Plasmodium falciparum infections is typically high among school-aged children (SAC). Nonetheless, SAC are often less likely to be covered by malaria interventions, making them a group with an untreated reservoir of parasite transmission. This study aimed to assess the effects of asymptomatic and clinical malaria infections on biochemical markers of iron status among SAC in Malawi.Iron status is considered as a continuum from an iron deficiency with anaemia, without anaemia, varying amounts of stored iron to iron overload. The burden of Data from the 2015–2016 Malawi Micronutrient Survey (MNS) was used and multivariable logistic regression models using a generalized estimating equation to account for the complex cluster survey design were constructed. Blood samples of 684 children aged 5 to 14 years old were evaluated for clinical and asymptomatic malaria infections. Furthermore, blood samples were used to estimate haemoglobin (Hb), serum ferritin (SF) and, soluble transferrin receptors (sTfR) concentrations.Of the 684 SAC analysed, approximately 42% had asymptomatic malaria, while 41.0% had clinical malaria. Anaemia (low Hb levels), iron deficiency (low SF concentration), and functional iron deficiency (high sTfR levels) were found in 20%, 5%, and 30% of the children, respectively. School-aged children with asymptomatic malaria had increased odds of being anaemic  and increased levels of sTfR . Similarly, SAC with clinical malaria had increased odds of being anaemic  and increased levels of sTfR .Both asymptomatic and clinical malaria were independent risk factors for anaemia and functional iron deficiency (FID). The notion that asymptomatic and clinical malaria were associated with both anaemia and FID underscores the need for public health programmers to consider adding mass screening and treatment for malaria to existing school-based health programmes.The online version contains supplementary material available at 10.1186/s12936-022-04297-1. Iron status is considered as a variety of conditions ranging from iron deficiency (ID) with anaemia , to ID without anaemia , to normal iron status with varying amounts of stored iron, to iron overload –4. Iron . On the other hand, analytical challenges may affect iron status interpretation due to the lack of assay standardization, common reference ranges, and common cutoffs [Indicators of iron status encompass a continuum of parameters and can be confounded by factors varying from inflammation to analytical challenges . General cutoffs . The Wor cutoffs , 25. Som cutoffs . They ad cutoffs , ZPP was cutoffs . At the  cutoffs . Further cutoffs , and SF  cutoffs .Plasmodium species; namely, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax and Plasmodium knowlesi [P. falciparum infections in SAC are associated with compromised health, diminished cognitive function, and lower educational achievement [P. falciparum infections involve an increased removal of parasitized and unparasitized RBCs through several mechanisms that may result in anaemia in under-five children [P. falciparum infections on biomarkers of iron status among this under-appreciated age group in Malawi.Malaria is a life-threatening disease caused by five knowlesi . Globallknowlesi , while oknowlesi . Despiteknowlesi , 34. Uniknowlesi , 36. Howknowlesi , 38. Henknowlesi , 39. Sevknowlesi , 41. Speievement , 43. Furchildren , 45. Thechildren , 39. HowThis was a cross-sectional study that used data from the 2015–2016 Malawi Micronutrient Survey (MNS). The MNS was conducted jointly with the 2015–2016 Malawi Demographic and Health Survey (MDHS) between December 2015 and February 2016. The comprehensive methods used in this study can be obtained from elsewhere , 47. In ssThe MDHS teams collected survey data electronically using tablets, while the MNS teams used paper-based questionnaires pre-translated into two common Malawian languages, i.e. Chichewa and Tumbuka. Further, the MDHS teams completed data collection in each cluster before the arrival of the MNS team. The MDHS teams pre-selected the MNS participants through an algorithm pre-programmed into tablets. After finalizing data collection in each selected household, the MDHS supervisor completed the cover sheet of the MNS questionnaire booklet with the names and ages of all eligible individuals selected for the MNS. The MDHS enumerators placed a household label with a unique barcode on the questionnaire and entered the barcode number into the tablet for each eligible household. Eventually, the barcode number allowed the data collected separately by the MDHS and the MNS teams to be linked , i.e. from 22% in 2009 to 16% in 2015–16. Considering a confidence level of 95%, power of 80%, design effect of 2.0, and 90% household and individual response rates, data had to be collected on a minimum of 1452 PSC. Calculations assumed a 90% household-response rate, 90% individual-response rate, and an average household size of 4.3 persons. The 2015–16 MNS was conducted in 2262 residential households, including 480 households in urban areas and 1782 households in rural areas. Thus, the sample size calculated was expected to result in data collected from about 750 eligible women of reproductive age, 252 eligible men, 700 eligible SAC.n = 6) were also excluded from this study. A final sample of 684 was analysed.The present study focused on SAC (children aged 5 to 14 years old). In order to achieve quality data, the MNS excluded those deemed too ill to participate and those with a physical disability that would prevent them from having accurate height and/or weight measurements. All SAC with missing information in any of the variables  rapid diagnostic test (RDT) was used [Another 10 μL of the blood sample from the EDTA vacutainer was used to test for malaria Fig. . The Stawas used .Regarding biochemical tests, 100 μL of serum from the trace free elements test tube Fig.  was tranThe dependent variable of the current study was iron status measured using three domains namely; anaemia, ID, and FID. Using the WHO recommendations, anaemia was defined as Hb concentration < 11.5 g/dL for children 5–11 years, and < 12.0 g/dL for children 12–14 years. Hb concentration was adjusted for altitude . Serum fP. falciparum parasitaemia of any density and axillary temperature ≥ 37.5 °C [P. falciparum parasitaemia of any density with an axillary temperature < 37.5 °C or without any apparent clinical manifestations at the time of collecting the blood [P. falciparum positive blood smear [P. falciparum infection for both clinical malaria and asymptomatic malaria was confirmed using the RDT [Asymptomatic and clinical malaria were the two main independent variables considered in this study. Clinical malaria is defined as  37.5 °C . On the he blood . Usuallyod smear . However the RDT . Table 1The covariates included in this study were, sex of the child, age of the child, fever, and diarrhoea in the last 2 weeks, acute and chronic inflammation, any inflammation, household hunger, type of place of residence, and region of residence. Sex , the age of the children was categorized as 5–10 years and 11–14 years. Fever (Yes/No), the respondents were asked if their child had a fever in the last 2 weeks. Similarly, diarrhoea was also reported for episodes that occurred in the last 2 weeks. Basically, diarrhoea is defined as the passage of three or more loose or liquid stools per day  . Acute aAll statistical analyses were conducted using SAS software version 9.4 . The survey-specific SAS procedures for weighting, clustering, and stratification were used to account for the complex survey design. Baseline characteristics were reported as weighted frequency and percentages stratified by sex of the child. The differences in sociodemographic, clinical outcomes, biochemical markers, morbidity outcomes, and community factors according to the sex of the child were compared using Rao-Scott Chi-Square test. The effects of asymptomatic and clinical malaria on the markers of iron status were performed using univariate and multivariable logistic regression analyses. Analyses were adjusted for clustering effects of individuals from the same households by use of generalized estimating equations. The results of multivariable analyses were reported as adjusted odds ratio (aOR) with their p-values and 95% confidence intervals (CI). P-values < 0.05 were considered to indicate statistical significance.A permission was obtained from the Monitoring and Evaluation to Assess and Use Results Demographic and Health Surveys (MEASURE) and/or the Demographic and Health Survey (DHS) program for the use MNS 2015–16 datasets before the primary purpose. The MNS survey protocol was approved by the Institutional Review Board of ICF Macro and the Malawi National Health Sciences Research Committee (NHSRC). The survey was implemented by the National Statistics Office, the Community Health Sciences Unit of the Ministry of Health, Department of Nutrition, HIV, and AIDS, and jointly with the 2015–16 MDHS. Informed consent for MNS took place at several levels. Firstly, community leaders from each cluster were informed regarding the MDHS and MNS, and communal consent was sought prior to the arrival of the MDHS teams. Secondly, the MDHS enumerators asked each MNS-eligible household for permission to participate in the MNS where consent for each household was documented on the MNS paper-based questionnaires. Finally, the paper-based questionnaires were then handed over to the MNS team. The nurse asked for informed consent from each individual for anthropometry measurements and biological samples.Of the 684 SAC analysed, about 42% had asymptomatic malaria while 41.0% had clinical malaria. Furthermore, anaemia, ID, and FID were found in 20%, 5%, and 30% of children, respectively. Figure P < 0.0001 and increased odds of developing functional iron deficit—high of sTfR levels , P < 0.0001. Similarly, compared to children without clinical malaria, SAC with clinical malaria had increased odds of being anaemic , P < 0.0001, and increased odds of developing functional iron deficit , P < 0.0001.Tables P < 0.0001 and increased odds of developing functional iron deficit , P < 0.0001. Similarly, compared to children without clinical malaria, SAC with clinical malaria had increased odds of being anaemic , P < 0.0001, and increased odds of developing functional iron deficit , P < 0.0001.Tables The current study aimed to assess the implications of asymptomatic and clinical malaria on biochemical markers of iron status among SAC in Malawi. The study found that the prevalence’s of both asymptomatic and clinical malaria were high and reported at 42% and 41%, respectively. After adjusting for various background characteristics, the current study found that both asymptomatic and clinical malaria infections were significantly associated with anaemia (low Hb levels) and functional ID (high levels of sTfR) but not ID, which is characterized by low SF.P. falciparum infections and thus increased demand for iron for erythropoiesis [Increased levels of sTfR could be exclusively explained by the elevated rate of RBCs haemolysis associated with opoiesis . sTfR aropoiesis . Generalopoiesis . It is kopoiesis . Increasopoiesis . Thus, wopoiesis . Even thopoiesis . In the opoiesis . Hepcidiopoiesis , 71. Henopoiesis . Prior ropoiesis . Thus, sopoiesis . The resopoiesis  and Tanzopoiesis , where mPlasmodium life cycle, infection with malaria parasite increases the rate of haemolysis of RBCs directly while suppressing the erythropoiesis process, or in part due to inflammatory cytokines [P. falciparum may also induce loss of functional body iron and Hb through haemozoin and through urinary excretion [P. falciparum infection may induce anaemia and a decline in Hb levels through altered cytokines balance and inflammation [In line with previous studies conducted in the other age groups , 45, 75,ytokines , 77. Evexcretion , 79. Preammation , 80, 81.ammation , 83.P. falciparum brings about an extensive deformity in the RBC population [P. falciparum infection, the spleen reconstitutes and expands rapidly. As such, this phenomenon results in elevated clearance capability and decreased splenic activities for the removal of abnormal erythrocytes [Other studies have reported that infection with pulation , 85. Thepulation –88. Furtpulation , 90. Addhrocytes .In this situation, the spleen eliminates great numbers of relatively rigid red cells, which is a healthy uninfected subject that would be allowed to remain in the circulation , 93. ThuP. falciparum infections and the outcomes of interest of the current study. Secondly, the use of RDT to diagnose asymptomatic and clinical P. falciparum infections may not be able to detect some infections with lower numbers of malaria parasites circulating in the patient’s bloodstream. Thirdly, the use of the self-reported nature of collecting clinical information, such as fever and diarrhea in the last 2 weeks can lead to recall bias. Fourthly, participants that deemed too ill and those with a physical disability were excluded from the survey. As a result, this would have introduced selection and measurement biases. Lastly, it is documented that indicators for iron status comprise the concentrations of Hb, SF, sTfR, ZPP, reticulocyte, serum iron, and hepcidin and total iron [The use of the nationally-representative sample could permit these results to be generalized among SAC in Malawi. Assessments of Hb, sTfR, and SF objectively minimized the risk of measurement bias. However, the results of the current study should be interpreted with caution. Firstly, the use of a cross-sectional study design cannot provide a causal relationship between asymptomatic, clinical tal iron , 94. HowThe current study found that both asymptomatic and clinical malaria were independent risk factors for anaemia and FID but not ID. The notion that asymptomatic malaria was associated with both anaemia and FID underscores the need for public health programmers to consider adding mass screening and treatment for malaria to existing school-based health programmes in order to improve nutrition status especially iron status among SAC.Additional file 1. Questionnaire for micronutrients survey module.Additional file 2. Questionnaire for the main Malawi Demographic and Health Survey.


