Plasmodium falciparum genetic diversity can add information on transmission intensity and can be used to track control and elimination interventions.P. falciparum malaria therapeutic efficacy trial in three malaria endemic sites in Ethiopia from October to December 2015, and November to December 2019. qPCR-confirmed infections were subject to amplicon sequencing of polymorphic markers ama1-D3, csp, cpp, cpmp, msp7. Genetic diversity, the proportion of multiclonal infections, multiplicity of infection, and population structure were analysed.Dried blood spots (DBS) were collected from patients who were recruited for a e across all markers was 0.730. Population structure was moderate; populations from Metema and Metehara 2015 were very similar to each other, but distinct from Wondogent 2015 and Metehara 2019.Among 198 samples selected for sequencing, data was obtained for 181 samples. Mean MOI was 1.38 (95% CI 1.24–1.53) and 17% (31/181) of infections were polyclonal. Mean HThe high level of parasite genetic diversity and moderate population structure in this study suggests frequent gene flow of parasites among sites. The results obtained can be used as a baseline for additional parasite genetic diversity and structure studies, aiding in the formulation of appropriate control strategies in Ethiopia.The online version contains supplementary material available at 10.1186/s12936-023-04814-w. Malaria caused an estimated 247 million cases and 619,000 deaths worldwide in 2022 .Plasmodium falciparum populations and multiplicity of infection (MOI) in humans might vary according to transmission intensity in different geographical regions [Genetic diversity of  regions –9. The s regions –14.Next-generation sequencing (NGS) tools are increasingly being applied to evaluate diverse markers in the parasite genome , 7–10. CAmplicon sequencing studies reveal population structure and genetic relatedness among parasite populations, which can be used to understand the spread of the parasite within a region and to evaluate the effectiveness of ongoing control efforts , 15–22.P. falciparum from northern, southern and eastern part of Ethiopia were genotyped by amplicon sequencing at 5 highly diverse markers to assess parasite diversity and malaria transmission in the country.In this study, The study obtained ethical approval from the Institutional Review Board (IRB) of College of Natural and Computational Sciences, Addis Ababa University, certificate reference number IRB/033/2018. Written informed consent/assent was obtained from participants or parents/guardians for minors. Malaria positive cases were treated as per the national treatment guidelines for malaria .Samples for this study were collected in three sites: Metema, Wondogent and Metehara. Metema is in northern Ethiopia, 925 km northwest of Addis Ababa, with altitudes 1608 m above sea level River include the Genda Wuha, is a notable mosquito habitat near the study area. Wondogent in southern Ethiopia is in the Rift Valley 261 km southern of Addis Ababa at an elevation of 1,723 m. It is surrounded by primary forests and water bodies which provide suitable habitats for mosquitos. Metehara is situated in eastern-central Ethiopia in the Rift Valley area 128 km southeast of Addis Ababa at an elevation of 947 m. Nearby rivers and an irrigated sugarcane farms provide breeding sites for malaria mosquitoes  were collected on Whatman 3 mm™ filter and stored in individual plastic bags containing desiccant.2 was cut from each DBS and DNA was extracted using the NucleoMag Blood kit (Macherey–Nagel). All samples were screened by qPCR for P. falciparum using a varATS assay, targeting a multicopy gene with approximately 20 copies per genome [P. falciparum culture strain was used as a positive control and to generate a standard curve for quantification. Primers and probes of qPCR are in Additional file A piece roughly 5 mmr genome . The 3D7Plasmodium falciparum-positive samples with Ct values < 30 based on the varATS assay were selected for parasite genotyping at 5 highly diverse markers: apical membrane antigen 1 , circumsporozoite surface protein , a conserved Plasmodium protein , a conserved Plasmodium membrane protein , and merozoite surface protein 7  [198 1335100) . Amplico1335100) . The libama1-D3 major clone sequences, the K80 nucleotide substitution model, and the neighbour-joining method [Haplotype calling and population analyses were conducted using R software version 4.2.2  and Haplg method . Principg method  and Bayeg method .e) was calculated using the formula SPSS  was used to conduct additional statistical analyses. MOI was determined by the maximum number of alleles observed at an individual locus and the average MOI was calculated for each sub-population. The Student’s t-test was used to assess the relationship between MOI, parasite density, and patient age, and the spearman’s rank correlation coefficient was used to see association between age and MOIs. A chi-squared test for independence was done to compare the proportion of polyclonal infections at each site. The expected heterozygosity  of samples  were collected in 2015 and 413 (62%) were collected in 2019. qPCR screening identified 280 (42%) samples positive for ama1-D3, csp, cpp, cpmp, and msp7 were successfully sequenced in 177 (89.3%), 179 (90.4%), 177 (89.3%), 176 (88.9%), and 158 (79.8%) of 198 samples, respectively. All five markers were sequenced in 150/198 (75.8%) samples. Between 11 (csp and msp7) and 23 (ama1-D3) different alleles per marker were observed  in Metema, 24.4% (10/41) in Wondogent, 32% (8/25) in Metehara in 2015, and 13.6% (6/44) in Metehara in 2019 and 0.716 for p = 0.48). No significant association between MOI and age or relative parasitaemia was observed (p > 0.05). Mean He across all sites was 0.730, indicating a moderate to high level of genetic diversity.Mean MOI across all markers was 1.38 Detailed monoclonal and polyclonal infections by marker are given in Table Bayesian clustering analysis of admixed indicated that the data best fit three genetic clusters. STRUCTURE analysis revealed moderate population structure among parasites. Parasites from Metema-2015 and Metehara-2015 likely share ancestry from a single genetic group. Parasites from Wondogent-2015 largely belong to a second ancestral group. Roughly 25% of parasites from Metehara-2019 share ancestry with the parasites from Wondogent, and the remaining parasites belong to a third ancestral group  [In this study, Hnd Ghana , 25, 31, in Asia . The He 7, 0.54) –35. This7, 0.54) –35.P. falciparum analysed in this study were genetically diverse and generally did not cluster by collection site. This is indicative of gene flow among the geographic locations sampled, likely due to immense human traffic through the country as part of routine socio-economic activities. Additionally, this study revealed evidence of a shift in parasite populations between 2015 and 2019 in Metehara. Between the two time points MOI, heterozygosity, and the proportion of polyclonal infections decreased, although the differences were not statistically significant. Structure analysis showed a shift from a diverse parasite population arising from three ancestral groups in 2015 to a more structured population comprising just two ancestral groups in 2019. Additionally, this decrease in genetic diversity may represent a decrease in malaria transmission or a reduction in gene flow to and from Metehara by 2019. In this study, there is a reduction of MOI similar to the study done in Indonesia 1.6 [The isolates of esia 1.6  and a stesia 1.6 . In thisesia 1.6 .The low to moderate level of genetic differentiation of parasites between the study sites is likely a consequence of immense human traffic as part of routine socio-economic activities. Further sampling of more populations along this North, South and East of Ethiopia including asymptomatic infections, will refine the boundaries of gene flow and inform the Ethiopia National Malaria Control Programme (NMCP) on local approaches to tackle malaria elimination.P. falciparum diversity and structure across malaria-endemic sites in Ethiopia. Overall, parasites were highly diverse, with little distinction by collection site. This is indicative of gene flow among parasite populations around the country. Further sampling of more populations along this North, South and East of Ethiopia including asymptomatic infections, will refine the boundaries of gene flow and inform the Ethiopia National Malaria Control Programme (NMCP) on local approaches to tackle malaria elimination. Therefore the study will help as baseline for further study.This study employed amplicon sequencing of five polymorphic markers to characterize Additional file 1: Table S1. Primers and probe used for qPCR assay.Additional file 2: Table S2. Detailed procedure used for deep amplicon sequencing of five highly polymorphic markers.


