the likelihood of chance imbalances between study arms. All entomological, epidemiological, and social studies will be conducted following the same rule described above i.e., stratified randomization. The randomization will be done by ZA to prevent selection bias by concealing the allocation sequence from the field researchers assigning sub-kebeles into the four treatment arms until the moment of assignment. Thus, both the chief investigator and the principal investigator will not be involved in the randomization process.As the study area is composed of different localities (and elevations) of the district, the risk of malaria infection also varies among . (2016) . StratifScreening of windows and doors will not be blinded as it is difficult to conceal them. However, we will follow the World Health Organization 2015  guidelinDuraNet®  will be provided to all households (except control arm) at the rate of one bed net for two people following the NMCP and as per WHO recommended universal net coverage , 39. TheThe house screening work will be undertaken by pre-trained artisans of about 4 to 5 people to be recruited from the study area. The training will be done by an in-country contractor familiar with screening of houses. The screening of 500 houses is expected to take about 2 months. Household owners will be trained on the care needed to keep the screens intact and effective and avoid activities that could result into making holes in the mesh or cause the screen to slide and create spaces that could allow mosquito entries into the houses. Other routine procedures to be used to reduce mosquito entry into houses such as closing windows and doors early will be emphasized and adherence to this practice monitored passively by the study team. As part of the intervention all the windows, doors and eaves will be screened using polyethylene material (POLYTEX INTERNATIONAL (UK) LIMITED, 14 Rutherford Way, Drayton Fields, Daventry, Northants, NN11 8XW, United Kingdom).Desmodium uncinatum. The pull component refers to the brachiaria grass, Brachiaria cv mulato, which is usually planted at the periphery of the plot and the intercropped plant which is our target plant to be protected (Maize or Sorghum). While the desmodium is rich in protein, the brachiaria is rich in carbohydrate, which will serve as an important source of animal feed. The detail application of PPT is described elsewhere [Push pull technology is a biological method of controlling pests of cereals: stem borer and striga. It is a novel pest control method in which a repellent intercrop and attractant trap plant is used simultaneously. It has three components which include the push, the pull, and the intercropped plant. The push component refers to desmodium plant, a.k.a. lsewhere . The deslsewhere , 41. Botlsewhere .Desmodium intortum 3-5 kg/Ha from the same company. The amount of Kg provided to each farmer will be decided on the size of plot made ready by the farmer.Thus, as part of the intervention training will be provided to selected farmers on the concept of PPT and how to use it. These include plot preparation, sowing, establishing, managing, animal feed preparation and seed harvesting. A total of 425 farmers who successfully completed the training and who been selected to house screening treatment will be provided PPT plant seeds namely Mulato II Hybrid Brachiaria,  and There is no risk of any infection as we do this study, however, finger pricking for blood test is usually with mild pain and discomfort.For this study, each household will be provided with a specific identification (ID) number. The child included in the study from each household will receive a unique personal, three-digit ID number (village number/household number/person number). All forms and datasets will identify participants by their unique identifier numbers, and names will not be used. The geographic coordinates of all study households will be documented at the beginning of the study and used in mapping the study points. Questionnaires and forms used will be initially prepared in English and translated into Amharic for data collection and then it will be translated back to English later. The questionnaire has already been validated. We will use the questionnaire for collection of the malaria KAP, socio-economic, socio-demographic, the acceptance of HS, challenges related to HS utilization, LLINs ownership and utilization. There are questions pertaining to PPT acceptance, utilization, and other opinion. Databases will be password protected and accessible only to authorized personnel. All documents will be securely stored in locked filing cabinets and accessible only to authorized personnel.Awareness creation on the purpose of the study, on the type of the interventions, and the need to involve in the study will be communicated to the community members through existing channels such as local government centres and through health extension workers. Farmers will be randomly selected to be part of the trial, and this could create some confusion among community members and lead into unnecessary misinformation. In this regard, the randomization process will be clarified to the community members to clear the misinformation and further explain that the community members at the receiving end of the intervention do not have any role whatsoever in the process of selection.Artemether-lumefantrine (AL) and chloroquine by a trained health worker following national guidelines. In Ethiopia, CHWs are trained and mandated to test and treat malaria.A rapid diagnostic test (RDT), Microscopy and dry blood spot (DBS) will be used to measure the epidemiological parameters. In addition, prevalence of anaemia and splenomegaly among the study participants (children) will be graded according to their Hb level and by observation respectively. The axillary temperature will be taken fortnightly by health extension workers from all enrolled study children and if the child shows ≥37.5 °C or history of fever in the past 48 h, then, a