Plasmodium falciparum malaria infection and disease among children under five years of age in Benin, West Africa.Despite a global decrease in malaria burden worldwide, malaria remains a major public health concern, especially in Benin children, the most vulnerable group. A better understanding of malaria’s spatial and age-dependent characteristics can help provide durable disease control and elimination. This study aimed to analyze the spatial distribution of A cross-sectional epidemiological and clinical survey was conducted using parasitological examination and rapid diagnostic tests (RDT) in Benin. Interviews were done with 10,367 children from 72 villages across two health districts in Benin. The prevalence of infection and clinical cases was estimated according to age. A Bayesian spatial binomial model was used to estimate the prevalence of malaria infection, and clinical cases were adjusted for environmental and demographic covariates. It was implemented in R using Integrated Nested Laplace Approximations (INLA) and Stochastic Partial Differentiation Equations (SPDE) techniques.P. falciparum infection was moderate in the south (34.6%) of Benin and high in the northern region (77.5%). In the south, the prevalence of P. falciparum infection and clinical malaria cases were similar according to age. In northern Benin children under six months of age were less frequently infected than children aged 6–11, 12–23, 24–60 months, (p < 0.0001) and had the lowest risk of malaria cases compared to the other age groups (6–12), (13–23) and (24–60): OR = 3.66 [2.21–6.05], OR = 3.66 [2.21–6.04], and OR = 2.83 [1.77–4.54] respectively (p < 0.0001). Spatial model prediction showed more heterogeneity in the south than in the north but a higher risk of malaria infection and clinical cases in the north than in the south.The prevalence of Plasmodium falciparum infection and clinical cases will make interventions more evidence-based by showing progress or a lack in malaria control.Integrated and periodic risk mapping of The online version contains supplementary material available at 10.1186/s12889-022-14032-9. Between 2000 and 2019, malaria incidence rates in the World Health Organization (WHO) African Region reduced from 368 to 222 per 1000 population at risk, but increased to 232 in 2020 , 5. HoweEvaluating the impact of interventions is essential to designing more efficient and sustainable strategies for malaria control and elimination . FrequenAnopheles vectors resistance to insecticides were also described by many studies .The prevalence of 61, Fig. a. The prp = 0.3918) . A total of 267 (58.4%) clinical cases were confirmed with RDT and attributed to malaria (positive RDT plus signs). The prevalence of clinical malaria cases did not vary according to age group . Contrary to the south, the prevalence of infection increased with age in the DCO district (p < 0.0001). Children aged less than six months were less frequently infected than children aged 6–11, 12–23, 24–60 months OR = 4.92 [CI95% 3.75–6.45], OR = 6.99 [CI95% 5.33–9.17], and OR = 18.75 [CI95% 14.67–23.96] respectively  [CI95% 62.46%– 65.57%] in the DCO.The prevalence rate of p < 0.0001 . A total of 527 (95.3%) were attributed to malaria (RDT + signs). The prevalence of clinical malaria cases varied according to age groups. Children aged less than six months had a low risk of suffering from malaria compared to other age groups (6–12), (13–23) and (24–60): OR = 3.66 [CI95% 2.21–6.05], OR = 3.66 [CI95% 2.21–6.04], and OR = 2.83 [CI95% 1.77–4.54] respectively, From the raw maps of the malaria prevalence at the observed villages in the OKT region Fig. , there iIn the OKT health district, a clustering of observed data points (positive autocorrelation) around 1 km and 4.5 km and repulsion around 8.5 km (negative autocorrelation) for the prevalence of infection was noticed . Exposures such as annual rainfall "bio12_wc30s", moisture index of the moist quarter “mimq_wc30s’’, distance to the coastlines “dst_coastlin”, topography “srtm_topo” and land cover influence significantly the prevalence of malaria infection within the OKT health district since the 95% credibility intervals of their mean estimates do not contain 0 Regarding the DCO region, apart from the local mean prevalence , the population density "ben_ppp" is the only risk factor that significantly affects the prevalence of malaria infection. None covariate significantly affects the estimated prevalence of malaria cases since the 95% credibility interval of their effects contains 0 Table . ContrarBayesian Generalised linear spatial models implemented using stochastic partial differential equations (SPDE) combined with the INLA approach revealed that the prevalence of malaria infection varies locally within villages throughout the two study regions , and others (extreme north of Djougou) had an estimated prevalence greater than 75% (i.e. holoendemic areas). However, some areas located at Ouaké and the south-eastern part of Djougou in the DCO health district has a mesoendemic transmission of malaria infection (10 to 50%)  and northern Benin (one rainy season).Half part of care-seeking is attributable to malaria in Benin despite the high intervention packages implemented throughout the country. Routine data exist and help decision making but seem insufficient. Spatial modelling can help to observe disease indicators from new angles, allow for finer analysis and thus help to make more relevant decisions. The main objectives of this study were to address the spatial distribution of WHO recommended ensuring access to malaria prevention, diagnosis and treatment as part of universal health coverage by determining the most effective mix of interventions according to the local context and needs. This induce data collection or using existing data that reflect a need for subnational level . Recent P. falciparum infection has already been described in the OKT district in a previous study and was not surprising [P. falciparum was very high in northern Benin. The DCO district remained a hyperendemic area. The Malaria Atlas Project confirmed a similar pattern in the spatial epidemiology of P. falciparum [P. falciparum infection was also predicted in the north compared to the south [P. falciparum prevalence between northern and southern regions. National malaria programmes should determine the appropriate package of interventions for local level considering transmission intensity, as well as a good understanding of each area’s ecological, epidemiological, and social features. Interventions should be adapted and tailored to specific geographical areas within each country [This is the first-time spatial analysis of malaria infection and morbidity focused on the OKT and DCO health districts. The two ecological regions do not have the same epidemiological profile. The OKT district was a mesoendemic and/or hyperendemic area with a perennial transmission . Heterogrprising , 35. A clciparum , 37. In he south . Vectors country . The lacA 2010 study conducted at the national level in Benin showed that 34.5% of households in the north versus 28.3% in the south owned at least two LLINs. In opposite, 55.9% of children under five years slept under the LLINs the night before in the north compared to 68.1% in the south . This coAnopheles gambiae, the main malaria vector, is highly resistant to the standard insecticides [The vector resistance to insecticide was high in all the studied health districts. cticides , 13. Datcticides .During the 2011 and subsequent campaigns, community awareness of LLIN use was mainly addressed to mothers, pregnant women and children. The understanding or perception induced by the communication campaigns is that the LLINs is to be used by women of childbearing potential and the children with whom they usually sleep. A large part of children was kept out from intervention by the messages delivered during awareness campaigns. The malaria awareness campaigns should therefore be reviewed in order to improve LLINs use rates among elder children and adult males.In Benin, malaria control tools  are available at the health facilities. The index of availability of malaria control tracers among health facilities offering this service provided by SARA survey was 82% in Donga department , compared to 71% in Atlantique department . However, stock-out of anti-malarial drugs were often noticed in certain health centres. Care-seeking at health centres and utilization of recommended treatment of malaria remains a challenge. One major reason is to be self-medication by herbal tea and other non-recommended drugs such as chloroquine, quinine and paracetamol .P. falciparum [P. falciparum infection and the proportion of clinical patients were higher than in the south. The prevalence rate of infection was lower among children under six months of age than in the oldest. According to knowledge, the prevalence of parasitemia increases sharply beginning at about 20 weeks of age [Studies suggest a protective effect of maternal breastmilk against lciparum , 43. So,lciparum . In the s of age .Plasmodium antigens, as IgG levels decrease from birth over the first year of life. Another assumption is that the protection of infants may be associated with parasite growth-inhibitory factors such as lactoferrin and secretory IgA found in breast milk and maternal and infant sera [P. falciparum clinical cases decreased after 23 months, suggesting the beginning of immunological premonition acquisition [Nevertheless, children remain extraordinarily resistant to high parasitemia, fever, and severe malaria until about six months of age. This protection is associated with maternal immunoglobulin G (IgG) antibodies specific to ant sera , 45. Furant sera . Howeverant sera . We did ant sera . Thus, buisition , 48. Theuisition , 43, butuisition . In soutuisition , 18. To This study assessed the spatial heterogeneity of malaria risk in two ecological regions in Benin by using 2011 database. It estimated the disease prevalence adjusted for environmental and population covariates and found a significant relationship between temperature seasonality, rainfall, population density, moisture index and distance to the nearest waterways. However, the data were aggregated at village location in place of household. When GPS coordinates are available at the household level, similar models could be implemented to improve the smoothness of malaria risk prediction in the study regions. Considering household or individual-level data can allow the inclusion of other socio-demographic or personal characteristics  that are likely to influence disease transmission but not easy to obtain for aggregated data collected in village or county-level. The climate changes and the variations of temperature and precipitations change significantly with a cycle of 30 years average. However, mayor events like catastrophes and crisis can affect diseases epidemiology. Benin country did not experience this kind of event the last decade. Finally, more complex models could be implemented by assuming a non-stationary spatial process to evaluate its influence on models' outcomes .The spatial analysis concerns malaria infection and disease distribution among children under five years across two endemic and various areas in Benin. A higher risk of malaria infection and clinical cases were found in the north than in the south. The surveillance by geospatial approach and capacity building in modelling needs to be systematic and periodic to facilitate decision-making on the cost-effectiveness and global impact of malaria control efforts.Additional file 1: Figure A1. Correlation between preselected covariates (c & d) and their similarity groups (a & b) for the OKT region (top panel) and DCO region (bottom panel) – Cells with crosses are correlation coefficients that are not statistically significant.Additional file 2: Table A1. Simple binomial GLM fitted to the OKT data.Additional file 3: Table A2. Simple binomial GLM fitted to the DCO data.Additional file 4: Table A3. Bayesian binomial model with random noises fitted to the OKT dataAdditional file 5: Table A4. Bayesian binomial model with random noises fitted to the DCO data