Emotional distress experienced by patients with diabetes (PWD) can affect glycemic control and quality of life. However, limited tools are available in Indonesia to detect emotional distress in PWD in clinical setting or research. This study aimed to evaluate the validity and reliability of the Indonesia version of the Problem Areas in Diabetes (PAID-5) scale.After the cross-cultural adaptation method was conducted, psychometric tests were done from August to November 2019 at affiliated hospitals in Yogyakarta by involving 100 adult PWD. All PWD with no medical records of mental health problems or cognitive disorders were voluntarily included. Content and construct validity and internal consistency measurements were used to evaluate the psychometric properties.The mean age was 61.2 years of the men and women who equally participated in the study and mostly were non-working patients. The PAID-5 resulted in five question items to identify the emotional distress of PWD in the Indonesian language. Some minor modifications were done in items four and five after discussing them with the original authors and experts in Indonesia. The results showed item content validity index for item and scale were 0.6-0.8 and 0.72, respectively. The calculated r-values ranged from 0.751 to 0.888, which were higher than the r table (0.197). The Cronbach alpha of the Indonesia version of PAID-5 was 0.87 with inter-item and item-total correlations of 0.43-0.71 and 0.61-0.79, respectively.The results of the study indicate that PAID-5 is considered valid and reliable to assess emotional distress among PWD and can be useful in clinical setting or for research purposes. Continued assessment of emotional distress is applicable and helps patients to better deal with their emotional distress. Diabetes is reported to be among the top 10 causes of death globally. In 2017, the number of patients with diabetes (PWD) in Indonesia was ranked 6th in the world with the prevalence of 10.3 million. In 2045, the estimated prevalence will increase to about 16.7 million.9A valid, reliable, and brief evaluation tool is required to assess emotional distress for PWD. The Problem Areas in Diabetes (PAID) is known as a specific instrument to measure emotional distress among patients with type-1 and type-2 diabetes, originally consisting of 20 items. It is applicable either in clinical settings or for research-based purposes.The emotional distress assessment is a necessary part of the comprehensive approach to treatment interventions during medical visits.Several versions of translated PAID-5 are available. The Korean and Norwegian versions exist with valid and reliable outcomes.All research processes were approved by the Medical and Health Research Ethics Committee . Each participant received a printed version with an explanation of the study as well as verbal instructions. Their involvement in the study was voluntary and they had the full right to withdraw from the study at any time. To maintain their privacy, no identification information was required and they were asked to choose the ideal meeting situation.A cross-sectional design was used for psychometric testing and data were collected in August to November 2019 at affiliated hospitals in Yogyakarta. Patients were included using the consecutive sampling technique based on daily registration lists. Considering the minimum 20 participants for each item in the questionnaire,11The cross-cultural adaptation of PAID-5 was done before the data collection stage. The PAID-5 was specifically developed to detect emotional distress in patients with type-1 and type-2 diabetes and is an existing valid instrument with a brief version. The PAID-5 consists of five items, which are rated on a 5-point Likert scale from 0-4 . All items are added together and the total score ≥ eight indicates emotional distress. The possible total score of PAID-5 ranges from 0 to 20. The greater obtained score indicates the greater experience of emotional distress.17As a part of content validity testing, the CVI for item (I-CVI) and scale (S-CVI) were calculated. In addition, the experts could give feedback about the questions which were discussed during the expert panel review with the authors. Following the step, a pilot testing of the pre-final version was tested on ten type-2 diabetes patients to see the feasibility of PAID-5. The study was continued through testing of the final version of PAID-5 to the eligible patients in several diabetes clinics at affiliated hospitals. This step was aimed to assess the construct validity and internal consistency. Nurses in charge assisted in the identification of potential participants using their list of diabetic patients through consecutive sampling technique. The medical records confirmed the criteria and the researchers approached the patients to explain the study to them during waiting for their routine visit. The full set of patient information Form and self-administered questionnaire was provided to each participant including demographic characteristics . The researchers accompanied the participants while they completed it then checked the questionnaire for completion. The completed questionnaire was stored in a closed envelope after self-completion by each of the participants.11Data analyses were done using Statistical Package for Social Science-SPSS version 22 . The univariate analysis was used to summarize the participant characteristics such as age, sex, level of education, employment status, income status, and marital status. Moreover, the Polit and Back model was used to evaluate the I-CVI and S-CVI. The criterion of an adequate item of CVI was more than or equal to 0.78 and more than or equal to 0.80 for subscale of CVI.18A total of 100 patients with type-2 diabetes participated in the study (response rate 100%). The mean age of the participants was 61.2 years (standard deviation = 9.6 year), ranging from 39-82 years. Men and women had equal distribution by chance (50%), with one-third of them having a diploma/bachelor degree (37%), and who mostly were non-working/retired 74%, .The results of the pilot testing on 10 participants showed 1 of the 5 items (item five) was difficult to understand, while 80% of participants declared that the instrument was easy to understand. In total, they required about one minute and four seconds to complete it.An expert panel evaluated the content validity of PAID-5. The clarity, readability and equivalence of the PAID-5 were 84%, 84%, and 88%, respectively, indicating a promising outcome. The I-CVI ranged between 0.60 and 0.80. The S-CVI score was 0.72. Two items (items 4 and 5) had I-CVI below 0.70. Accordingly, some modifications were done after discussing the original content of the items together with the research team and the experts. One word in item 4, “power” was modified into “energy” (synonym). For item 5, the statement of “facing complication of diabetes” was modified to “ability to face complications of diabetes” (making it clearer and straightforward). This change corresponded with the pilot result in which participants had some difficulty to understand the content of item 5.The construct validity analysis used a comparison between r-value and r-table. The R-values ranged between 0.751-0.888 , which indicated that all of the items on PAID-5 were valid. Coefficient correlation (r) was categorized as 0.9-1.0 indicating very highly correlated, and 0.7-0.9 indicating highly correlated.16The score of Cronbach’s alphas of PAID-5 was 0.87, showing a highly reliable outcome. This score indicated that the PAID-5 was consistent in measuring emotional distress.To the best of our knowledge, this paper provided the first information about the validity and reliability of the PAID-5 Indonesian version. This evidence supports the early detection of emotional distress among PWD, even as early as pre-school children.The study provides information about the validity and reliability of PAID-5. The results ensure that the PAID-5 is applicable to assess and evaluate the emotional distress among PWD. The tool is brief and requires short time (one-two minutes) thus it is useful for clinical setting where there is limited available time. By detecting the emotional distress among PWD, clinicians can do early treatment and management before the patient develops more serious problems.The study was conducted in a tertiary clinic that receives referral patients from several hospitals to enlist more diabetic patients who are experiencing emotional distress. However, they only covered type-2 diabetes patients. The original version of PAID-5 is also applicable for type-1 diabetes. Further analysis and study for type-1 and gestational diabetes may be required to see the similar evidence of psychometric properties.The PAID-5, an Indonesia Version of PAID-5, can be considered as a valid and reliable instrument to measure emotional distress among patient with diabetes. The results of this psychometric testing confirmed the validity and reliability of the PAID-5 Indonesian version in identifying the emotional distress among patients with diabetes. Therefore, this tool may be applicable in terms of research or clinical purposes.