rapid diagnostic test  will be conducted to further cross check the presence of malaria parasite. In line with this, blood spot sample (DBS) will be collected for later testing using PCR-based methods. In this regard we propose to collect two set of malaria case data. The first one is monthly epidemiological data that is collected based on clinical symptoms during the house-to-house visit and the second set of epidemiological data is obtained through active annual malaria prevalence survey. Monthly case follows up and testing of febrile cases will be conducted using malaria RDT kit whereas the annual active parasite screening will be done using diagnostic tools, Microscopy and DBS. The combination of diagnostics will be used to fill the gap among the tools. For instance, asymptomatic and sub-microscopic cases will be detected using the DBS PCR approach. The monthly house to house follows up will be made by health extension workers. The annual prevalence survey will be made by licensed laboratory technicians and health extension workers. In all instances, malaria positive children will be provided with a full dose of Anopheles mosquitoes (Indoor and Outdoor) will be identified using taxonomic keys of Gilles and Coetzee (1987) [Anopheles gambiae s.l., and Anopheles pharoensis collected by CDC light traps from each study villages (Kebeles) will be carefully separated from the abdomen and tested for P. falciparum and, P. vivax-210 and P. vivax-247 circumsporozoite proteins (CSPs) simultaneously following the protocol developed by Beier et al. 1988 [A total of 10 houses, (five houses for indoor and five houses for outdoor LTs) catches will be selected from each study village. Indoor and outdoor catches will be collected from each study arm. Mosquitoes will be collected indoor and outdoor from 6:00 pm to 06:00 am from each selected house using standard battery-operated CDC light traps. Traps will be hung from the ceiling or from roof support at the foot end of the bed where people sleep at night and each trap will be suspended about 1.5 m from the floor. Traps will be also hung outdoor under the eaves of the house for outdoor mosquito collection. Each trap will be set by trained research team members. Collection bags will be retrieved from traps in each house in the morning between 06:00 am and 07:00 am. All unfed, fed, half gravid and gravid adult female e (1987) . Dried hal. 1988 . On agriSemi-structured questionnaire will be prepared to collect socio-demographic data at the beginning and end of the study. Each study household will be observed for durability of the screening material, the presence of holes, tears, losses and the fitness of windows and doors. The presence or absence of LLINs and the number and age of people who slept under bed nets in the previous night will be assessed in every visit to mosquito sampling (before and after screening). The impact of screening intervention on bed net use rate will be measured by observing bed net use among household members before and after house screening intervention, and between intervention and control groups. For the social science component of this impact evaluation, the team will collect data using household surveys, focus group discussions and key informant interviews. A baseline survey, midline survey and end line surveys will be undertaken using a structured household survey questionnaire and trained enumerators.The main outcomes of the clinical study will be the malaria incidence, splenomegaly, and anaemia. Thus, the baseline clinical survey of all study children will take place between Sept and Oct 2020 to determine the clinical parameters. As part of the follow up, starting from Nov 2020, trained health extension workers will visit the house of each child and take data of clinical symptoms, temperature, and splenomegaly (sign of spleen enlargement). While monthly follow up of the child continues throughout the 2 years of study period (Sept 2020 to Dec 2022) for documentation of any malaria incidence, blood testing of the entire cohort of the study children will be repeated at the end of each rainy season (Sept/Oct 2021) and Sept/Oct 2022). In between the annual clinical surveys , blood testing will be done only to children who develop fever to minimize community fatigue development. Temperature measurement, RDT testing, blood haemoglobin measurement, blood sample collection for microscopy will be conducted by a licensed laboratory professional following the WHO guideline .The major entomological outcomes of the study are changes in mosquito densities, rates of mosquito infection (sporozoite rates), and the entomological inoculation rate (EIR). Both indoor and outdoor mosquito collections will be made using the Centres for Disease Control (CDC) light traps to estimate the mosquito density. Collections will be made twice per year (July to Sept and January to March) starting from July 2020 to Oct 2022. All mosquito collections will be preserved in silica gel and transported to the International Centre of Insect Physiology and Ecology (ICIPE) Kenya, Nairobi, ICIPE molecular laboratory, where they will be identified to species level and examined for sporozoite infection using Polymerase chain reaction (PCR) technique. Blood meal analysis of fed females will be done using Enzyme linked immunosorbent assays (ELISA).In line with the investigation of vectors of medical entomology, the agronomics team will evaluate the efficacy of PPT against stem borers, fall armyworm (FAW) and striga infestation. Visual observation will be made in selected plots from each treatment arm (PPT plot and control) and the degree of infestation will be measured using scale scores where 1 attributes to clean plant with no infestation and 5 attributes to very severely damaged plant .The actual outcomes for the economic and social evaluations are discussed in detail in Abro et al. 2020. “Social networks, incentives, and diffusion of house screening and push-pull technology interventions in rural Ethiopia.” AEA RCT Registry. April 06. 