Entamoeba histolytica is usually diagnosed based on its clinical symptoms, medical imaging abnormalities of the liver, and serological tests, the most common being the enzyme-linked immunosorbent assay (ELISA). For more than three decades, no investigation has evaluated the diagnostic performance of immunoglobulin G (IgG) subclasses in the serodiagnosis of ALA. Herein, we assessed the efficiencies of anti-amebic IgG and IgG subclasses for diagnosing ALA.Amebic liver abscess (ALA) caused by n = 14), healthy individuals (n = 40), and patients with other diseases (n = 276).A serological ELISA-based test was performed to assess its diagnostic performance using a total of 330 serum samples from ALA patients  and a slightly higher specificity (99.1% 95% CI [97.3–99.8]). The other IgG subclasses  displayed a lower sensitivity and specificity. The sensitivity and specificity did not significantly differ between tests measuring total IgG and IgG1 , with a concordance of 98.2%, represented by a Cohen’s kappa of 0.83 (p < 0.001), indicating almost perfect agreement.ELISA targeting the total IgG antibody to ELISA targeting IgG1 can provide valuable information to clinicians in differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human total IgG is cheaper than anti-human IgG subclasses. Therefore, we suggest that total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis. Entamoeba histolytica, is the second leading cause of death from protozoan infections worldwide  assay, the gold standard method, helps diagnose ALA if aspirated pus is available, but this technique is costly and only used in some laboratories, particularly in developed countries, where infrastructure and skillful staff are available , which is associated with significant morbidity and mortality . Trophozvailable . Therefovailable , like imvailable , indirecvailable , enzyme vailable , and latvailable .The enzyme-linked immunosorbent assay (ELISA) is still the most common test in many countries for diagnosing ALA due to its high sensitivity, specificity, ease of use, and cost-effectiveness. In addition, detecting antibodies can be beneficial when fecal microscopy or antigen detection tests are ineffective for diagnosis. In endemic settings, however, significant background measurements are produced; consequently, the original protocol’s cut-off may need to be adjusted for each individual situation , particuE. histolytica-specific antibody detection tests indicate using immunoglobulin G (IgG) in the detection system , which were proven to be free of any intestinal parasitic infections at the time of blood collection; (2) samples from cases of ALA caused by E. histolytica (n = 14), which were described in detail together with the clinical courses and diagnostic evidence of ALA (n = 276). No mixed parasitic infection sera were used in this study. The frozen sera stocks of fourteen Japanese ALA patients whose illnesses that occurred between 1980 and 1997  negative control samples from healthy volunteers from northeastern Thailand and 1997  were useion test  which stion test . Serologuncommon , includiuncommon . Strongyuncommon . Trichinuncommon . Angiostuncommon . Gnathosuncommon . Clonorcuncommon  and ELISuncommon . Cysticeuncommon . Schistouncommon . Sparganuncommon . Blastocuncommon . Malariauncommon . Viral hThe study protocol was approved by the Human Research Ethics Committee of Khon Kaen University  and the study was conducted in accordance with the Declaration of Helsinki and the ICH Good Clinical Practice Guidelines.E. histolytica HK-9 strain was cultivated in Diamond’s medium as per previously published protocols  and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was dialyzed against distilled water containing proteinase inhibitors  and stored at −20 °C until further used as crude E. histolytica HK-9 antigen in the ELISA. The protein content of the antigen was determined using the standard method  in citrate phosphate buffer, pH 5.0, was used as a substrate and incubated for 30 min at RT in the dark. The enzyme reaction was stopped by adding 50 µL of 8 N sulfuric acid. The optical density (OD) was measured at 492 nm using a microplate reader . Duplicates of each serum sample were included in the same plate to ensure reproducibility of the tests. The precision of the ELISA was investigated by performing the test on different days using the same pooled positive and negative sera, and the same batch of antigen under the same conditions. Consistent data were obtained from all the tests indicating no day-to-day variation.A standard ELISA protocol was followed. Antigen, serum, conjugate, and substrate were used at volumes of 100 µL/well and the optimum condition to be finally used in ELISA was determined by checkerboard titration. A 96-well microtiter plate  was coated overnight with the antigen at an optimum concentration of 2.5 µg/mL  and 5.0 µg/mL (for IgG subclasses) in carbonate buffer, pH 9.6, at 4 °C. The plates were washed five times with 0.05% Tween 20 in 0.1 M phosphate-buffered saline, pH 7.4 (PBST). After blocking nonspecific binding sites with 300 µL of PBST-3% bovine serum albumin (BSA) for 1 h at RT, the plate was then washed with PBST, incubated with diluted serum in PBST-1% BSA for 1 h at 37 °C, and washed again with PBST. The optimum serum dilution was 1:800 for detecting total IgG and 1:100 for IgG subclasses. This was followed by incubating with either horseradish peroxidase (HRP)-conjugated monoclonal anti-human IgG  diluted 1:100,000 for total IgG, or HRP-conjugated anti-human IgG subclass antibodies diluted 1:6,000 for IgG1 , 1:4,000 for IgG2 (Zymed), 1:500 for IgG3 (Invitrogen), and 1:1,000 for IgG4 (Invitrogen) for 1 h at 37 °C. After thorough washing, κ) were graded as almost perfect agreement (κ = 0.81–1.00), substantial (κ = 0.61–0.80), moderate (κ = 0.41–0.60), fair (κ = 0.21–0.40), slight (κ = 0.00–0.20), and poor agreement (κ <0.00) . Continuous variables were compared using the nonparametric Kruskal–Wallis method. Quantitative data were presented as mean ± standard deviation. The cut-off values that gave the best balance of sensitivity and specificity for ELISA were established using the receiver operating characteristic (ROC) curve with sera from ALA patients and from healthy individuals. The standard diagnostic indices, including accuracy, sensitivity, specificity, and likelihood ratios, were calculated as previously described κ <0.00) .E. histolytica HK-9 antigen (p < 0.001). According to the ROC analysis, the best cut-off OD values were 0.412, 0.079, 0.200, 0.093, and 0.139 for total IgG, IgG1, IgG2, IgG3, and IgG4, respectively. The sensitivities for detecting total IgG, IgG1, IgG2, IgG3, and IgG4 were 100%, 100%, 92.9%, 71.4%, and 85.7%, respectively (p > 0.05), with a concordance of 98.2% represented by a Cohen’s kappa of 0.83 (p <  0.001), indicating almost perfect agreement  and specificity (97.8%), comparable to the values of 80–100% sensitivities and 93–100% specificities reported for commercial and in-house IgG ELISA kits in cases of ALA  in group 3, which is not very high compared with the average for developing nations. These results demonstrate that our IgG-based ELISA method is valuable in the context of a real-world investigation conducted in a developing nation. Our in-house ELISA has two significant flexibility advantages over commercially available assays: (1) shelf life, freshly prepared ELISA components are dependent on the physician’s estimated monthly requests, and (2) the assay conditions, including serum dilution, conjugate dilution, type of conjugate and blocking buffer, etc., can be optimized to each endemic zone.The present study was designed to assess the diagnostic potential of ELISA, targeting total IgG and IgG subclasses produced against crude soluble antigens of s of ALA . InteresCross-reactions in the IgG ELISA test were observed in some samples from patients infected with other parasites: hookworm infection, capillariasis, clonorchiasis, fascioliasis, schistosomiasis, and blastocystosis. The occasional cross-reactions with these other parasite species do not pose a significant problem in the clinical setting. Hookworm infection, capillariasis, and blastocystosis normally show a diagnostic stage in fecal specimens and have clinical symptoms that differ from those of ALA. Although clonorchiasis, fascioliasis, and schistosomiasis can elicit clinical symptoms and liver inflammation/abscess similar to ALA, they also present with a history of infection, microscopic examination of the stool, peripheral blood eosinophilia, medical imaging abnormalities of the liver, and medication responses that are different from those in ALA . MoreoveDuring infection, different IgG subclasses are produced but, as we have shown here, all of them were not equally elevated in the humoral response to ALA. This situation is likely to be consistent and predictable . In thisWe realize that ELISA is time consuming. However, as a routine laboratory service, this is the first step taken in developing countries, like Thailand and nearby Asian countries. Our in-house ELISA can be manipulated more easily than the PCR assay. Cost also significantly limits the use of current PCR methods, particularly in developing nations. In such nations, positive antibody testing, along with a patient’s clinical manifestations, risk history, other laboratory findings, and/or abdominal imaging, are currently used for diagnosis . TherefoThe present study has some limitations. First, our study was carried out using a small sample size of ALA patients, and the PCR assay was not used to confirm the ALA diagnosis. So, there was some bias in estimating the sensitivity for ALA patients. Second, the sera were obtained from banks in Thailand and Japan; the results may not apply to other populations. Third, cross-reactions should be investigated with pyogenic liver abscess, leishmaniasis, and other infectious diseases primarily found in tropical regions. Addressing these limitations will strengthen the clinical laboratory application of this study.No previous studies have investigated the diagnostic effectiveness of total IgG and IgG subclasses in ALA. We found that IgG1 is an excellent diagnostic antibody for ALA. ELISA targeting this subclass of IgG can provide valuable information to clinicians by differentiating ALA from other parasitic diseases, cancers, cirrhosis, and viral hepatitis. However, enzyme-conjugated anti-human IgG subclasses are quite costly. Considering no significant differences in sensitivity and specificity between IgG1 and total IgG detection and almost perfect agreement between them, we recommend that a total IgG-based ELISA is sufficient for the routine serodiagnosis of human ALA and possibly other clinical manifestations of invasive amebiasis.