In the multivariate regression model age, blood pressure, nocturnal indices and FPG were shown as independent predictors of PWV values. Conclusion: Significantly higher PWV values were found in subjects with prediabetes and non-dipping profiles in all three examined hypertension groups.Background: Pulse wave velocity (PWV) is a known predictor of target organ damage, cardiovascular disease and overall mortality. The aim of this study was to compare the PWV values in subjects with prediabetes, a non-dipper profile and arterial hypertension with their values in healthy subjects. Methods: A total of 301 subjects, aged 40–70 years, without diabetes mellitus were included in this cross-sectional study (150 with prediabetes). They underwent a 24 h ambulatory blood pressure monitoring (ABPM). Subjects were divided into three hypertension groups . Dipping status was determined according to ABPM results, and PWV was measured by an oscillometric device. Prediabetes was defined as having 2 separate fasting plasma glucose (FPG) measurements between 5.6 and 6.9 mmol/L. Results: The highest PWV values were found in group C (9.60 ± 1.34 vs. 8.46 ± 1.01 in group B vs. 7.79 ± 1.10 in group A;  To have PreD, it is necessary to meet one or more set of criteria in at least two separate laboratory measurements : fasting plasma glucose (FPG) 5.6–6.9 mmol/L, or HbA1c 5.7–6.4%, or blood glucose values by oral glucose load test 75 g glucose (OGTT) after 2 h 7.8–11.0 mmol/L [PreD is associated with a 20% higher risk of cardiovascular disease (CVD), especially carotid atherosclerosis and coronary artery calcification, while the association declines with aging, probably as a result of the influence of other risk factors ,4,5. AltMicro- and macrovascular damage are associated with PreD and are presented by greater arterial stiffness and faster progression of atherosclerosis. The gold standard for determining arterial stiffness is pulse wave velocity (PWV) using tonometry or oscillometric devices; arterial stiffness is a known predictor of target organ damage (TOD), CVD and overall mortality ,7,8. An The absence of a drop in BP at night (non-dipping profile) is also a significant risk factor in the development of CVD, and it is known that an increase in systolic blood pressure (SBP) of 10 mmHg at night increases the risk of death by 21% . Non-dipHypertensive crisis is defined as a SBP above 180 mmHg and/or diastolic blood pressure (DBP) above 120 mmHg, which includes uncontrolled AH  and hypertensive emergency (with acute TOD) ,18,19. IPreD and non-dipping profiles are significantly underestimated in clinical practice, leaving many patients with high cardiovascular risk and already existing TOD without adequate and timely therapy, especially if they have normal BP values ,22. PWV The aim of this study is to compare the PWV values in subjects with PreD, non-dipper profiles and arterial hypertension with their values in healthy subjects. We hypothesize that higher FPG values, higher BP levels and a non-dipper profile are associated with higher PWV values and higher CV risk, i.e., TOD. This cross-sectional study was conducted in four family medicine practices (FMP) of the Health Center Zagreb-West (groups A and B) and at the Emergency department of the Clinical Hospital Merkur (group C) in Zagreb, Croatia from January 2019 to December 2022. We completed an a priori power analysis and the power analysis resulted in 251; however, we recruited 301 Caucasian subjects (153 women and 148 men). Information about a patient’s medical history and used medication was collected from the electronic medical record  in Clinical Hospital Merkur). Subjects were divided into three groups using the 24 h ambulatory blood pressure monitoring (ABPM) findings.Group A —subjects were selected by simple random sampling from the list of insured persons in four FMPs without a diagnosis of AH (ICD-10: I10-I15) and with average BP values lower than 130/80 mmHg measured by ABPM.Group B (patients with AH without HU)—subjects from four FMPs with a diagnosis of AH (ICD-10: I10-I15) confirmed by ABPM without measured BP values higher than 180 mmHg and/or 120 mmHg in more than five individual measurements and data about HU in the medical history. AH is defined by exceeding the average of all measured BP values above 130 and/or 80 mmHg by APBM and/or taking at least one antihypertensive drug ,22.Group C (patients with HU)—was formed by simple random sampling of patients from the database of patients from Clinical Hospital Merkur who sought emergency medical help due to high BP values. BP values upon arrival at the institution were measured after five minutes of quiet sitting with a one-minute interval between each measurement (three times), and ABPM was set upon discharge. Only those patients whose BP values were measured by a correctly performed ABPM of more than 180 and/or 120 mmHg in more than five individual measurements for 24 h were included.2, atrial fibrillation, diabetes mellitus, chronic kidney disease G3b, G4 and G5, pregnancy, immunosuppressive, corticosteroid and/or biological therapy, chemotherapy regime in the last five years and expected life expectancy less than six months.Exclusion criteria included hypertensive emergency, CV event in personal medical history, incomplete data, age below 40 and above 70 years, BMI > 50 kg/m®  device using an adequate cuff placed on the lower half of the subject’s upper arm determined by the circumference of the middle part of the distal half of the upper arm . All subjects were divided into three groups according to the percentage change in night BP value  [A session of 24 h ABPM was performed on a special schedule  using the BTL Cardiopointper <0%) ,24. In cper <0%) .® B900  oscillometric device at a room temperature of 20–25 °C with a suitable cuff in the same previously described manner as in the performance of ABPM. A total of 2 measurements were taken according to specific schedules with subjects (working days between 2 and 6 pm), between which there was a break of 30 s. At the first measurement, brachial SBP and DBP were determined, and at the second, PWV, central SBP (cSBP), central DBP (cDBP), pulse pressure (PP) and AIx. In case of imprecise measurement, the device reports an error, and the procedure was repeated within five minutes of the first measurement.PWV was measured with an AgedioParticipants were recruited during their regular provider visits and then scheduled for ABPM, PWV measurements (within ten working days) and a blood draw. A blood draw was performed during the visit (emergency laboratory parameters for group C) or/and the morning after at 7 to 8.30 am (for groups A and B and other parameters for group C). Biochemical parameters in the examinee’s serum were determined by sampling 2 tubes of 8–10 mL of venous blood in the emergency hospital laboratory: the first was to determine hematocrit value, the concentration of creatinine, sodium ions, potassium ions and glucose, while the second was for triglycerides, HDL, LDL, total cholesterol and urate (since they could not be performed in an emergency laboratory). For other subjects, the same laboratory parameters were determined through the primary health care laboratory (including lipid status and urate concentration), and the fasting glucose value was repeated after two weeks. Acute organ damage in the case of HU was ruled out by standard biochemical and radiological examinations of the clinic  and ECG. It includes acute kidney damage, ischemic or hemorrhagic stroke, acute coronary syndrome, acute pulmonary edema, acute left heart failure, hypertensive encephalopathy and aortic dissection. The glomerular filtration rate was assessed using CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula . ® ) and had their height and waist circumference measured with a tailor’s tape measure. Prediabetes was defined according to the 2017 ADA criteria [On the first visit, chronic antihypertensive and hypolipemic therapy was also recorded. Due to the possible effect on the measurements, the medications were not changed until all procedures were completed (if needed). Additionally, all participants were weighed in the office  and the Ethics Committee of the Merkur Clinical Hospital (Number 03/1-6063). All participants signed an informed consent to participate in the research.The sample size was calculated according to the expected arithmetic means of the PWV values and their standard deviations. Power analysis was performed a priori for ANOVA for special effects and interactions. With significance level α = 0.05, effect size (f) = 0.25, and a total of 251 respondents with equally large groups, ANOVA has a test power of β = 95% to recognize differences in two dependent variables between the three observed groups with one predictor . Finallyt-test, or Mann-Whitney U test, and categorical variables with Pearson’s χ2 test. ANOVA was used in the analysis of dependent variables. In post hoc analysis, we performed Tukey’s HSD test to check the differences between the groups. Variables that showed a statistically significant Pearson correlation with PWV were included in the linear regression model. The final linear regression model included PWV as a dependent variable and age, SBP, DBP, heart rate, nocturnal indices, FPG, and GFR as independent variables. A value of p < 0.05 is considered statistically significant. The program used for statistical analysis is Statistica v.12.0.The normality of the data distribution was checked with the Kolmogorov-Smirnov test. Continuous variables (depending on the distribution) were analyzed with Student’s p < 0.001), had higher PWV  and AIx values , but lower SBP , DBP , triglycerides , uric acid  and BMI values (27.24 vs. 28.45 kg/m2). Differences in FPG  and other variables between sexes were not found. In the studied groups, women were significantly older than men  compared to the other groups  and a significantly higher number of non-dippers  compared to other groups . Other differences are shown in column A in The division of subjects according to the results of ABPM resulted in 77 subjects in group A, 138 in group B and 86 in group C. Group C had a significantly higher number of patients with PreD . The highest PWV values were measured in all inverse dippers , especially in group C (10.12 m/s). All differences between the examined dipping profiles and hypertension groups are shown in All dippers had the lowest average PWV values (8.23 m/s), followed by non-dippers with higher average PWV values . Although statistically insignificant, PWV values were slightly higher in non-dippers without PreD than in dippers with PreD (p = 0.370). In PWV analysis among hypertension groups, subjects with PreD and a non-dipper profile had the highest values of all other group subjects without noticeable differences between genders  in which as many as 35% of them had PreD and 28% were non-dippers since their PWV values were even higher than in patients with well-controlled AH (group B) in our study. These subjects are often neglected in clinical practice because they seem to have a negligible CV risk, according to SCORE2 charts .If we consider that higher PWV equals higher CV risk (TOD), then patients with PreD and non-dipping profiles in all hypertensive groups had the highest CV risk than all other subjects, which increases with aging ,30,31. KBesides increased FPG, glycemic variability (GV) is associated with accelerated atherosclerosis even in non-diabetics, independently of conventional risk factors . A studyA non-dipping profile is associated with impaired modulation of vascular smooth muscle tone during the night, increased PWV and CV risk ,44. The A great problem of everyday clinical practice is the underutilization of CVD prevention. ESH-ESC Guidelines recommend using the SCORE2 model as a minimum requirement. However, TOD is not included in it, making the CV risk stratification imprecise . For exaThe strength of this study is that PWV was also measured in healthy subjects included by strict criteria, always at a similar time of day (2–6 pm) with little differences in air temperature (20–25 °C), which can be extremely important for the accuracy of the measurement .This study has several limitations. For defining PreD, we used average FPG from two measurements. HbA1c and values of albuminuria (an important parameter of TOD) could not be obtained due to practical reasons. In a small number of patients, the ABPM and PWV measurements were not performed on the same day due to the oscillometric device’s unavailability. The number of patients with an inverse dipping profile in different groups  was too small  for good analysis using ANOVA. Accordingly, inverse dippers were not included in the ANOVA analysis.Our findings showed significantly higher PWV values in subjects with PreD and non-dipping profile in all three examined hypertension groups, meaning these subjects already have higher CV risk and a greater probability of TOD. This study highlights the relevance of BP evaluation with ABPM in all subjects with PreD and the timely introduction of lifestyle changes and medication when needed. Future studies should assess the impact of lifestyle and medication interventions on PWV values in otherwise healthy subjects with PreD.