10.1257/rct.5642-1.0. The primary outcomes are PPT knowledge score, PPT and HS adoption productivity of maize (kg/ha), milk productivity , cost of illness (USD/household), malaria prevalence (%), lost working days due to malaria, and lost school days due to malaria for children. We have also other secondary outcomes such as willingness to pay for PPT and HS, women and children dietary diversity score, and household food insecurity access scale (HFIAS) .Data will be stored in two forms, i.e., hard, and soft copies in compliance with the principles of good clinical practice protecting the confidentiality of participants. Specimens of mosquitoes, blood film slides and genotype print of PCR outcomes will be maintained by the principal investigator and be available up on request from authorized representatives, regulatory bodies. The results of the study will be made publicly available through peer reviewed journals.Change in clinical malaria incidence over 2 years period among the treatment and control arms will be determined using Poisson distribution. In this trial we will follow modified ITT protocol and if a child is lost from follow up, or withdraw from the study, or refuse to be included in the treatment, it will be immediately replaced by a child from the reserve list. The Relative Risk (RR) and RR Reductions (RRR) will be calculated with corresponding 95% confidence intervals to compare dichotomous variables, and difference in means will be used for additional analysis of continuous variables. Mixed effects Poisson model will be used to test the difference in incidence rates among the study arms, to determine effects of the repeated measurements within house, village, the effect of year and village-intervention interaction effects. To control the effect of clustering or village and individual level confounding factors such as gender and age, these covariates will be fitted in to random effects during analysis. If a child is diagnosed with malaria case within 28 days of the first episode with the same plasmodium species, then it will be put on treatment as part of the safety protocol, however, the case will not be included in the analysis. The prevalence of anaemia among the study participants (children) will be analysed using the guideline developed by world health organization . AccordiAnopheles densities will be compared for each study arm using a student t-test. Overall mosquito density among treatment arms will be compared using one-way analysis of variance (ANOVA) and if there is significance difference among treatment arms mean separation test will be done using turkey’s range test. The sporozoite rate will be determined as the proportion of malaria vectors positive for CSPs over the total number tested for CSPs.Indoor and outdoor The durability test of screening material will be conducted following the guideline developed by Kinde et al. (2018)  and modiThe socioeconomic data analysis will be done using descriptive and econometric approaches. An incremental cost effectiveness ratio (ICER) will be calculated for each outcome and arm using standard Disability Adjusted Life years (DALYs). The interventions will be ranked according to cost effectiveness, whereas inequality in terms of health outcomes, will be measured by the Gini coefficient and the concentration index.The right of participants to withdraw from the study at any time without giving a reason will be communicated prior to recruiting each participant. If the first child from the house is moved to other place for various reasons, the second child will be replaced. In unlikely scenario of the situation in which the entire family moves , the follow up will be discontinued and other related data collection activities  will be replaced by reserve house.House to house visit, children follow up and RDT testing during monthly follow up period will be conducted by the trained health extension workers (HEWs). Yearly blood sample collection and processing will be handled by professional. Blood samples for RDTs, microscopic examination and Haemoglobin test measurements will be collected using aseptically disposable lancets. Positive, cases found during the monthly follow up will be treated by the HEWs according to the national guidelines  and any The implementation of this clinical trial will be overseen by Amhara regional public health institute  and NORAD, sponsoring the trial. A contract agreement will be signed among the three parties . As part of the contract the PIs will submit annual reports detailing the progress of the trial, the safety procedures put in place and the overall impact of the study. Blood sample collection and treatments are part of routine malaria control in Ethiopia and will be undertaken in collaboration with the health workers at the health posts and therefore, no need to oversee the routines. Side wastes that result from the trial such as used insecticide nets, empty sachets, cartons, plastic bags, used gloves, pricking needles and other contaminated materials will be properly disposed following the guideline set by Robertson et al. (1995) .www.pactr.org With registration No: PACTR202006878245287. The copy of ethics approval letter is prepared in separate document  and renewed up on evaluating the trial progress with ref.: H/R/T/T/D/5/3 and date 20/08/2021. The protocol was registered online on Date 28/05/2020 on site Informed consent will be obtained from each study participant. In case of children enrolled into the study package the family or guardian will be consulted to get their oral and written consent. In addition to consent from family/guardian, informed consent will be sought for children above 12 years old. For illiterate participants the purpose and detail content of the consent form will be read by the PI and their consent to participate in the study will be requested. Detail consent form which includes the title, the purpose, intervention procedure, benefits, risks, benefits, right to refuse and to discontinue, and