We also demonstrated that XN-31 was a reliable method for patient follow-up on days 3, 7, and 28. Malaria diagnosis can be improved in non-endemic areas by the use of dedicated haematology analysers coupled with standard microscopy or other methods in development, such as artificial intelligence for blood slide reading. Given that XN-31 provided an accurate diagnosis in 1 min, it may reduce the time interval before treatment and thus improve the outcome of patient who have malaria.Malaria diagnosis based on microscopy is impaired by the gradual disappearance of experienced microscopists in non-endemic areas. Aside from the conventional diagnostic methods, fluorescence flow cytometry technology using Sysmex XN-31, an automated haematology analyser, has been registered to support malaria diagnosis. The aim of this prospective, monocentric, non-interventional study was to evaluate the diagnostic accuracy of the XN-31 for the initial diagnosis or follow-up of imported malaria cases compared to the reference malaria tests including microscopy, loop mediated isothermal amplification, and rapid diagnostic tests. Over a one-year period, 357 blood samples were analysed, including 248 negative and 109 positive malaria samples. Compared to microscopy, XN-31 showed sensitivity of 100% (95% CI: 97.13–100) and specificity of 98.39% (95% CI: 95.56–100) for the initial diagnosis of imported malaria cases. Moreover, it provided accurate species identification as Plasmodiumtransmitted through the bite of an infectedAnophelesmosquito. In 2020 there were more than 240 million infections with an estimated 627,000 deaths reported globally [Plasmodiumspecies are regularly identified in infected humans:Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale curtisi,Plasmodium ovale wallikeri(while the distinction between those two species is uncommon), andPlasmodium malariae[Plasmodium knowlesiis the most frequently observed in humans [Malaria is a life-threatening disease caused by the protozoan parasiteglobally . The cou malariae,21. Amonn humans .P. falciparum(86.5%), followed byP. ovale(6.8%),P. vivax(2.5%),P. malariae(2.2%), and multiplePlasmodium species(1.3%) [Currently, France ranks first for the number of imported malaria cases per country followed by Great Britain, the United States, and Italy . Patientes1.3%) . Early a% . Earlyiparum86.%, followes(1.3%) . Rapid des(1.3%) ,39. Conses(1.3%) ,16. Thuses(1.3%) .Haematology analysers have been used for at least a decade for malaria diagnosis ,26,29,33Plasmodiumspecies identification and parasitemia assessed by XN-31 to those performed by microscopic examination of the thin smear, and to study the repeatability over time of the XN-31 method after blood storage under different conditions.The first objective of this study was to evaluate the diagnostic accuracy of the XN-31 automated haematology analyser  for the initial diagnosis and/or follow-up of imported malaria cases compared to the reference malaria tests including RDT, LAMP, and microscopy . The secThis was a non-interventional study, without any additional procedures. Data were collected during routine patient care. Provided data were centrally checked for completeness, plausibility, and integrity before analysis. To ensure reproducibility and completeness of data extraction, an excel spreadsheet  compiling all variables to be extracted was used.http://ec.europa.eu/research/swafs/pdf/pub_archive/textbook-on-ethics-report_en.pdf). Electronic records were stored and processed in compliance with the conventions of the French National Committee for Data Protection and Freedom of Information. Ethical clearance was obtained from Lyon University hospital. The following data were collected using hospital databases: malaria final diagnosis based on RDT, LAMP and microscopic test, date of sampling at admission and follow-up if any, and drug used for malaria treatment.This research involved anonymised data sets in which personal identifiers were permanently and completely removed from data, meaning that the data can no longer be associated with an individual in any manner. Anonymisation and removing of protected health information from clinical narratives were performed according to the European Textbook on Ethics in research  hosted by the Enhancing the Quality and Transparency of Health Research (EQUATOR) network were used as methodological support ,17.n = 365 participants).Eligibility criteria were either suspicion of non-severe imported malaria based on fever and history of travel to a malaria endemic area or follow-up of a malaria-positive case. A venous blood sample was collected for malaria diagnosis on two EDTA tubes and rapidly transferred to the diagnostic facility at room temperature. All patients who were suspected of having malaria or followed up after a malaria-positive diagnosis were assessed for eligibility. According to the requirements for confirmatory diagnostic accuracy studies, sensitivity and specificity were considered co-primary endpoints, and the prevalence of the target population was taken into account . The samPlasmodiumspecies. Thin and thick blood stained smears  were performed in compliance with the national guidelines for malaria diagnosis and certification requirements of the National Certification Program. Proficiency testing and external quality assessment for microscopy were used once a week and bi-monthly, respectively. They were read by two independent trained and certified biologists and checked by a senior microscopist. Microscopic examination allowed for the identification ofPlasmodiumspecies and the determination of parasitaemia, recorded as the number of infected red blood cells (RBCs) with asexual parasites compared to non-infected RBCs. Malaria microscopic diagnosis was considered to be negative if no parasites were found in 100 microscopic fields of 200 red blood cells for thin smears and 25 microscopic high power fields for thick smears. Cases of discrepancy between XN-31 and microscopic or molecular testing were resolved by a real-time PCR test as described [LAMP  and RDT  Ag Combo RDT, Access Bio Inc., Somerset, NJ, USA) were performed as screening methods according to the manufacturer’s specifications on fresh blood samples . If one The same fresh blood samples were tested with the Sysmex XN-31 analyser, according to the manufacturer’s recommendations ,37. Two P. falciparum(n = 97), followed byP. malariae(n = 4),P. ovale(n = 2), andP. vivax(n = 1). Five of them were mixed infections ofP. falciparumandP. malariae were suspected of having non-severe malaria or followed up after a positive malaria diagnosis, leading to 392 blood samples eligible for inclusion. Due to MI-RBC Abnormal Scattergrams according to the XN-31 analyser, 5 positive and 30 negative samples were excluded, according to the manufacturer’s recommendations. The final study sample size was 357, including 109 positive and 248 negative samples. Among them, 288 blood samples were collected on admission and 69 during patient follow-up. Among the 109 malaria positive samples, most of them were mono-infections caused by malariae.n = 357), XN-31 showed very high sensitivity of 100% (95% CI: 97.13–100) and specificity of 98.39% (95% CI: 95.56–100) compared to microscopy , XN-31 showed sensitivity and specificity of 98.98% and 100%, respectively. In this sub-group, the VPN and VPP were 99.48% and 100%, respectively.Using only the samples collected at admission  were detected as “malaria: others” by XN31, leading to an excellent distinction betweenP. falciparumand other species. This identification was confirmed by real-time PCR as described (data not shown). One sample withP. malariae(RT-PCR confirmed) with low parasitaemia of 0.01% (0.0294 MI-RBC %) did not lead to species identification (“UNC?”). This sample showed 85% gametocytes and the patient had already received antimalarial treatment for two days. Considering the low parasitaemia, the high ratio of gametocytes/trophozoites, and the impact of treatment on parasite morphology, the “UNC?” comment was considered acceptable.All theP. falciparum + P. malariae) were also detected by XN-31, with a double indication of “P. falciparum”and “others” species. The mixed infections were confirmed by RT-PCR (data not shown). Additionally, four samples collected during the recrudescence episode from D28 to D34 of a patient presentingfalciparummalaria and treated with artemether-lumefantrine on admission, and then with quinine IV during the recrudescence episode, were qualified as “P. falciparum + others” and “others”, while confirmed asP. falciparumusing real-time PCR. This discrepancy may be attributed to the abnormal morphology of parasites after treatment as confirmed by microscopy. Gametocytes appeared during the last three days of the follow-up after quinine and were correctly detected by XN-31.Interestingly, 5/5 mixed infections , H24, H48, and H72. All tests were repeated three times. There was no significant difference in MI-RBC count between results of samples kept at room temperature and at 4 °C from H0 to H72 for all the samples, except one (sample 5) presenting unexplained increased values at H48 at 4 °C that disappeared at H72 .During the 12-month study period, 357 blood samples were studied, including 109 positive and 248 negative samples. This prospective study was conducted in Lyon University hospital during the COVID-19 pandemic that led to a dramatic reduction in travel to and from malaria endemic areas, and consequently to a reduced number of suspected and confirmed malaria cases. The number of malaria cases decreased by more than 80% during 2020 and the first 3 months of 2021. Later in 2021, the number of travelers increased again significantly, allowing us to include more patients, although the number of included travelers from Asia and South America remained low.The objective of this study was to test the XN-31 hematology analyser method for malaria diagnosis in routine conditions. To this end, data were compared to the standard diagnostic procedure which includes a molecular test (LAMP) and an antigenic test (RDT) for screening, and microscopic examination of thin and thick blood smears for parasite identification and parasitaemia determination.PlasmodiumDNA