Plasmodium falciparum could play a role in preventing clinical disease. BK-SE36 is a promising malaria vaccine candidate that has shown a good safety profile and immunological responses during field evaluations. It was observed that repeated natural infections could result in immune tolerance against SE36 molecule.A vaccine targeting the erythrocyte stages of Plasmodium falciparum infection by microscopy. Blood samples were further collected one month post each vaccination to evaluate immunogenicity.The primary trial was conducted to assess the safety and immunogenicity of the BK-SE36 in two cohorts of children aged 25-60 months (Cohort 1) and 12-24 months (Cohort 2). Immunization was at full dose (1.0 mL) administered at 0, 1, and 6 months. Blood samples were collected before each vaccination for immunological assessments and detection of vs. 92.8 (95% CI: 34.9-246.6), p = 0.002. There was a 14.3 (95% CI: 9.7-21.1) and 2.4 (95% CI: 1.3-4.4) fold-change, respectively, in uninfected and infected participants between one-month post Dose 2 and booster. The difference was statistically significant (p < 0.001).Of seventy-two (72) subjects that have received BK-SE36 vaccination, 71 had available blood smears during vaccination days. One month post Dose 2, the geometric mean of SE36 antibodies was 263.2 (95% CI: 178.9-387.1) in uninfected individuals compared to 77.1 (95% CI: 47.3-125.7) in infected participants. The same trend was observed one-month post booster dose. Participants uninfected at the time of booster vaccination had significantly higher GMTs compared to those who were infected (424.1 (95% CI: 301.9-595.8) P. falciparum during BK-SE36 vaccine candidate administration is associated with reduced humoral responses. However, it is to be noted that the BK-SE36 primary trial was not designed to investigate the influence of concomitant infection on vaccine-induced immune response and should be interpreted cautiously.Concomitant infection by WHO ICTRP, PACTR201411000934120. Malaria due to P. falciparum is the deadliest form and predominates in Africa.Malaria is a huge global public health problem with around 40% of the world’s population at risk of infection. In 2020, there were approximately 241 million clinical episodes of malaria and more than 627,000 deaths  was selected for clinical development based on (a) epidemiological studies that show correlations of high antibody titers and malaria symptoms and severe disease –13; (b) hibition –17; and nfection , 17, 18.nfection  with limnfection . SE36 wanfection .Phase I safety and immunogenicity trials of BK-SE36 were conducted in healthy, malaria-naïve Japanese adults , in malaP. falciparum infection had interfered with the immune responses to the BK-SE36 malaria vaccine candidate in line with previously reported malaria-induced immunosuppression . The inclusion of a third dose at Week 26  was intended to increase the immune response and evaluate the effect of a booster dose. The first and second vaccine doses were given during the rainy season ; the third dose during the dry (low transmission) season. In Cohort 1, the primary doses (28 days apart) were given in July-Aug 2015 and the booster dose (6 months from the first dose) given January 2016. For Cohort 2, primary vaccinations were given Oct-Nov 2015, and the booster on April 2016.The full study methodology as well as the trial protocol were published elsewhere . In brieia cases , 26. CoiAnti-SE36 IgG antibody titers were measured by ELISA before vaccination , 4 weeks after each vaccination , and at D365 and D477. ELISA was outsourced to a GLP certified testing facility  and used a standardized methodology. Results were expressed in titers calculated using an equilibrium line assay (Geometric mean titers (GMTs) were calculated at each time point. IgG1 and IgG3 subclasses were determined  for thosP. falciparum, P. malariae or P. ovale), and counts for P. falciparum were made for both sexual and asexual parasites. The parasite presence and density were determined independently by two readers for the same slide; if readings were judged to be discordant, a third independent read was organized. The parasite density (parasites/μL) was calculated as the geometric mean of the two positive readings . Microscopist competency was evaluated through two external quality control (EQC) programs. The first EQC was carried out by the College of American Pathology; proficiency testing with a set of 5 slides provided to each microscopist for reading thrice a year. Only a performance graded above 80% was considered satisfactory for the laboratory. The second EQC was performed by WHO  and involved the reading of a set of 20 slides every quarter by each microscopist. Only those with a score of at least 80%, graded as ‘competent’, were tasked in the reading of trial participants’ slides.Prior to each vaccination, capillary blood was also collected to prepare malaria smears. The presence of parasites was assessed by 100 x bright field microscopic examination, assuming 8,000 leukocytes/µl of blood. The count was made by species  includes subjects who received Dose 1 and 2 (termed “primary immunization”) and had a blood smear taken at these two occasions with results available. The subgroup considered subjects who received 2 doses since the interest is to see the effect of infection on the full vaccination status; however, the difference in GMTs between infected and non-infected subjects after one dose is provided in P. falciparum count are presented in terms of the geometric mean with 95% confidence interval. Because SE36 antibody titers are not normally distributed , we use the Wilcoxon rank sum test. All the comparisons are made between infected and uninfected groups. A p-value of 0.05 was the level of statistical significance.Antibody titers and www.stata.com) and GraphPad (Prism ver. 9.4.0).Statistical analyses were performed using Stata ver. 14  Good Clinical Practice (GCP) guidelines. The clinical trial protocol and associated documents were reviewed and approved in Burkina Faso by CNRFP institutional bioethics committee , the Ministry of Health Ethical Committee for Biomedical Research (Ref: 2014-12-144) and the National Regulatory Authority . The trial package was also approved by the Scientific Committee/Institutional Review Committee of the Research Institute for Microbial Diseases (Ref: 26-6), Osaka University (Ref: 574) in Japan; and the London School of Hygiene and Tropical Medicine Research Ethics Committee (Ref: 9175) in United Kingdom.33.1The vaccination of Cohort 1 started on July 4, 2015 (Dose 1), and the last vaccination (Dose 3) was completed by January 11, 2016. Vaccination of Cohort 2 began on October 12, 2015 (Dose 1) and was completed by April 18, 2016 (Dose 3). In total 72 participants received 2 primary doses of BK-SE36 , howeverThe median age was 25.4 (IQR 28.2) months, and 38.9% of subjects were male.3.2P. falciparum infection at the time of primary immunizations is shown in P. malariae, P. vivax and P. ovale infections.The proportion of individuals carrying p = 0.002).For subgroup 1, 64.8% (46/71) participants were free of infection at both Dose 1 and 2. At either dose, 35.2% (25/71) were harboring malaria parasites. The mean age was 39.6 ( ± 14.9) and 27.9 ( ± 14.6) months in infected and uninfected children, respectively. The difference in the proportion of children infected during vaccination days was statistically significant  of the vaccinees at the time of the booster vaccination. The mean age was 45.2 ( ± 14.6) and 29.5 ( ± 14.9) months in infected and uninfected children, respectively. The difference was statistically significant (p = 0.001).In subgroup 2, no P. falciparum infection (subgroup 3). The mean age was 39.7 ( ± 15.8) and 26.7 ( ± 13.8) months in infected and uninfected children, respectively. Again, the difference was statistically significant (p < 0.001).In total, 58.8% (40/68) receive their primary and booster doses in the absence of any P. falciparum parasites density was 1077.6 trophozoites/µL , 1811.4 trophozoites/µL  and 2359.9 trophozoites/µL  respectively at Dose 1, 2 and 3. Individual P. falciparum count is provided in The geometric mean of 3.3p = 0.877). One-month post-primary vaccinations (after Dose 2), the geometric mean of SE36 antibodies was 263.2  in uninfected individuals compared to 77.1  in infected participants. The difference was statistically significant (p < 0.001). In terms of fold change for each subject, there was a 2.2  and 1.6  fold change respectively in uninfected and infected participants from baseline to one-month post Dose 2. The difference, however, was not statistically significant  (p = 0.002). There was a 14.3  and 2.4  fold change respectively in uninfected and infected participants between one-month post Dose 2 and Dose 3. The difference was statistically significant , the GMT was 34.6  in uninfected individuals compared to 25.4  in infected participants. The difference was not statistically