PI contact information are prepared in separate document  the combined effect of HS and PPT on improving the human health with specific focus on clinical malaria, 2) the combined effect of HS and PPT on improving animal health and maize crop production 3) the cost of malaria burden and cost effectiveness of house screening as reflected by the end-users. In addition to the publications planned stakeholder workshop will be conducted in the district where the project is being implemented with objective disseminating the major findings of the project.Timelines of activitiesCountries and inter-state actors who work in public health sectors are running out of options to contain vectors of malaria vectors as existing interventions become less effective due to growing insecticide resistance  and adapScreening houses for the purpose of excluding mosquito vectors into residential structures is well established practice in Europe , AmericaDesmodium intortum plant releases the semiochemical which repels stem borer moths, and attracts their natural enemies, while Napier grass (now replaced by Bracharia mulatotu-II) attracts the pest. In addition, the Desmodium plant can effectively suppress striga weeds and improve soil fertility through nitrogen fixation. Both plants provide animal fodder thereby increasing the health and productivity of livestock. Commercialization of these companion crops can be used as a source of income generation through seed production. By integrating the push-pull technology and malaria vector control interventions, we hypothesize that the PPT will improve the livelihood of farmers through increased production and productivity thereby the synergy of the technologies contributing to improved overall life standard including the fight against malaria.Push pull technology on the other hand is a semiochemical based intervention in which two plant species are being used in integrated approach with primary objective of increasing the quality and quantity of crop yield per unit area. It is developed at ICIPE research center, Kenya, in collaboration with Rothamsted research center UK and scaled up throughout Africa to support smallholder farmers . The strWe expect some mild risks arising from this study. Screening of windows and doors may lead to reduced aeration in houses which may result in respiratory infection of children as it was hypothesized in similar studies conducted in The Gambia . RoutineDesmodium and Bracharia. Agricultural development agents (DAs) will visit each household regularly to support the establishment of the technology on their farm. In addition, children enrolled in this study will benefit from regular check-ups and treatment whenever there is malaria incidence.People involved in this study will benefit from freely available house screening materials and push pull technology. Their houses (windows and doors) will be screened, and they will be trained on how to use the push pull technology. As part of the technology transfer, each farmer will be provided with training and seeds of This trial will provide critical information on whether combining LLINs, HS and PPT will have additive value in malaria control efforts. This study will also generate useful information on cost effectiveness of the intervention in combination and when applied singly. In addition, the acceptability of both HS and PPT interventions by the community will be carefully assessed. Thus, the findings of this study will be used for an effective planning and implementation of vector control interventions by national malaria control programs.Additional file 1. Ethics Review approval letter.Additional file 2. Ethics information sheet and consent form.


Alpha-lipoic acid (ALA) is extensively utilized in multivitamin formulas and anti-aging products. The purpose of this study was to investigate the potential protective benefits of ALA on 5-fluorouracil (5-FU)-induced gastrointestinal mucositis in Wistar albino rats. Tissues from the stomach, small intestine, and large intestine were excised, and blood sera were obtained to identify biochemical indices such as TNF-α, IL-1β, MDA, GPx, SOD, MMP-1, -2, -8, and TIMP-1. A histopathological study was also performed. The results revealed mucositis-elevated TNF-, IL-1, MDA, MMP-1, -2, -8, and TIMP-1 levels in both tissues and sera, and these values dropped dramatically following ALA treatment. Reduced SOD and GPx activities in mucositis groups were reversed in ALA-treated groups. The damage produced by mucositis in the stomach and small intestine regressed in the ALA-treated group, according to histopathological evaluation. Consequently, the implementation of ALA supplementation in 5-FU therapy may act as a protective intervention for cancer patients with gastrointestinal mucositis. In light of the findings, ALA, a food-derived antioxidant with pleiotropic properties, may be an effective treatment for 5-FU-induced gastrointestinal mucositus, and prevent oxidative stress, inflammation, and tissue damage in cancer patients receiving 5-FU therapy. Alpha-lipoic acid (ALA) is present in a wide range of foods, including vegetables  and meats , as well as several nutritional supplements ,2. ALA hCancer is the leading cause of mortality in every country and a major impediment to longevity . The sidThe most often used chemotherapeutic drug in gastrointestinal malignancies is the pyrimidine analogue 5-fluorouracil (5-FU). 