or antigens during the patient’s follow-up, which are in fact not relevant for active malaria infection. In fact, the standard screening methods (molecular tests such as LAMP and antigenic tests such as RDT) frequently remained positive for 2–4 weeks post-infection, while parasite clearance was obtained [The results for specificity and sensitivity (100% (95% CI: 97.13–100) and 98.39% (95% CI: 95.56–100), respectively) allowed us to conclude that the XN-31 haematology analyser was as effective as conventional methods for the screening of patients who were suspected of having malaria. While almost 10% of the tested samples were excluded due to an abnormal scattergram, as required by the study protocol, all these 35 samples (30 negative and 5 positive) showed correct malaria results. For patients who were followed up after a positive malaria diagnosis, few false-positive and false-negative results were found, compared to LAMP or RDTs. This was attributed to the persistence of circulatingSimilarly, XN-31 was found to be highly correlated to microscopy for species identification and parasitaemia assessment. Moreover, based on the repeatability study, there was no impact on XN-31 results of storage temperature (room temperature or 4 °C), nor of the time interval of 72 h for blood sample analysis.Apart from its excellent technical characteristics for initial malaria diagnosis or follow-up, XN-31 has excellent practicability, and while it requires specific training, its accessibility is straightforward for non-expert users of haematology devices. When the machine is ready for the working day, the time to a result including parasitaemia is very short (within a minute), whereas RDT and LAMP as screening methods require 25 and 45 min, respectively to obtain a result without a parasitaemia count. The short timeframe to obtain a malaria result with XN-31 is a major advantage compared to other screening methods, considering that the clinical prognosis is improved with early diagnosis and treatment.In view of the excellent performance and practicability of XN-31, we need to consider the place of XN-31 in our malaria diagnostic strategy. Some guidelines  from nonPlasmodiumspecies as “falciparum” and “non-falciparum”. Acute determination of non-falciparumspecies is not available today. Moreover, XN-31 is an expensive method that will be possible to implement only in major health facilities. This problem of cost could be overcome given the excellent results of our repeatability study. One can imagine that healthcare facilities that did not have direct access to XN-31 will use conventional screening methods, including LAMP and RDTs, and will send their blood samples for diagnostic confirmation and parasitaemia assessment to laboratories where an XN-31 system is in use. Although XN-31 is an attractive method for malaria diagnosis, it is not possible to base the malaria diagnostic strategy on this method alone. Microscopy is still needed to confirm the identification ofPlasmodiumspecies and to describe complex diagnostic scenarios. This would also prevent any problems due to reagent shortage, machine breakdown, or computer issues. Automatic staining of blood smears combined with artificial intelligence (AI) for species identification and parasitaemia count appears to be the best partner for XN-31 in the near future [Compared to microscopy, XN-31 also has value regarding its capability to efficiently assess parasitaemia. Importantly, expert microscopists are not always available at the diagnostic facilities and expertise in malaria diagnosis is decreasing with the decline in the number of malaria cases in many countries. Thus, the accuracy of microscopic examination of stained blood smears is not that high for many laboratories. Here, we provide evidence that XN-31 has the same performance as microscopy to assess parasitaemia. This excellent correlation between XN-31 and microscopy gives a strong rationale for the use of XN-31 for the assessment of parasitaemia. Additionally, XN-31 can also determiner future ,24. ManyIn our study, we demonstrated that XN-31 has the technical requirements to change the diagnostic malaria strategy in well-equiped laboratories. Thus, it is of utmost importance to work with key players to make this transition possible. This task may also be supported by antimalarial stewardship programs [


Malaria rapid diagnostic tests (mRDTs) have played an important role in the early detection of clinical malaria in an endemic area. While several mRDTs are currently on the market, the availability of mRDTs with high sensitivity and specificity will merit the fight against malaria. We evaluated the field performance of a novel One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits in Pwani, Tanzania.In a cross-sectional study conducted in Bagamoyo and Kibiti districts in Tanzania, symptomatic patients were tested using the SD BIOLINE, One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits, microscope, and quantitative Polymerase Chain Reaction (qPCR). An additional qPCR assay was carried out to detect Histidine-Rich Protein 2 (HRP-2) gene deletion on mRDT negative but microscope and qPCR positive samples. Microscope results confirmed by qPCR were used for analysis, where qPCR was used as a reference method.The sensitivity and specificity of One Step P.f/P.v Tri-line mRDTs were 96.0% (CI 93.5–97.7%) and 98.3% (CI 96.8–99.2%), respectively. One Step P.f mRDT had sensitivity and specificity of 95.2% (CI 92.5–97.1%) and 97.9% (CI 96.3–99.0%) respectively. Positive predictive value (PPV) was 97.6% (CI 95.4–98.7%) and negative predictive value (NPV) was 96.2% (CI 95.5–98.3%) for the One Step P.f/P.v Tri-line mRDTs respectively, while One Step P.f mRDT had positive predictive value (PPV) and negative predictive value (NPV) of 97.0% (CI 94.8–98.3%) and 96.7 (CI 94.9–97.9%) respectively. 9.8% (CI 7.84–11.76) of all samples tested and reported to be malaria-negative by mRDT had HRP-2 gene deletion.One Step Malaria P.f/P.v Tri-line and One Step Malaria P.f rapid test kits have similar sensitivity and specificity as the standard mRDT that is currently in the market, demonstrating the potential to contribute in the fight against malaria in endemic settings. However, the identified malaria parasites population with HRP-2 gene deletion pose a threat to the current mRDT usability in the field and warrants further investigations. Malaria continues to be a major public health concern in the endemic area despite enormous efforts and funding invested in the fight against malaria to date  occurred globally WHO . These sDiagnostic tools are, however, among the important pillars of elimination strategies as it could be used for the clinical management of the disease , rendering them undetectable by HRP-2-based RDTs  circulating in the blood. These proteins are specific to Plasmodium falciparum and other Plasmodium species. However, the field performance evaluation of these kits in terms of sensitivity and specificity was yet to be established. This study aimed to evaluate the field performance sensitivity and specificity of the new One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits by comparing with microscopy and qPCR as reference methods.An addition of new mRDT with improved performance sensitivity and specificity will merit the current battle against malaria. The InTec PRODUCT INC Company from China has introduced new mRDT kits for detecting the A cross-sectional study was conducted on symptomatic participants from July 2020 to September 2021. Participants aged 5 years and above who presented to the health facilities with fever or a history of fever within 48 h or an illness that the attending doctor suspected might be due to malaria infection were targeted. Participants who met the enrollment criteria consented to participate in the study. The study involved three arms; clinical, analytical and blood type. Clinical study aimed to determine operation performance, sensitivity and specificity of the (P.f) and One Step (P.f/P.v) mRDTs test kits. A blood type study aimed to investigate the performance of independent operators when blood collected in different anticoagulants were used on the One Step Malaria (P.f) and One Step (P.f/P.v) Tri-line test kits, while the analytical study aimed to investigate whether co-infections could interfere with the performance accuracy of the One Step Malaria (P.f/P.v) and (P.f) tests.A total of 1630 (1175 P.f negative and 455 P.f positive) patients were tested for malaria using the registered SD BIOLINE, One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits. The current sample size calculation was based on the requirement for Technical Specifications Series for submission to WHO Prequalification Diagnostic Assessment TSS-3 Malaria rapid diagnostic tests, which requires at least 1500 participants to be enrolled for this type of analysis  under the national malaria control program. In the current study area, malaria transmission intensity varies within geographical region and seasonality despite that infections are acquired all year-round. The study was implemented in two districts of the Pwani region. These included the peripheral dispensaries located in Kibiti (Bungu and Mtawanya) and Bagamoyo  districts. Kibiti district representing stable and high malaria transmission where most cases are caused by Both direct finger prick (capillary) and whole venous blood collected in EDTA, Heparin, and Sodium citrate were parallel tested with mRDTs One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits in the blood type study. The blood slides were examined using the World Health Organization (WHO) protocol as described elsewhere  serially diluted into a different range of concentrations  up to the lowest concentration of 0.001 p/µl) were used to generate the relative standard curves for parasite density quantification. Samples with Ct < 40 on the target gene and Ct < 35 in the housekeeping gene were considered to be positive.Genomic DNA was extracted from the whole blood using Quick-Gdna™ Blood MiniPrep Kit  following the manufactures instructions. Master Mix was prepared using Lunar Universal Probe qPCR Master Mix (New England Biolabs NEB M3004X.) as per Ifakara Health Institute laboratory protocol. The real-time qPCR assays were performed using the Bio-Rad CFX96 detection system and analysis done by using CFX Manager Software . Six categories of malaria parasite density were used to characterize mRDT performance accuracy. These included: < = 10 p/µl, 11–50 p/µl, 51–100 p/µl, 101–200 p/µl, 201–2000 p/µl, and > = 2000 p/µl. The number of positives in each range was established and discrepancies were recorded to obtain the percentage agreement among the tests. Blood smear results that were only confirmed by qPCR were used as reference densities for this comparison.