significant  compared to those who were found with P. falciparum infection at some point in time during vaccination days . The difference between uninfected and infected subjects was statistically significant (p < 0.001). This difference was maintained towards the study end with GMTs of 101.0  vs 27.21  (p = 0.006) at visit 32 (D365); and 96.7  vs. 25.6 , (p = 0.001) at visit 36 (D477) (uninfected vs infected subjects). A subgroup analysis by cohort is provided in Study participants who did not have any microscopically detectable Anti-SE36 IgG antibody subclasses levels in uninfected and infected children are presented in 4In this study, we report the immune responses post-vaccination with BK-SE36 blood-stage malaria vaccine candidate in 12-60 months old children in Burkina Faso. The study findings suggest that concurrent blood-stage infection during immunization/vaccination days has reduced the magnitude of the immune responses to the vaccine. This low response to vaccination in infected individuals has also been reported for pre-erythrocyte vaccine candidates including PfSPZ  and ME-TP. falciparum malaria is characterized by an important immunoregulatory blood transcriptional signature with the upregulation of various pathways involved in the inhibition of CD4+T-cell function  and longevity of antibodies are also important , 42. AntP. falciparum density, respectively for subgroup 1, 2 and 3. Whether clearing the infections before immunization could mitigate this immunosuppression also needs further studies.Of note, a multivariate analysis showed, as expected, a negative correlation between parasite density and anti-SE36 antibody GMT. Adjusted for baseline antibody titer, age, and the interaction between age and baseline antibody response there was a 1.93%, 133.9% and 11.3% decrease in antibody GMT for each 1000 parasites/μl increase in P. falciparum infection was only defined based on microscopy. Owing to the detection limit of optical microscopy (~ 100 parasites/µL), we could not rule out the possibility that submicroscopic infections were missed , the Ministry of Health Ethical Committee for Biomedical Research (Ref: 2014-12-144) and the National Regulatory Authority ; Japan: the Scientific Committee/Institutional Review Committee of the Research Institute for Microbial Diseases (Ref: 26-6), Osaka University (Ref: 574); and United Kingdom: London School of Hygiene and Tropical Medicine Research Ethics Committee (Ref: 9175). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SS, EB, AT, IN, SH, FD, NP, and TH contributed to the conception and design of the study. AT, EB, IN, AO, SS were responsible for study implementation at the study site. AT performed the statistical analysis. NA and NP for materials/reagents/assay development and analysis tools. AT and NP wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.


The role of cytokines such as interleukin-5 (IL-5) in the pathogenesis of malaria remains unclear. This systematic review sought to synthesize variations in IL-5 levels between severe and uncomplicated malaria, as well as between malaria and controls not afflicted with the disease.This systematic review was registered at the International Prospective Register of Systematic Reviews . Searches for studies that reported IL-5 levels in patients with malaria (any severity) and/or uninfected individuals were performed in Web of Science, PubMed, EMBASE, Scopus, CENTRAL, and MEDLINE, between 1st and 10th October, 2022. The risk of bias among all included studies was minimized using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting observational studies. The differences in IL-5 levels between malaria and uninfected controls, and between severe and uncomplicated malaria were synthesized by narrative synthesis.Among 1177 articles identified in the databases, 23 matched the eligibility criteria and were included in this systematic review. Qualitative syntheses showed the heterogeneity of IL-5 levels between different severities of clinical malaria and uninfected controls. The majority of the included studies  found no change in IL-5 levels between malaria cases and uninfected controls. Similarly, most studies found no difference in IL-5 levels between severe (regardless of complications) and uncomplicated malaria . The qualitative syntheses revealed that most studies found no difference in IL-5 levels between severe and non-severe malaria.The comprehensive review suggests that IL-5 levels are unchanged in patients with different levels of clinical severity of malaria and uninfected controls. Given the limited number of published studies on IL-5 levels in malaria, there is a need for additional research to determine the function of this cytokine in the pathogenesis of malaria.The online version contains supplementary material available at 10.1186/s12936-023-04659-3. Plasmodium parasites: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. These parasites are transmitted by the bite of Anopheles spp. mosquitoes [P. falciparum, but other Plasmodium species can also cause severe disease, albeit at lower rates [Plasmodium species, plays a crucial role in the underlying mechanisms of illness and the pathogenesis of malaria [Malaria in humans is caused by five species of squitoes , 2. Seveer rates –6. Cytok malaria , 8. Pro- malaria –15.c-Myc, c-Fos, c-Jun, Cis, Cish1/Jab, and pim-1 genes in B cells, which induce cell proliferation and have anti-apoptotic effects [Interleukin-5 (IL-5) was first discovered as an interdigitating homodimeric glycoprotein because of its ability to promote the in vitro growth and differentiation of mouse B cells and eosinophils . Eosinop effects , 19. In  effects , 21. In  effects . Further effects . Previou effects –26, the  effects , or the  effects . Given tThis systematic review was registered with the International Prospective Register of Systematic Reviews . This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocol (PRISMA 2020 Checklist).Searches were conducted on six databases, namely, Web of Science, PubMed, EMBASE, Scopus, CENTRAL, and MEDLINE, between 1st and 10th October, 2022. The search strategy was constructed using the keywords “malaria” and “interleukin 5.” A search for synonyms of both keywords was performed using the MeSH terms in the National Center for Biotechnology Information database. The searches were not limited to a particular language or publication date. The details of the search strategy for each database are given in Additional file After removing duplicates, two review authors (MK and KUK) independently screened the title and abstract of all of the articles. Then, the full texts of selected studies were examined by the same two authors based on the eligibility criteria. Reasons for the exclusion of any studies were recorded. Any disagreements between the two reviewers were resolved by discussion until a consensus was reached. A third review author (AM) also reviewed and validated the final list of included studies. Two review authors (MK and KUK) independently extracted data from the included studies using a data extraction sheet. Information extracted included name of the first author, study setting (country), year of publication, study period, study design, participant characteristics , IL-5 levels in malaria cases of different severity and uninfected controls, parasite density, method of malaria diagnosis, method of IL-5 quantification, and company/brand of IL-5 reagent. After resolving any disagreements, the extracted data were compared and finalized by the third review author (AM).Two review authors (MK and TD) assessed the risk of bias among all included studies using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for reporting observational studies . This toThe main outcomes of the systematic review included (i) the difference in IL-5 levels between malaria and uninfected controls, and (ii) the difference in IL-5 levels between severe and uncomplicated malaria. Outcome data were synthesized by narrative synthesis as a few studies reported quantitative levels of IL-5 among groups of participants. The significance of difference in IL-5 levels among groups of participants was indicated in the included reports. The percentage of data was calculated using Microsoft Excel 2019 (Version 16.0) .A total of 1177 articles were identified from Embase (n = 700), Scopus (n = 190), Web of Science (n = 107), MEDLINE (n = 89), PubMed (n = 82), and CENTRAL (n = 9). After the removal of duplicate records (n = 442), the remaining articles were screened for titles and abstracts (n = 735). After irrelevant records were excluded (n = 594), the remaining articles were sought for retrieval (n = 141), and were assessed for eligibility (n = 124). Twenty-three articles that met the eligibility criteria were included in the systematic review. Meanwhile, 101 articles were excluded for the following reasons: in vitro studies (n = 19), no data on IL-5 (n = 54), gene expression/polymorphism studies (n = 11), review