5-FU exerts its anticancer effects by inhibiting thymidylate synthase (TS) and integrates its metabolites into RNA and DNA . HoweverTo date, several studies have reported the use of 5-FU and ALA together in different cancers; these include cervical and colorectal tumors ,30. FurtThe animals were taken from the Animal Experiments Units of the Near East University. The rats  were maintained under a 12:12 h dark/light cycle from the start at a room temperature of 22  ±  2 °C in a humidity-controlled facility (50 ± 5%). Feeding was ad libitum without any restriction to their standard rat chow and drinking water. Rats were housed in a plexiglass cage (4 rats per cage with dimensions of 60  ×  40  ×  40 cm). The study protocol was authorized by the Near East University Local Animal Experiments Ethics Committee , and ethical guidelines mentioned in the 1964 Declaration of Helsinki and its later amendments were followed.n = 8): Control, ALA, Mucositis, and Mucositis + ALA groups. The Control group received only intraperitoneal (i.p.) saline (SF) once a day for one week. The ALA group received only ALA at a dose of 100 mg/kg i.p. once a day for one week from day 1 [Thirty-two Wistar Albino rats of both sexes weighing 200–250 g were allocated into four groups of eight rats each . Then, after being delivered to the laboratory, they underwent a homogenization procedure. Tissue homogenization was performed according to the manufacturer’s protocol using RIPA buffer  and Dounce tissue grinder set  on ice. After homogenization, samples were centrifuged at 1600× g for 10 min at +4 °C. The Bradford protein determination technique was utilized to quantify protein levels in tissue samples, and supernatants were then frozen at −80 °C for subsequent investigation.Blood samples were obtained and submitted to the laboratory in serum separator tubes. The serum samples were separated at 2000× Activities of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), lipase (LIP), amylase (AMY), and creatinine, blood urea nitrogen (BUN), total protein (TP), and albumin levels were measured as biochemical indices in serum samples using an automated BS-240 VET Clinical Chemistry Analyser .TNF-α and IL-1β concentrations in sera and tissue samples were measured using commercially available rat-specific enzyme-linked immunosorbent assay (ELISA) assay kits  following manufacturer’s instructions.MMPs and TIMP-1, which are involved in a variety of biological processes, such as cancer and inflammation, were measured in both tissue homogenates and sera using the manufacturer’s instructions and recommendations of commercially available rat-specific ELISA assays . Serum concentrations of the MMPs and TIMP-1 were expressed as pg/mL, while tissue levels were expressed as pg/mg protein.MDA levels in tissues were evaluated using commercially available assay kits to determine the lipid peroxidation state . The measuring principle is based on the reaction with thiobarbituric acid (TBA) in boiling water for 60 min in an acidic medium and the measurement of the absorbance of the reaction mixture at 532 nm . A VersaMax Tunable Microplate Reader was used to measure the absorbance . MDA concentrations in tissues were expressed as nmol MDA/mg protein.In tissues, GPx and SOD activity were evaluated using ready-to-use assay kits.  by using an automated BS-240 VET Clinical Chemistry Analyser . The quantities of GPx and SOD in the tissue samples  were expressed as U/mg protein.v/v) neutral formalin for 24 h at room temperature. After fixation, the tissues were placed on a tissue tracking device, Leica TP1020 . Tissues taken from the tissue tracking device were then embedded in paraffin. Sections of 5-μm thickness were cut from the tissues with the help of a microtome Leica RM2255 , and routine hematoxylin & eosin (HE) staining was performed. Sections were examined histomorphologically with a Leica DM500 light microscope coupled with the Leica Microsystem Framework integrated digital imaging analysis system .The tissues of the stomach, small intestine, and large intestine obtained immediately after the experimental period were fixed in 10% . The data were all expressed as mean ± standard deviation (p < 0.05–0.0001). However, there was a significant difference in albumin, AST, ALT, ALP, LDH, BUN, creatinine, amylase, BUN, and creatinine levels between the ALA + Mucositis group and the Mucositis group (p < 0.05–0.001). ALP, ALT, AST, LDH, and amylase levels were also substantially different between the Mucositis and ALA groups (p < 0.05–0.0001). Accordingly, the levels of liver enzymes, pancreatic enzymes, and metabolites were observed to be dramatically reduced in the Mucositis group when ALA was added to the treatment.p < 0.05–0.0001). However, TNF-α and IL-1β levels in the sera and all tissues reduced significantly in the Mucositis + ALA group compared to the Mucositis group (p < 0.05–0.0001). Between the Control and Mucositis + ALA groups, there was a substantial difference in the levels of TNF-α in the small and large intestines. (p < 0.01). There were also significant differences in TNF-α levels in the large and small intestines between the ALA and Mucositis + ALA groups. . Furthermore, there were statistically significant differences between the Control and Mucositis + ALA groups in terms of IL-1 levels in the stomach (p < 0.05). Therefore, ALA appeared to significantly suppress the levels of pro-inflammatory cytokines in mucositis.The levels of pro-inflammatory cytokines TNF-α and IL-1β in serum, stomach, small intestine, and large intestine samples can be seen in p < 0.05–0.0001). In addition, the levels of MDA in the large intestines of the Mucositis + ALA group were substantially different from those of the Control and ALA groups (p < 0.0001).MDA levels, GPx and SOD activities were quantified to assess peroxidation and antioxidant status at the tissue level . MDA levp < 0.05–0.0001). SOD levels in the large intestine samples were significantly different in the ALA and Mucositis + ALA groups (p < 0.01). In the stomach samples, the SOD levels were significantly lower in the ALA and Mucositis + ALA groups (p < 0.05). Furthermore, SOD levels in the small intestine of rats from the Mucositis group were substantially lower than SOD levels in the ALA group (p < 0.05). However, SOD levels in the stomach and large intestine of animals in the Mucositis and Mucositis + ALA groups were substantially different (p < 0.05–0.0001).Rats in the Control and ALA groups had significantly higher SOD levels in all tissues than rats in the Mucositis group (p < 0.05–0.0001). The increase in