∘C, 45 cycles, 15 s at 95∘C, and 35 s at 57.5 °C.The HRP-2 gene deletion was identified from samples that were mRDTs negative but positive for microscopy and qPCR. To evaluate the effectiveness of DNA extraction and qPCR amplification, the internal control gene for pfrnr2  with sequences of AGTATCCAAAACACTATAATTCCAAGTAC forward and ATTTTCTCCTTTCTTAACAGTTTCTTCC reverse respectively were used. Also gene deletion marker, pfrp2  with sequences of GTATTATCCGCTGCCGTTTTTGCC forward and TCTACATGTGCTTGAGTTTCG reverse were chosen, respectively. Utilizing the Bio-Rad CFX96 detection system and the CFX Manager Software, real-time qPCR assays were performed. Master Mix was prepared and following the thermal profile was used for qPCR: 5 min at 95 To determine the impact of malaria co-infections in the test kits, hospital guideline was used to select patients with other disease Table  and wereData were entered in an excel database (Microsoft Excel) and used to assess clinical performance of One Step Malaria (P.f/P.v) Tri-line line and One Step Malaria (P.f) rapid test kits, as well as microscope and qPCR results from different malaria transmission intensities. The STATA software version 15 was used for establishing sensitivity and specificity of the tests.P.f negative and 455 P.f positive) patients seeking medical care and presented with fever, met the enrollment criteria at health care facilities, were tested for malaria using both the registered mRDT (SD BIOLINE), the One Step Malaria (P.f/P.v) Tri-line One Step Malaria (P.f), microscope and qPCR as reference. SD BIOLINE was used to manage patients at the health care facilities whenever needed. 62.1% (1012/1630) of participants were female and 34.5% (562/1630) were males. 10.98% (179/1630) and 23.5% (383/1630) of male participants were malaria positive and negative respectively and 15.5% (253/1630) and 46.6% (759/1630) of female participants were malaria positive and negative respectively. Gender of 3.4% (56/1630) of participants was not specified. The characteristics of the study demographic characteristics are summarized in Table A total of 1630  Tri-line and One Step Malaria (P.f) rapid test kits, sensitivity and specificity of the kits were calculated with reference to blood smear results that were confirmed by qPCR. The results showed that, there was no different in sensitivity and specificity of One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) rapid test kits when finger prick blood were used [95.7% 93.2–97.6% CI) and 97.9% (96.3–99.0% CI)], respectively, and when venous blood were used sensitivity and specificity were [96.0% (93.5–97.7% CI) and 98.3% (96.8–99.2% CI)], respectively. Similar finding was observed for the One Step Malaria Pf test RDTs  when finger prick blood was used and  when venous blood were used. Additionally, the sensitivity and specificity of SD Bioline mRDTs that are currently in the market and used in the health facilities was [94.95% (92.22–96.93% CI) and 97.75% (96.10–98.83%)]  Tri-line, One Step Malaria (P.f) rapid test kits and SD Bioline mRDTs performance were similar across the parasite density ranges Tables  and 6. TThree hundred and seventy-six samples were malaria positive by both blood slides and qPCR. 44 out of 376 samples that were positive by both qPCR and microscopy were negative for One Step Pf/Pv Tri-line, One Step Malaria Pf only and SD Bioline mRDTs and were further analyzed for HRP-2 gene deletion by qPCR assays. 7/44 (15.9%) samples gave positive results for HRP-2 gene, implying that the gene was there. However, 37/44 (84.1%) were negative for the HRP-2-gene. The general proportion of HRP-2 gene deletion reported from the current study was 9.8% (7.84–11.76)  Tri-line and One Step (P.f) mRDT could detect all cases detected by blood smears and qPCR, when blood collected from juvenile patients who had co-infected with other diseases were tested. Twenty-eight 28) patients who had malaria positive results as detected by both BS/qPCR and RDTs were also positive by other diseases infections  Tri-line and (P.f) mRDTs were similar to that of the standard kit (SD Bioline) in the field evaluation. The sensitivity and specificity of other novel mRDT have also been compared to the standard mRDT kits (SD Bioline) which have been to the market for decade  is produced in abundance by tion WHO . One postion WHO . Other stion WHO  that leaFurthermore, blood collected in different anticoagulants did not affect the performance of One Step Malaria (P.f/P.v) Tri-line and One Step (P.f) mRDTs test kits as compared to standard mRDT kits (SD Bioline). The scored sensitivity and specificity were similar to sensitivity and specificity reported by other mRDT kits available in the market  to determine whether there was presence of HRP-2 proteins in individuals who had HRP-2 gene deletion. Although the qPCR itself is sufficient to confirm gene deletion, the results from antibodies could have been a positive supplement for the finding. Lastly, the detection of other co-infection was sorely depended on clinical judgments made by clinicians using national treatment algorithms and not laboratory testing.First, the current One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) mRDTs test kits were designated to detect antibody for The current study demonstrated that the sensitivity and specificity of novel One Step Malaria (P.f/P.v) Tri-line and One Step Malaria (P.f) mRDTs test kits were similar to that of SD Bioline which is the standard kit used routinely in the health care facilities in Tanzania and elsewhere. The proportional of samples with HRP-2 gene deletion reported in the study population and causing false negative mRDT results warrants further studies to map the prevalence of gene deletion and determine how it affects the current malaria control strategies.


Plasmodium parasites by mosquitoes. TB31F is a humanised monoclonal antibody that binds the gamete surface protein Pfs48/45 and inhibits fertilisation, thereby preventing further parasite development in the mosquito midgut and onward transmission. We aimed to evaluate the safety and efficacy of TB31F in malaria-naive participants.Malaria elimination requires interruption of the highly efficient transmission of Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. The trial is registered with Clinicaltrials.gov, NCT04238689.In this open-label, first-in-human, dose-escalation, phase 1 clinical trial, healthy, malaria-naive, adult participants were administered a single intravenous dose of 0·1, 1, 3, or 10 mg/kg TB31F or a subcutaneous dose of 100 mg TB31F, and monitored until day 84 after administration at a single centre in the Netherlands. The primary outcome was the frequency and magnitude of adverse events. Additionally, TB31F serum concentrations were measured by ELISA. Transmission-reducing activity (TRA) of participant sera was assessed by standard membrane feeding assays with Between Feb 17 and Dec 10, 2020, 25 participants were enrolled and sequentially assigned to each dose (n=5 per group). No serious or severe adverse events occurred. In total, 33 grade 1 and six grade 2 related adverse events occurred in 20 (80%) of 25 participants across all groups. Serum of all participants administered 1 mg/kg, 3 mg/kg, or 10 mg/kg TB31F intravenously had more than 80% TRA for 28 days or more, 56 days or more, and 84 days or more, respectively. The TB31F serum concentration reaching 80% TRA was 2·1 μg/mL (95% CI 1·9–2·3). Extrapolating the duration of TRA from antibody kinetics suggests more than 80% TRA is maintained for 160 days (95% CI 136–193) following a single intravenous 10 mg/kg dose.P falciparum parasites from humans to mosquitoes. In areas of seasonal transmission, a single dose might cover an entire malaria season.TB31F is a well tolerated and highly potent monoclonal antibody capable of completely blocking transmission of PATH's Malaria Vaccine Initiative. Plasmodium falciparum infections.3Malaria is a global health priority with over 200 million yearly cases worldwide, including more than half a million deaths resulting primarily from P falciparum malaria transmission. Despite its important value in reducing malaria morbidity and mortality, deployment of RTS,S/AS01 will be insufficient to prevent all episodes of malaria. Moreover, this vaccine does not directly prevent onward transmission of parasites to mosquitoes and remains susceptible to selection and spread of escape mutants. The high efficacy of monoclonal antibody CIS43LS in preventing infections in a human challenge model underscores the clinical potential of monoclonal antibodies as a complementary tool to combat malaria, as shown for other infectious diseases.7In 2021, landmark progress was achieved in the field of malaria vaccines. WHO issued a recommendation for widespread use of RTS,S/AS01, the world's first approved malaria vaccine, among children living in regions with moderate to high Anopheles mosquitoes in whose midgut gametocytes activate into gametes, fertilise, and ultimately render the mosquito infectious to the next human host. The long duration of (asymptomatic) P falciparum infections in humans, the abundance of mosquitoes that bite multiple hosts, and efficient transmission of parasites from humans to mosquitoes and vice versa, all contribute to reproduction rates (R0) for malaria that exceed 100 in many African settings.Malaria transmission depends on the uptake of male and female gametocytes by blood feeding Evidence before this studyPlasmodium falciparum infections. In addition to strategies focusing on preventing and treating individual cases of malaria, interrupting the highly efficient transmission