articles (n = 5), in vivo studies (n = 3), inability to extract IL-5 data (n = 2), not participants of interest (n = 2), poster/news (n = 2), study using the same groups of participants (n = 2), and IL-5 measurement was performed after treatment of malaria (n = 1) , followed by those with P. vivax infection (17.4%). Children (43.5%) made up the majority of the study participants in the included studies, followed by adults (21.7%), pregnant women (17.4%), and both children and adults (17.4%). Most of the included studies used microscopy (69.6%) to detect malaria parasites. The majority of the included studies used plasma (65.2%) and bead-based assays (78.3%) for the quantification of IL-5 levels  between 2011 and 2022. The largest segment of the included studies employed a cross-sectional design, accounting for 30.4% of the total. This was followed by prospective observational and prospective cohort studies, each comprising 26.1%, and finally case–control studies, which made up 17.4% of the total study designs. The majority of included studies (65.2%) were carried out in Africa, followed by Asia, America, and then Europe . One study was carried out in five different countries  . PatientAmong the cross-sectional studies, six were of high quality , 30–34, Fifteen studies reported IL-5 levels in both malaria and uninfected controls , 45, 46.Eight studies reported IL-5 levels in severe malaria cases , 37, 42.IL-5 in malaria co-infections was demonstrated by four studies , 43, 44.P. falciparum, except for the studies by Dobaño et al. [P. vivax malaria. These two studies were conducted in overlapping regions, but they enrolled different participants. Specifically, while Dobaño et al. [Plasmodium spp. or the method used for IL-5 measurement. In terms of the methods used to determine cytokine levels, Young et al. suggested that ELISA and bead-based assay yielded similar results for rat IFN-γ, TNF, and IL-6. ELISA was found to be more sensitive in the low range of the standard curve, whereas the bead assay could detect higher protein concentrations [Cytokines have a systemic effect during the immune response of the host to malaria and are gaining popularity in the development of point-of-care diagnostics, not only for malaria but also for other inflammation-mediated diseases , 48. In o et al.  and da Co et al. , which eo et al.  enrolledo et al.  enrolledo et al. , 43, 46.o et al. , 39, 45.o et al. , 35, 38.trations . Additiotrations . Neverthtrations , 43, 46.trations , 38, 45.P. falciparum infection; as such, there was no heterogeneity of Plasmodium spp. on the outcome of the synthesis analysis herein. Heterogeneity of IL-5 levels between severe malaria and uncomplicated malaria was observed with respect to geographical area. In Africa, IL-5 levels were either similar or higher in severe malaria cases relative to the levels in uncomplicated malaria [The qualitative syntheses revealed that most studies found no difference in IL-5 levels between severe and non-severe malaria cases –26. None malaria , 36, 37. malaria , 25, 42. malaria –25 or hi malaria , 37 IL-5This systematic review had some limitations. First, there were few studies investigating IL-5 in malaria, so the results of this review were limited. Second, the meta-analysis to pool the mean difference in IL-5 levels among severe malaria, uncomplicated malaria, and uninfected controls could not be performed due to the few studies reporting quantitative data for IL-5 levels between groups of participants.This comprehensive review revealed that IL-5 levels did not differ in patients with malaria of various levels of clinical severity and uninfected controls based on the included studies. In the literature, there is only a small number of studies investigating IL-5 levels in malaria; thus, further research is required to determine how this cytokine contributes to the disease’s pathophysiology.Additional file 1: Table S1. Search terms.Additional file 2: Table S2. Details of the included studies.Additional file 3: Table S3. Quality of the included studies. PRISMA abstract checklist. PRISMA 2020 checklist.


Acute febrile syndrome is a frequent reason for medical consultations in tropical and subtropical countries where the cause could have an infectious origin. Malaria and dengue are the primary etiologies in Colombia. As such, constant epidemiological surveillance and new diagnostic tools are required to identify the causative agents. A descriptive cross-sectional study was conducted to evaluate the circulation and differential diagnosis of six pathogens in two regions of Colombia. The results obtained via multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR-ELISA) were comparable to those obtained using rapid tests conducted at the time of patient enrollment. Of 155 patients evaluated, 25 (16.1%) and 16 (10.3%) were positive for malaria and dengue, respectively; no samples were positive for any of the other infectious agents tested. In most cases, m-RT-PCR-ELISA confirmed the results previously obtained through rapid testing. Acute febrile syndrome (AFS) is defined as a morbid state with sudden onset of fever that evolves in less than seven days in patients between 5 and 65 years of age in whom no specific signs or symptoms of infection are identified . AFS is Epidemiological surveillance and diagnostic searches have focused upon a few causative agents, e.g., malaria, for which diagnosis is more readily accessible. It is estimated that in some malaria-endemic regions, 90% of patients with AFS receive antimalarial treatment based upon a presumptive diagnosis, although only 20 to 40% are confirmed cases . As suchIn Choco and the Uraba region of Antioquia, conditions are favorable for the transmission of viral infectious agents that cause both vector-borne and non-vector-borne febrile illnesses. Like other regions of the country, the availability of diagnostic tests for etiologic agents other than malaria is limited .Most infectious diseases that cause AFS are associated with socioeconomic factors such as inequality and poverty in areas far away from population centers. These factors hinder access to healthcare services, timely diagnosis, and adequate treatment . In addiThere are regions in Colombia that are simultaneously endemic for diseases like malaria and circulating arboviruses, e.g., dengue (DENV), chikungunya, and Zika. The latter exhibit endemic–epidemic patterns, as well as ecological and epidemiological characteristics similar to other circulating arboviruses that have been described in America, but not in the national territory. The prevalence of these infections has not been studied to any degree using precise diagnostic methods, e.g., molecular biology ,9.The symptomatology of AFS is usually non-specific, impeding clinical identification of the causative agent. The most common symptoms in addition to fever are myalgia, chills, asthenia, and arthralgia; symptoms such as headache and abdominal pain, however, can also present at the time of anamnesis . The simAccording to the Columbian Clinical Practice Guidelines, Diagnosis and Treatment of Malaria, published in 2022, malaria should be diagnosed via microscopy  or using rapid diagnostic tests. Molecular tests are not indicated for the clinical diagnosis of malaria; however, they are recommended for the confirmation of cases  where microscopy can underestimate and the sensitivity of rapid tests can be low (~75%) .In the case of dengue, rapid tests are only indicated for clinical diagnosis and should be performed within the first five days after symptom onset. The only tests accepted as confirmatory, however, are DENV-specific IgM antibody tests performed after the fifth day, or virus-specific IgG tests performed 15 days after the onset of symptoms . Virus-sRT-PCR molecular tests are recommended for the clinical diagnosis of Zika, chikungunya, yellow fever, and West Nile viruses ,15,16. SIn recent years, diagnostic methods that allow the simultaneous detection of several pathogens have shown great potential, especially in tropical and subtropical areas where co-infections are common . The mulIn Choco, 23,544 cases of malaria and 888 cases of dengue were reported in 2022; no cases of Zika, chikungunya, yellow fever, or West Nile virus infection were reported. In Antioquia, 7180 cases of malaria, 2229 cases of dengue, 3 cases of Zika virus disease, and 6 cases of chikungunya, but no cases of yellow fever or West Nile virus, were reported .Plasmodium and arbovirus infections using m-RT-PCR-ELISA in cases of AFS, which occurred in two regions of Colombia that are difficult to access diagnostically.Tropical diseases represent approximately 17% of infectious diseases worldwide. Conducting epidemiological surveillance studies and continuing the search for sensitive and specific diagnostic methods that are easy to implement, low-cost, and reliable should continue to be a priority. This is true particularly in regions where access to healthcare services is