MMP-1, MMP-2, MMP-8, and TIMP-1 levels in Mucositis rats’ sera and stomach, small intestine and large intestine were significantly suppressed in Mucositis + ALA rats (p < 0.05–0.0001), although there was no significant difference in serum MMP-8 levels between the Mucositis + ALA and Mucositis groups.MMP-1, MMP-2, MMP-8, and TIMP-1 activities  in serumSurface epithelial cells, foveola gastrica sections, gland epithelial cells, submucosa, muscularis, and serosa layers were all normal in the Control and ALA groups’ gastric sections a,b. Degep > 0.05)  . The tun > 0.05) a,b. Irre > 0.05) c. Villus > 0.05) d. Cancer incidence and death rates are rising dramatically across the world, reflecting both a growing and an aging population, together with changes in the prevalence and distribution of the main cancer risk factors, many of which are linked to socioeconomic development ,35. Chem5-FU is a commonly used in anticancer medication since its inception in 1957 and has played a significant role in the treatment of colon cancer. It is also utilized in patients with other malignancies, such as breast and head and neck cancers . The nucAnticancer medications such as 5-FU may induce a significant decrease in antioxidant enzyme activity, such as SOD and GPx, as well as harmful impacts on non-enzymatic antioxidants, such as sulfhydryl groups. These findings fully support prior research indicating that 5-FU therapy is associated with oxidative imbalance ,42,43. EA high rate of destruction of the general architecture of the stomach and intestines is a known important feature of gastrointestinal mucositis, expressed mainly as rupture and atrophy of intestinal villi, loss of crypt structure, degeneration of gastric epithelial cells, increased goblet cell emptying, and infiltration of inflammatory cells . The mosThis study showed that proinflammatory cytokines IL-1β and TNF-α are elevated in Mucositis group’s sera and tissues . Earlier studies have demonstrated that 5-FU administration increases plasma IL-1β and TNF-α levels, which are in line with our plasma findings ,64. HoweThe five subgroups of the MMP family of calcium-dependent zinc-containing enzymes include collagenases, stromelysins, gelatinases, membrane-type MMPs, and other endopeptidases . They arIn comparison to the Control and ALA groups, 5-FU treatment enhanced MMP-1, MMP-2, MMP-8, and TIMP-1 activation in sera and tissues in the Mucositis + ALA group. Several models of inflammation have been utilized to examine the effect of ALA on the expression levels of MMP-1, MMP-2, MMP-8, and TIMP ,73. ALA’Although some studies indicate that TIMP-1 activation plays a protective role by inhibiting MMP activation, some other studies emphasize that increased TIMP-1 activation leads to an inflammatory response by increasing proinflammatory cytokine expression ,78. EnhaChemotherapeutics, which are often advised as first-line treatment for cancer, pose a significant challenge due to their adverse side effects. 5-FU, which is used to treat malignancies of the breast, colon, rectum, stomach, and pancreas, has the notable side effect of causing gastrointestinal mucositis. Therefore, the use of medicines that may be provided alongside chemotherapeutics to avoid or decrease these adverse effects is critical, and this area of study is still under investigation. We feel that by using ALA in our research, we have contributed to the corpus of knowledge in this area. As a result of our research, we have discovered that the combination of ALA and 5-FU induces favorable changes in the parameters involved in the control of inflammation, together with an oxidant–antioxidant balance. Our findings indicate that the incorporation of ALA into 5-FU therapy is a potential option for cancer patients with gastroenteritis.


Plasmodium falciparum severe malaria, but for severe P. vivax and P. knowlesi malaria, similar evidence is missing. The validation of these biomarkers is hindered by several factors such as low sample size, paucity of evidence-evaluating studies, suboptimal values of sensitivity/specificity, poor clinical practicality of measurement methods, mixed Plasmodium infections, and good clinical value of the biomarkers for concurrent infections (pneumonia and current COVID-19 pandemic). Most of these biomarkers are non-specific to pathogens as they are related to host response and hence should be regarded as prognostic/predictive biomarkers that complement but do not replace pathogen biomarkers for clinical evaluation of SM patients. This review highlights the importance of research on diagnostic/predictive/therapeutic biomarkers, neglected malaria species, and clinical practicality of measurement methods in future studies. Finally, the importance of omics technologies for faster identification/validation of SM biomarkers is also included.Globally, malaria is a public health concern, with severe malaria (SM) contributing a major share of the disease burden in malaria endemic countries. In this context, identification and validation of SM biomarkers are essential in clinical practice. Some biomarkers  have yielded interesting results in the prognosis of  Plasmodium protozoan parasite transmitted to humans through the infectious bite of female Anopheline mosquitoes during a blood meal. Five Plasmodium species can elicit human malaria viz. P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), P. ovale spp (Po), and P. knowlesi (Pk), with Pf and Pv responsible for the major bulk of malaria burden worldwide [Tremendous efforts are being made worldwide to fight against malaria, but the disease remains an important public health concern, with 241 million cases and 627,000 deaths estimated in 2020 [Pf and Pv [Pm, Po, and Pk to also elicit SM [The clinical spectrum of malaria is diverse, going from asymptomatic carriage of parasites to clinical malaria with varying degrees of signs/symptoms that define non-severe and severe malaria . A largef and Pv , but reclicit SM .In clinical practice, a good management of SM is