of malaria is key to reducing the global burden of this disease. We searched PubMed on March 8, 2022, for clinical trials testing transmission blocking vaccines and monoclonal antibodies against P falciparum malaria, using the search terms:  AND ((transmission blocking) OR (transmission-blocking) OR (transmission reducing) OR (transmission-reducing)) AND . No language or date restrictions were applied. No records were present of clinical trials involving transmission-blocking monoclonal antibodies; although, one study reports the clinical testing of a monoclonal antibody that targets the pre-erythrocytic stage of the parasite and hence can prevent infection. Among transmission-blocking monoclonal antibodies evaluated to date in preclinical studies, the humanised antibody TB31F has shown the most potent functional activity.Malaria is a global health priority with over 200 million cases and over 500 000 deaths that result primarily from Added value of this studyP falciparum transmission blocking monoclonal antibody (TB31F). We report TB31F administration to malaria-naive healthy participants to be safe and well tolerated at all doses tested. TB31F in participants' serum showed potent activity, reaching the benchmark of at least 80% reduction in transmission of parasites to mosquitoes in the standard membrane feeding assay at serum concentrations as low as 2·1 μg/mL. Moreover, such activity was estimated to last for up to 5 months after a single administration.This is the first clinical trial assessing the safety and efficacy of a Implications of all the available evidenceP falciparum parasites to mosquitoes, underscore its clinical potential. Current malaria control measures, even when complemented by the widespread deployment of the pre-erythrocytic malaria vaccine RTS,S/AS01, as recommended by WHO in October, 2021, will be insufficient to prevent all episodes of malaria and subsequent onward transmission of parasites. TB31F represents a promising complementary tool to be deployed in several use-scenarios, including in areas of seasonal malaria transmission, where a single administration might have transmission-blocking activity for the duration of an entire season.The preliminary safety and highly promising efficacy profile of TB31F, in particular its ability to potently block transmission of P falciparum gametocytes and early gametes, that are being evaluated in phase 1 (NCT04862416) and phase 2 (NCT03917654) clinical trials, respectively. The effect of these vaccines will depend on their capacity to induce and maintain effective antibody titres against specific key epitopes,P falciparum NF54 gametocytes.Malaria transmission-blocking vaccines aim to induce antibodies that target the surface of sexual stage parasites and are coingested with gametocytes during the mosquito bloodmeal, consequently preventing parasite development in the mosquito midgut and hence onward transmission.e 2 NCT0317654 cliThis single-centre, open-label, first-in-human, dose-escalation, phase 1 clinical study evaluating the safety and efficacy of humanised monoclonal antibody TB31F  was done25 eligible participants were included and upon enrolment sequentially allocated from group 1 to group 5 .Adverse events were recorded and graded by the attending clinical investigator as mild , moderate  or severe . All participants received a memory aid booklet to register symptoms throughout the study and an oral thermometer to register their daily body temperature for 6 consecutive days after TB31F administration. Solicited local adverse events were defined as pain, redness, and swelling at the injection site up to 7 days after TB31F administration. Solicited systemic adverse events were defined as fever, headache, myalgia, fatigue, chills, and rash up to 28 days after TB31F administration. Unsolicited and serious adverse events were recorded through to the end of the study (day 84). Clinical laboratory results assessed as adverse events were scored for severity with the adapted Food and Drug Administration Toxicity Tables. Antidrug antibody levels in participants were assessed by sandwich ELISA at the Human Immunology Laboratory in London, UK, as an exploratory outcome .Serum concentrations of TB31F were measured by ELISA against the recombinant protein R0·6C, which contains the 6C fragment of the Pfs48/45 antigen to which TB31F binds.max), the area under the concentration-time curve from 0 to 12 weeks, and from time zero to infinity with the trapezoidal rule, and the elimination half-life. The apparent clearance and volume of distribution were additionally determined for individuals receiving intravenous TB31F administration. The bioavailability and the time of Cmax (Tmax) were additionally determined for individuals in the subcutaneous administration group. Projected TB31F concentrations beyond day 84 were estimated for each participant with concentrations from day 7 to day 84 after administration assuming first-order kinetics.For the intravenous and subcutaneous groups, the following pharmacokinetic parameters were determined by non-compartmental analysis for each individual curve: the maximum concentration  mosquitoes. Time series of individual participants were grouped in the same SMFA experiment. Additional SMFA with sera from participants receiving 10 mg/kg TB31F was done as a post-hoc analysis to determine TRA against the genetically distant Asian P falciparum isolate NF135.19TRA was determined at predefined intervals by SMFA with participants' sera.80) was estimated by linear regression, regressing the square root of serum concentration on the log-mean oocyst ratioStudy sample size was chosen pragmatically on the basis of the assessment of safety and in line with similar first-in-human dose-escalation trials. The study's original sample size of 20 (four intravenous groups of five participants each) was determined to allow a power of 90% to observe at least one event if the true rate of such an event is 10·9% or more; and a 90% chance of observing no events if the true rate is 0·5% or less. The fifth (subcutaneous) group was added later and pragmatically included the same number of participants as the other groups. Safety data are shown as frequencies and percentages are tabulated. TB31F pharmacokinetic parameters were assessed by standard non-compartmental methods, with multiple observations per group and per study participant. TB31F serum concentration  at which 80% TRA is expected  and Prism 9 (version 9.2.0). SMFA data analysis was done in R (version 4.1.1) and R studio (version 1.4.1717). Pharmacokinetic parameters were determined with non-compartmental analysis in Phoenix WinNonlin (version 8.3). The trial is registered with This study was funded by PATH's Malaria Vaccine Initiative, Washington, DC, USA. The funder was involved in study design, data collection, analysis, and interpretations, and contributed to the writing of the report.2 (range 19·5–29·5). All participants remained in follow-up until the end of the study and were included in the safety analysis.From Jan 23 to Dec 8, 2020, 39 participants were screened for eligibility. In total, 25 participants were enrolled between Feb 17 and Dec 10, 2020 . BaselinBoth intravenous and subcutaneous administrations of TB31F were well tolerated. No serious or grade 3 (severe) adverse events occurred. Furthermore, no infusion reactions occurred and no dose adjustments or temporary interruptions of TB31F administration were necessary. Overall, 20 (80%) of 25 participants had at least one adverse event that was possibly, probably, or definitely related to TB31F administration. Most solicited local adverse events took place in the subcutaneous group in which four (80%) of five participants had grade 1 (mild) pain of short duration (≤2 min) during subcutaneous administration . 13 52%. No sevemax, reported as geometric mean and 95% CI) of 2·94 μg/mL (95% CI 2·34–3·69), 28·3 μg/mL (14·4–55·5), 80·8 μg/mL (55·2–118), and 255 μg/mL (177–367), respectively, at end of infusion or administration (=Tmax). Subcutaneous administration of 100 mg TB31F resulted in a maximum concentration of 9·28 μg/mL (3·49–24·7) at day 4·3 (1·15–16·0), with a bioavailability of 52% (0·17–1·61), all reported as geometric mean and 95% CI. The overall terminal half-life was estimated to be 32·2 days.TB31F displayed dose-dependent peak concentrations and a dose-independent terminal half-life . Dose-prTB31F remained detectable in serum until end of study at day 84 in all study groups, except for one participant in the subcutaneous group. In this participant, in contrast to other group 5 participants, no or very low concentrations of TB31F were detected in serum at any timepoint. Therefore, the data from this participant were only included in the safety analyses  was determined to be 2·1 μg/mL  malaria, or in outbreak suppression. Mathematical modelling can inform future studies investigating the population-wide transmission-reducing potential of TB31F as an intervention strategy against malaria in different population groups at varying dosages. This includes standalone TB31F administration, and use in combination with interventions targeting other stages of the parasite lifecycle, including the recently reported CIS43LS monoclonal antibody for preventing In summary, this trial provides highly promising initial data on TB31F, the first malaria transmission-blocking monoclonal antibody to be assessed in humans. Its safety, tolerability, and high efficacy at interrupting human-to-mosquito transmission mark TB31F out to be an important potential tool for malaria control and elimination, advocating for its further assessment in naturally infected gametocyte carriers.ClinicalTrials.gov, NCT04238689. Deidentified participant data that underlie the results reported in this Article will be made available upon request. Proposals should be directed to the corresponding author. Proposals will be reviewed and approved by the sponsor, investigators, and collaborators on the basis of scientific merit. After approval of a proposal, data requestors will need to sign a data access agreement. Data can be requested indefinitely.The study protocol and statistical analysis plan can be accessed via We declare no competing interests.