difficult and conditions exist that increase the risk of contracting these diseases . The aimA descriptive cross-sectional study was conducted between October and December 2019 and April and May 2022 with patients selected at random from Hospital Ismael Roldan Valencia (Quibdo–Choco) and Hospitals Antonio Roldan Betancur and Francisco Valderrama Hospital (Turbo and Apartado–Antioquia) . The stu® Dengue Early ELISA  rapid tests were performed. The HemoCue® Hb 201 + System  was used to quantify hemoglobin. Drops of whole blood obtained from a tube without anticoagulant were immediately transferred to cuvettes used specifically for this purpose.Blood was collected from each patient, and SD BIOLINE malaria Ag P.f/P.v  and PanbioSubsequently, the tubes were centrifuged, and the serum or plasma was pipetted into a tube with Invitrogen RNA later™ Solution . The samples, stored initially at −20 °C, were transported in liquid nitrogen to the Malaria Working Group laboratory of University of Antioquia and stored at −80 °C. The samples were subsequently shipped and processed at the Center of Pediatric and Adolescent Medicine, University Medical Center, Mainz, Germany.Plasmodium, DENV, Zika virus, chikungunya virus, yellow fever virus, and West Nile virus, were evaluated.The nucleic acids were extracted from samples using the Roche high-purity viral nucleic acid kit and the protocol provided by the company . Six pathogens, Reverse transcription (RT), based upon the protocol established by Puppe et al., was performed prior to multiplex PCR . BrieflyFor PCR, an aliquot of the RT reaction product was diluted in a solution of nuclease-free water, PCR buffer I , Primer Mix A/B (Invitrogen), AccuPrime Taq DNA Polymerase (Invitrogen), and digoxigenin-11-dUTP (Roche Diagnostics). The protocol for PCR was 35 cycles at 94 °C for 30 s, 55 °C for 20 s, and 72 °C for 40 s. The primers and probes used were based upon the protocol previously published by us . After mFor ELISA, the PCR product was denatured and an aliquot was transferred to a well in a 96-welled plate that contained 3′-biotinylated capture antibody specific for the amplified target sequence . SubsequPlasmodium species using protocols published previously [Samples positive for either dengue or malaria were analyzed via single-primer RT-PCR to determine the DENV serotype and eviously ,21. Addieviously .A descriptive analysis of sociodemographic variables, signs and symptoms, and the results obtained by molecular and ELISA testing was performed. Medians and interquartile ranges were estimated for quantitative variables without normal distribution. Relative and absolute frequencies were estimated for qualitative variables. All statistical procedures were performed using R Statistical Software .One hundred and fifty-five patients with a history of fever during the preceding 72 h were enrolled in the study . Of thesThe median vital signs reported here were within normal values, except for the heart rates of dengue- and malaria-positive patients, in whom a significant increase in the median and interquartile ranges of the data was observed . The mosP. vivax and P. falciparum in Antioquia, respectively. M-RT-PCR-ELISA confirmed twenty-three (92%) of these positive cases. It was not possible, however, to determine the species for one of the samples that tested Plasmodium-positive. Five and four of the cases in Chocó were positive for P. falciparum and P. vivax, respectively. All of the cases reported as positive through rapid testing in Chocó were confirmed positive through m-RT-PCR-ELISA, identifying the same species in both tests.Twenty-five patients were found positive for malaria by the rapid test conducted on the day of inclusion . Nine anSixteen patients were found positive for DENV by the rapid test conducted on the day of enrollment: 10 cases were reported in Antioquia and 6 were reported in Chocó . One patDiagnosis of the pathogenic cause of AFS is based mainly upon anamnesis, physical examination, and laboratory confirmation when possible. An epidemiological characterization is often not sufficient to guide the diagnosis ,22. AlthCurrently, the methods used most often to diagnose AFS are based upon microscopic examination, rapid tests, molecular tests such as PCR, and serology . The avaM-RT-PCR-ELISA panels are increasingly available commercially to accelerate and simplify the diagnosis of a variety of diseases in standard clinical practice ,31. The P. vivax and P. falciparum species, as well as four DENV serotypes  in Antioquia, and two DENV serotypes (DENV1 and DENV4) in Chocó. These results are consistent with previous reports of the presence of four DENV serotypes in Colombia [The present study demonstrated the presence of both Colombia ,33. The Colombia ,35,36,37The concurrent circulation of DENV serotypes is a common phenomenon in Latin America . WhetherThe results presented in the current study indicate that the Dengue NS1 Panbio rapid test and the SD BIOLINE rapid test for malaria can play important roles in the early diagnosis of infections. Fluctuations in sensitivity and specificity can occur, however, depending upon the test manufacturer, study, and stage of the disease ,44. M-RTM-RT-PCR-ELISA also demonstrated the ability to identify parasitic and arboviral infections accurately. To make a reliable statement about the sensitivity and specificity of the method, however, requires analyses of a larger sample population. In order to determine the presence of previously undetected pathogens in a patient sample, the incorporation of additional pathogens into the m-RT-PCR-ELISA panel is currently under development.The transmission of diseases like malaria and dengue is influenced by a variety of factors that include the environment, migration, deforestation, climate change, and sanitary conditions. The presence of arboviruses such as DENV, zika, and chikungunya has been reported mainly in urban areas, while parasitic infections such as malaria are found principally in rural areas. Cases of malaria have been reported in peri-urban areas, however, due to large migratory changes and the ability of vectors to reproduce in optimal climates below 2200 m above sea level [There was no significant difference between men and women, and the greatest number of cases occurred in patients over 15 years of age, which could be explained by social, behavioral, or occupational factors, where they could be more exposed to the vector’s bite.An increased heart rate was observed among patients who were positive for dengue or malaria relative to individuals who were negative . This inIn the case of the malaria-positive patients enrolled in this study, heart rate was lower than the rate determined in dengue-positive patients, a finding that differs from that reported previously in a study conducted in 2013 in French Guinea . In the Patients positive for malaria had hemoglobin levels between 9.66 mg/dL and 14.54 mg/dL, similar to the 10.8–15 mg/d values determined for malaria-negative patients in the current study. Consequently, levels that fell within this range were not indicative of an ongoing malarial infection. It should be noted, however, that hemoglobin levels are an important indicator in cases of complicated malaria where values below 5 mg/dL (severe anemia) are suggestive of ongoing or severe disease .Although it is recommended in clinical practice guidelines that anamnesis be performed as the main basis for diagnosis, the results of the current study demonstrate that the signs and symptoms exhibited by disease-positive and -negative patients, or by malaria-positive and dengue-positive patients did not differ significantly. Symptoms such as arthralgia, myalgia, fever, vomiting, and rash, however, were more frequent in patients positive for dengue. In a previous study, retro-orbital pain and rash were reported as the symptoms most characteristic of malaria . These fThe limitations in the current study include the timing of sample collection, as the number of intervening days between the onset of fever and sample collection is often unspecified. Given the maximum level of detection for some arboviruses  requires sample collection within the first week of the first occurrence of symptoms, collection outside an optimal time interval could account for the failure to detect certain pathogens .In the case of a single patient found positive for dengue by the rapid test in the present study, for example, dengue was not detected by m-RT-PCR-ELISA. Conceivably, the length of time between disease onset, sample collection, and sample analysis was an issue; viremia could already have fallen below the limits of m-RT-PCR-ELISA detection. Moreover, in cases involving coinfections with DENV serotypes, it is possible that viremia declines prematurely ,52. AddiThe presence of malaria and dengue was confirmed in both studied regions. m-RT-PCR-ELISA identified parasitic and arboviral pathogens in high concordance with the results of rapid testing.