achievable in endemic areas. However, SM management is very tricky in some areas, especially those from sub-Saharan Africa (sSA) that have a proportionally high share (>90%) of world malaria morbidity and mortality , with a Pf accounts for ~500,000 deaths annually, where children aged below five years old, pregnant women, and nonimmune travelers are major victims [Pv mono-infection-related SM is 22.9% in India [SM mainly due to  victims . However victims . Using ain India . A recenin India .In the present review, we evaluated clinical biomarkers for their potential in the diagnosis, prognosis, prediction, and therapy of SM, identified the potential gaps, and proposed areas for future investigations/solutions, with a focus on omics technologies and their possible utility in the current research era.Pf-infected adults [Since the 1990s, the World Health Organization (WHO) defined guidelines to diagnose SM based on findings from field and clinical studies. These guidelines have been regularly revised from 2000 to 2015 . For SM d adults .Figure Plasmodium regardless of species is very high (>70%) in clinical malaria cases with majority being Pf cases , with Pf and Pv both involved in hospital admitted SM cases, but with a clear predominance of Pv in Brazil and Colombia  as compared to other states  or a substrate (e.g. proteins and metabolites) that can objectively be measured and evaluated as an indicator of normal biological/pathogenic processes or response to a therapeutic intervention . In malaviz diagnostic, prognostic, predictive, and therapeutic. Generally, a diagnostic biomarker allows the early detection of a disease in a noninvasive way, leading to its prevention. A prognostic biomarker is a clinical or biological characteristic that is objectively measurable and that provides information on the likely outcome of the disease in an untreated individual [predictive biomarker is a clinical or biological characteristic that reveals information, allowing one to forecast the response of patients to a given treatment and thus identify individuals who will benefit from the treatment [therapeutic biomarker is most commonly a protein that can be used as a target for disease therapy  to discriminate untreated asymptomatic malaria patients susceptible to manifest clinical signs/symptoms, ii) identify untreated clinical patients whose malaria infection could lead to a severe form, and iii) identify untreated SM patients . Thus, tThe characteristics of an ideal biomarker have been described previously with the following attributes: i) clinical relevance, ii) good sensitivity, specificity, and predictive values, iii) reliability, iv) simplicity, and v) practicality ,34.The clinical relevance of a biomarker refers to the evidence that supports a coherent basis of its utility . Sensitivity refers to the ability of a biomarker to correctly identify patients with the disease-related outcome , while specificity refers to the capacity of a biomarker to correctly identify patients exempted from the disease-related outcome. The determination of the area under curve (AUC) of the biomarker by plotting a receiver operating characteristic curve (ROC curve) can also be used as an alternative to sensitivity and specificity. In practice, a biomarker with sensitivity (Se) ≥85%/specificity (Sp) ≥75% or an AUC ≥0.75 is considered to be of good clinical utility . A reliaThe validation of biomarkers is the guarantee underlying the subsequent production of high-quality research data on diagnosis, prognosis, and prediction of the SM outcome. The procedure for the validation of biomarkers is multifaceted and more complex than their evaluation where validation takes into account the above-mentioned criteria on evaluation along with additional aspects such as the study design , reproducibility in varied settings, pharmacokinetics—pharmacodynamics data, establishment of the biomarker normal range, and biological plausibility ,34.The literature analysis of the related topic showed very few studies that evaluated the prognostic potential of few biomarkers for SM. One study addressed the evaluation of the platelet count and plateletcrit as SM prognostic markers without any distinction of the involved malaria species . A crossPf biomarkers evaluated were host proteins  patients at SM risk, ii) markers of SM outcomes, iii) patients at risk of CM, SMA, shock, ARDS/pulmonary edema, and renal failure, iv) patients with chances to recover from an SM episode, and v) CM patients at fatal risk or survival with the development of neurological sequelae  , cardiac disease creatine kinase muscle-brain type (CK-MB), uric acid, chronic kidney disease-epidemiology estimated glomerular filtration rate (CKD-EPI eGFR), total bilirubin, caveolin 1, parasite lactate dehydrogenase (pLDH), high mobility group box protein 1 (HMGB1), oxylipin and endocannabidome metabolites, D-dimers, cell-free DNA (cfDNA), and carboxyhemoglobin , and the absence of statistical significance. Such biomarkers are procalcitonin (PCT), Finally, biomarkers such as, for instance, pigment-contained neutrophils (PCNs), fibrinogen, copeptin, some chemokines , IP-10, and schizontaemia showed poor clinical performances for their ability to discriminate the above-mentioned SM outcomes .Pv-SM, although the number of biomarkers evaluated is far lower than that seen in Pf-SM. To date, no strong evidence of the clinical utility of biomarkers is available for Pv-SM even though some of them such as superoxide dismutase 1 (SOD), titin, vitronectin, TNF-α, and the Ang-2/1 ratio have shown interesting performances for the prognosis of malaria severity  [There are no studies for the evaluation of SM biomarkers for these two species, and this is likely due to a very low number of orldwide ,6. Based for Po) .Pf-SM disease [Pf-SM-related clinical recovery and time [The evaluation and validation of reliable biomarkers for SM are crucial to develop efficient strategies to manage this important public concern worldwide. In the present review, it