The multiple regression model showed that only age (p = 0.028), SBP z-score (p = 0.006), and heart rate (p = 0.001) were significantly associated with PWV. The results were superimposable when the DBP z-score replaced the SBP z-score in the model. Mediation models showed that the effect of eGFR on PWV was fully mediated by SUA (p = 0.015) and that the effect of SUA on PWV was totally mediated by HOMA-index (p < 0.001). Both SUA (p < 0.01) and HOMA-index (p < 0.01) had a significant association with higher SBP (DBP) z-scores. The double mediation model including both BP and HOMA-index showed that the SUA effect on PWV was totally mediated by both variables . The results were superimposable when the DBP z-score replaced the SBP z-score in the model. In conclusion, insulin resistance and BP are both important mediators of the association between SUA and vascular stiffness in pediatric age.Several studies describe the association between serum uric acid (SUA) and arterial stiffness in adults. Uric acid contributes through several mechanisms to the increase in blood pressure (BP) and adversely affects the insulin signaling pathway. Moreover, SUA predict the development of hypertension and insulin resistance up to type 2 diabetes. Early arterial stiffening, estimated by carotid-femoral pulse wave velocity (PWV), may already be present in pediatric age. Aim of our study was to investigate the relationship between SUA and PWV in a pediatric population and its interaction with insulin resistance and BP. In 322 children and adolescents , we measured weight, height, waist circumference, BP and PWV. We also assayed SUA and estimated glomerular filtration rate (eGFR) and calculated HOMA-index as a marker of insulin resistance. Simple and multiple regression analyses were performed to assess variables associated with PWV. Mediation models were applied to identify the direct and indirect effects of individual variables on PWV. On univariate analysis, age ( Hyperuricemia and insulin resistance (IR) are well known risk factors for hypertension (HT) , 2, evenLongitudinal studies performed in adults show that SUA and IR influence blood pressure (BP) and the risk of cardiovascular events , 9. ThisSerum uric acid and homeostatic model assessment (HOMA) index, a recognized indicator of IR, are also associated with vascular damage in adulthood . Due to Carotid–femoral pulse wave velocity (PWV) is the non-invasive gold standard measurement of arterial stiffness , and it While a joint effect of increased SUA and IR on the development of subclinical vascular damage has been described in adults , it has The aim of the study was to investigate the complex relationship between SUA levels, IR, BP and subclinical organ damage in children and adolescents at increased cardiovascular risk. In the attempt to optimize our methodological approach, in addition to classical statistical regression models, we used a mediation analysis, an efficient method to clarify the causal paths underlying the association between the explanatory factors and the outcome.We studied a cohort of 322 children and adolescents (3.7–18.4 years), consecutively referred from 06/08/2012 to 10/06/2020 to our Unit for Cardiovascular Risk Assessment in Children  by their primary care pediatricians, because of evidence of elevated BP values and/or excess weight. Exclusion criteria were: diabetes mellitus, any form of secondary HT and treatment with antihypertensive drugs.The study protocol was approved by the local institutional ethics committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki . An informed consent was obtained from parents or legal representatives before the enrolment in the study.2. Waist-to-height-ratio (WtHr) was calculated dividing WC by height and expressed as percentage. BMI z-scores were calculated using the Centre for Disease and Control prevention charts available at https://www.cdc.gov/growthcharts/clinical_charts.htm. Weight class was defined according to the International Obesity Task Force classification  were measured. Weight was approximated to the nearest 100 grams, and height to the nearest 1 mm. Waist circumference was measured to the nearest 0.5 cm by a flexible tape in standing position. Body mass index (BMI) was calculated as weight (kg)/height (m)fication  distingufication , consideBlood pressure was measured using an oscillometric device validated in children  with the appropriate cuff for the children's upper-arm size.z-scores were calculated according to the nomograms of the National High Blood Pressure Education Program (NHBPEP) Working Group on High Blood Pressure in Children and Adolescents  and the average of the last two measurements was considered. Systolic BP (SBP) and diastolic BP (DBP) percentiles and lescents . The chiBlood samples were taken from all subjects after a 12-h fasting period to measure serum concentrations of glucose, insulin, SUA and creatinine. Commercial kits, normally used for routine examinations of patients, were employed for all analyses. In detail: enzymatic method with hexokinase Glucose HK Gen.3 Cobas Roche, for glucose assay; immuno Assay in ElectroChemiLuminescence Elecsys Insulin Cobas Roche for insulin assay; colorimetric enzymatic test Uric Acid 2 Cobas Roche for uric acid assay; colorimetric kinetic test based on the Jaffé method Creatinine Jaffé Gen.2 Cobas Roche for creatinine assay. HOMA-index was calculated by dividing the product of serum insulin (μU/ml) and serum glucose (mmol/L) by 22.5 .Measurements were obtained in a stable room temperature after 10 min of rest. A validated, easy-to-use and high-fidelity PulsePen tonometer  was used in this study , 25. RepT-test and Chi-square test.Continuous variables were expressed as mean and standard deviation, the categorical variables as percentages on relative frequencies. Comparisons across strata, defined by gender, were obtained by The relationship between the outcome (PWV) and explanatory variables was investigated through univariate and multivariable linear regression models.A mediation analysis through Structural Equation Modeling (SEM) was then performed to more deeply explore the roles of explanatory variables as predictors and possible mediators of PWV. Structural Equation Modeling is a very powerful multivariate technique that allows to examine a set of relationships among a few variables at the same time. It is based on a system of linked regression-style equations. In a classic regression model framework, there is a clear distinction between dependent and independent variables. In a SEM framework, however, a dependent variable in one model equation can become an independent variable in other components of the SEM system . RelatioThe mediation model was applied according to the following strategy. We explored the possible presence of an impact of SUA, mediated by HOMA-index, on PWV. Similarly, we explored the possible presence of an impact of HOMA-index, mediated by SUA, on PWV. The same approach was used to explore a possible mediation between SUA and HOMA-index on SBP and DBP and of SUA and eGFR on PWV. Suggested mediation paths were then included in a comprehensive model of PWV.Three hundred and twenty-two children and adolescents  were enrolled. p < 0.001), onset of pubertal developmental (p = 0.002), WtHr % (p = 0.036), SBP and DBP z-scores (p < 0.001), and heart rate (p = 0.028). There was a significant and positive association between PWV values and SUA (p = 0.002) and HOMA-index (p < 0.001), while the relationship between PWV and eGFR was inverse (p = 0.014).Univariable and multivariable linear regression models on PWV are shown in z-scores, the variables that were shown to be significantly associated to PWV values were: age (p = 0.028), SBP z-scores (p = 0.006), and heart rate (p = 0.001) , DBP z-scores (p = 0.005), and heart rate (p = 0.002)  , Model A= 0.002) , Model Bp < 0.001, p = 0.075).In the mediation analysis with PWV as outcome , the effz-scores and DBP z-scores as outcomes instead of PWV, HOMA-index and SUA showed both significant direct and indirect effects in the causal paths , whereas the effect of SUA on PWV was not mediated by eGFR .In the mediation analysis with PWV as outcome , the impz-score as mediators  ediators . In thisSimilar results were obtained including the direct impact of other confounders on PWV  and on SUA (eGFR) .z-scores), age, and heart rate maintain a significant association with PWV values. However, when the data are analyzed using mediation analysis, IR maintains an effect on vascular stiffness that is independent of SBP and DBP, while the association between SUA and PWV values turn out to be totally mediated by HOMA-index and BP.Our study shows that, in a population of children and adolescents with high BP and/or excess weight, IR (quantified by the HOMA-index) and SUA values are strongly associated with increased aortic stiffness, as assessed by carotid-femoral PWV. This relationship seems to be lost when the statistical model is corrected for possible confounding factors. In fact, at classical multiple regression analysis, only BP . The distribution of risk factors, but especially the time these risk factors had to exert their negative effect, varies with increasing age of children. It is very likely that the metabolic risk factors considered in the study, even if present very early in life, exert their negative effect to an increasing extent as the time since their onset increases. In the present study, age is significantly associated with higher PWV values in the multivariable regression model. To try to mitigate this drawback, we performed an age adjustment of the final mediation model. Age maintains its impact on PWV values, however, the main results of the study remain unchanged. Importantly, eGFR, which is strongly inversely related to SUA values, does not appear to have a direct effect on PWV levels, but only an indirect effect mediated by SUA, which, in turn, exerts its indirect effect on PWV through the HOMA-index. This result is probably because none of the children of the study had chronic kidney disease. Therefore, while the inverse relationship between eGFR and SUA is present even with normal renal function, the impact that a reduction in eGFR might have on the development of organ damage is determined by factors  that begin to act when renal damage is clinically evident.To better understand our results, and to find out whether the effects of any of the variables considered were mediated by those attributable to another variable, in addition to performing a classic uni- and multivariate analyses, we made use of mediation analysis. The mediation analysis is a method that can be applied to explain the causal paths underlying the association between the exposure and the outcome. If the mediators are on the causal path, then the exposure affects the mediator, which in turn affects the outcome. The total effect of exposure can pass both through the mediators (indirect effect) and through a direct pathway (direct effect). The mediation analysis makes it possible to identify the proportion of the direct and indirect effects of the exposure on the outcome. If the mediators fully mediate the effect from the exposure to the outcome, it is a full mediation case. Alternatively, situations where the mediators do not fully mediate the effect of the exposure on the outcome, represent partial mediation cases. In the present study the mediation analysis showed that both SUA and HOMA-index had a direct effect on SBP and DBP Our study has some limitations as well as several strengths. Among the first ones, other factors that we did not consider in this study may alter endothelial function  and may play an important role in determining vascular stiffness in this age group , 52. On In conclusion, the interactions between BP, IR and SUA on one side and vascular stiffness on the other side are extremely complex, and a statistical analysis performed with classical methods  is likely to miss this complexity. Our data suggest that SUA through the effects of IR and BP may cause structural remodeling of the macrovascular wall, which may further trigger arteriolar narrowing and increased large artery stiffness. The subclinical alterations of the vascular phenotype may play a key role in mediating the incidence of deleterious cardiovascular outcomes later in life. For this reason, PWV may be useful as a non-invasive tool to identify the presence of arterial stiffness in young people at increased cardiovascular risk. The presence of insulin resistance and hyperuricemia are not always considered important cardiovascular risk factors in children. Our study shows that these two metabolic factors are not only associated with the presence of HT in children and adolescents, but are also mediators of the onset of early organ damage, such as increased vascular stiffness, both directly (HOMA index) and indirectly (SUA). These findings should suggest the need for interventions aimed at reducing HOMA index and SUA values as early as pediatric age. Nevertheless, the results of our study contribute to the comprehension of the relationship between SUA levels, BP, IR and the occurrence of subclinical organ damage, without the chance to give indications about the threshold levels at which starting treatment strategies might be useful for primary prevention. It is therefore important to study potential preclinical mechanisms that lead to early disease pathogenesis to facilitate the identification of high-risk patients and thereby implement early therapeutic prevention strategies. Being able to analyze these phenomena in a pediatric population prior to the onset of clinical complications represents a very important because it allows to avoid some confounding factors such as vascular aging, drug interference, or smoking. Indeed, investigating changes observed in children offers a fascinating model for understanding the early pathophysiological mechanisms underlying HT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethic Committee Name: Istituto Auxologico Italiano, Approval Code: 2015102002, Approval Date: 20/11/2015. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.SG conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. IP, GL, AO, and MG collected data. LM and LA performed data analysis. GP, PS, and FV reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.Research funded by the Italian Ministry of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.