is obvious that this research era is still in its infancy, given the paucity of data on potential biomarkers. The studies were mainly focused on the prognostic aspect of the evaluated biomarkers rather than on their predictive and therapeutic aspects. The forecasting of the probable occurrence of SM is important, but the identification of patients with less chances of recovery from an antimalarial therapy (i.e. predictive biomarkers) and identification of biomolecules susceptible to be targeted by this therapy (i.e. therapeutic biomarkers) are also important aspects of management of SM by practitioners. Some studies outlined an association of elevated levels of biomarkers such as Ang-1, sTREM-1, CXCL10, and sICAM-1, with prolonged clinical recovery times in patients having survived from  disease , while oand time . DespitePf and ii) SOD-1, TNF-α, protein carbonylation, lipid peroxidation, IL-10, and cell adhesion molecules for Pv  CRP, circulatory complement‑lysis inhibitor, Ang-2, HRP2, thromboxane 2, cfDNA, platelet count, TNF-α, and 10-kDa INF gamma-induced protein for small sample size of cases and controls impacting greatly on the data analysis, statistical test findings, and finally the interpretation of data. Thus, the implementation of adequately powered studies is a prerequisite to support the findings;findings on the accuracy of methods used to determine the levels of biomarkers in biological fluids are rarely given in the studies and absence of taking into account this aspect may give prejudices to results;Pv malaria [the presence of latent concurrent infections such as viral, parasitic, and bacterial infections or conditions like malnutrition, which also may modulate the levels of biomarkers investigated and severity of malaria disease. Few studies used concurrent infections such as dengue to identify and evaluate the potential of some biomarkers for severe  malaria ,65. Othe malaria ,66, whil malaria ,68.On studying individually, we noted that a large proportion of biomarkers did not qualify as prognostic markers for SM, as no evidence of statistical significance or performance criteria values below the acceptable thresholds was available. One way to improve the potential of biomarkers could be to use them in combination as seen in previous studies ,58–63. MPf-Pm co-infection from North-Western part of Cameroon [Plasmodium infections is also increasing in sSA as reported recently in a meta-analysis where 9% of mixed infections led to SM and that the proportion of pulmonary complications and severe anemia were higher in patients with mixed infections [The validation of SM biomarkers should also overcome the problem of mixed infections with plasmodial species. In some settings, the proportion of co-infections is often surprisingly high as reported for Cameroon . The profections . Even thPf parasites with pfhrp2 deletions could be an important concern on the long-term utilization of PfHRP2 as a diagnostic biomarker for Pf infections where it is highly predominant in settings as in sSA and some areas of India [The PfHRP2 protein has shown good performances for CM prognosis and distinction between CM malaria patients with positive and negative diagnosis for retinopathy (CM-RP and CM-RN) , flow cytometry, light microscopy, immunocolorimetric assays, co-oximetry, and indirect potentiometry . Routine implementation of these tests at clinical sites is greatly jeopardized by drawbacks such as high cost, time consumption, extensive training/expertise/workload for clinical staff, sensitivity/specificity, and reproducibility . It woulThe few potentially helpful biomarkers identified here are mainly related to host response and they are therefore more likely suitable as prognostic and/or predictive biomarkers that complement but do not replace or substitute for diagnostics in the clinical evaluation and management of malaria patients. Biomarkers of host response are pathogen non-specific as mentioned in bacterial and COVID-19 infections and do not indicate the pathogen responsible for the clinical syndrome of “sepsis,” as represented by SM. As such, the biomarkers of host response provide insight into the potential risk for end-organ injury and adverse clinical outcomes. For example, biomarkers can potentially provide risk of progression to SM upon presentation of a patient with malaria (e.g. Ang-2).The investigation of new potentially promising candidate biomarkers is also crucial, and with the advent of transcriptomic, peptidomic, and proteomic technologies, the process of identification, evaluation, and validation of biomarkers for different aspects of malaria severity as seen in previous studies can be accelerated ,65,78–80Plasmodium infections are major obstacles for a better understanding of the relationship between the candidate biomarker and SM, and their evaluation as a potential biomarker needs to be carried out in further research studies. The advent of omics technologies would also be helpful in enhancing the identification and validation of clinical SM biomarkers, but affordable alternative assay systems need to be developed for translation in the clinical context of malaria.The present review concluded that the identification and validation of biomarkers of SM are still in an early phase. Some candidate biomarkers seem to be promising for SM prognosis, but further studies are still required to confirm their prognostic value. Also, this review presented different aspects for which the prognostic value of candidate biomarkers was evaluated and summarized these findings from different studies. The authors also point out the need for further studies/research in the identification of predictive and therapeutic biomarkers, which are other critical links to be addressed for efficient control of SM. Although prognostic marker validation is relatively easy, more stringent criteria are required for the validation of predictive and therapeutic biomarkers for SM. Also, the concurrent and mixed Click here for additional data file.