Background: Seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine (SPAQ) is a malaria prevention strategy recommended since 2012 by the World Health Organization (WHO) for children under 5 years. In Senegal, the scaling up of SMC started in 2013 in the south-eastern regions of the country with an extension of the target to 10 years old children. The scaling up of SMC requires regular evaluation of the strategy as recommended by the WHO. This study was conducted to evaluate the effectiveness of SMC.Methods: A case-control study was conducted in some villages of the health districts of Saraya and Kedougou in the Kedougou region from July to December 2016. A case was a sick child, aged 3 months to 10 years, seen in consultation and with a positive malaria rapid diagnostic test (RDT). The control was a child of the same age group with a negative RDT and living in the same compound as the case or in a neighbouring compound. Each case was matched with two controls. Exposure to SMC was assessed by interviewing the mothers/caretakers and by checking the SMC administration card.Results: Overall, 492 children, including 164 cases and 328 controls, were recruited in our study. Their mean ages were 5.32 (+/- 2.15) and 4.44 (+/-2.25) years for cases and controls, respectively. The number of boys was higher in both cases  and controls . Net ownership was 85.80% among cases and 90.85% among controls . The proportion of controls who received SMC was higher than that of cases (98.17% vs 85.98% and p=1.10-7). The protective effectiveness of SMC was 89% (OR= 0.12 (CI 95%=0.04-0.28)).Conclusions: SMC is an effective strategy in the control of malaria in children. Case-control studies are a good approach for monitoring the efficacy of drugs administered during SMC. AQ             AmodiaquineCI              Confidence intervalCHWs       Community health workersD2             Day 2D3             Day 3HBCP        Home-based care providerLLIN         Long-lasting impregnated mosquito netOR            Odd RatioRDT          Rapid Diagnostic TestSMC         Seasonal malaria chemopreventionSP             Sulfadoxine-PyrimethamineSPAQ)      Sulfadoxine-Pyrimethamine plus AmodiaquineTBS          Thin blood smearTDS          Thick drop slideWBC        White blood cellsWHO        World Health Organization. It consists of intermittent full treatment with an antimalarial drug during the season of high malaria transmission to prevent the disease, with the objective of maintaining therapeutic levels of antimalarial drug in the blood during the period when the risk of contracting malaria is the highest . One single dose of Sulfadoxine-Pyrimethamine (SP) and 3 daily doses of amodiaquine (AQ) are administered monthly to obtain the preventive dose. The drugs should be administered from the beginning of the transmission season, up to a maximum of four monthly cycles. The effectiveness of SMC has been demonstrated by numerous studies, which have also shown that it is well tolerated and inexpensive . These studies, conducted mostly between 2002 and 2011, showed that SMC would have prevented about 75% of all uncomplicated malaria attacks and also about 75% of severe malaria attacks. It would also have reduced child mortality by about 1 per 1000 and reduced the incidence of moderate anemia. Then SMC has been well received as a new tool offering a high degree of personal protection at a moderate cost and was recommended since 2012 by the World Health Organization (WHO) as an additional prevention strategy for malaria control. Senegal, like many African countries in the south of the Sahara, has adopted and implemented SMC on a large scale since 2013 in the regions of the country, that are eligible according to WHO implementation criteria. SMC is administered through a door-to-door strategy based on the community system. In Senegal the target of SMC, since the beginning of its implementation and scaling up in the southern regions of the country, has been children aged 3 to 120 months. The expansion of the SMC target in Senegal was decided at the time the strategy was adopted by the country's health authorities. This choice was reinforced by the numerous studies on intermittent preventive treatment (IPT) of malaria in children, which had shown that children aged 5 to 10 years were just as vulnerable as those under 5. This idea was submitted to WHO, which endorsed it, thus enabling the country, with the support of its technical and financial partners, to implement SMC for this group age.Seasonal malaria chemoprevention (SMC) is a strategy for malaria prevention in children under 5 years of age living in areas of moderate to high malaria transmission in sub‐Saharan Africa. This study was therefore conducted to assess the effectiveness of SMC campaign in Senegal, using a case-control study.While recommending the implementation of SMC on a large scale, WHO also specifies the need to monitor several parameters such as pharmacovigilance, coverage rate, malaria morbidity and mortality, and the appearance of drug-resistant strains of parasitesNMCP. Epidemiological report, 2019,). These different conditions made this region eligible for SMC, which has been implemented there since 2013. This study was conducted in villages with either a head nurse, or with a community health worker (CHW), or also a DSDOM  in the health districts of Kédougou and Saraya  corresponding to the three health districts of the region . Malaria Saraya 11.Epi info 7.1.3.3 software (RRID:SCR_021682) estimated our study population at 152 cases and 304 controls. Being between 0 to 10 years of age, residing in our study sites and for whom the parents had given free and informed written consent, were the main inclusion criteria for this study. Any child who met the inclusion criteria, self-referred to a health facility in the study site and had a positive rapid diagnostic test (RDT) for malaria was considered as a “case”. The "control" was a child of the same age group, living in the same compound or in a neighboring compound within 10 meters. Controls were recruited at concession level based on an apparent good health  and a negative RDT. Each case was matched with two controls.A case-control study was conducted from July to December 2016. Assuming a two-sided confidence level of 95% with a power of 80% and a match of one case to two controls, and a percentage of exposed cases of approximately 50%, theThe purpose and objectives of the study were first shared with the health authorities in the region, prior to the training of the field staff including community health workers (CHWs) and the head nurses for data collection. Each case and control will therefore be visited at home to record the current level of mosquito bed net use ; other interventions like the rate of SMC dosing; and the coverage of mosquito bed net use and other protective measures at concessions in the vicinity of the person's home.NMCP. National diagnostic guidelines for malaria, 2018.).A capillary blood sample was also taken from the pulp of the finger from each subject included in the study, for a rapid diagnostic test (RDT) and the preparation of a thick and a thin blood smear. The slides were stained for 15 minutes with a 10% Giemsa R solution  and then read by technicians from two different facilities. The slides were read at objective 100 with immersion oil on LEICA DM500 microscopes. Parasite density was assessed by counting the number of asexual parasites per 200 white blood cells (WBC) and estimated by the number of parasites per µl using the following formula: number of parasites × 8,000/200 assuming a WBC count of 8,000 cells/µl. Thick and thin blood smears were considered negative after microscopic reading of 100 fields with no parasites detected. Their reading was done according to the recommendations of the national guidelines for biological diagnosis of malaria in the laboratory . Only patients included in the study were taken into account in the data analysis.The different questionnaires and biological results were entered on a data entry mask developed with Microsoft Excel 2019, 16.60 (22041000) (RRID:SCR_016137) software. Data was analyzed with Epi Info 7.1.3.3 software (RRID:SCR_021682). Quantitative variables were described in means and standard deviation. Inter-group comparisons were made using the ANOVA test or Student's t-test according to the conditions of application of these tests. When these tests were not applicable, nonparametric tests  were used. Categorical variables were presented in percentage with confidence interval (CI). Proportions were compared using Chi-square test or Fisher exact test . Risk factors were assessed by multivariate survey logistic regression models. The significance level of the different tests was 0.05 two-tailed. The effectiveness of the different malaria prevention methods investigated in this study was calculated using the following formula: Efficiency =1-OR  and controls  and there was no statistically significant difference compared to those of the opposite sex (p=0.1868). Recruitment was as follows for cases: 1.82% (CI 95%=0.38-5.25%) in July; 29.87% (CI 95%=22.99-37.51%) in August; 26.22% (CI 95%=19.67-33.65%) in September; 22.56% (CI 95%=16.41-29.73%) in October; 15.85% (CI 95%=10.63-22.36%) in November and 3.66% (CI 95%=1.35-7.79%) in December. For controls, there was 0.6% (CI 95%=0.17-2.20%) in July; 28.96% (CI 95%=24.32-34.09%) in August; 22.86% (CI 95%=18.65-27.71%) in September; 21.65% (CI 95%=17.53-26.42%) in October; 19.82% (CI 95%=15.86-24.47%) in November and 6.10% (CI 95%=3.98-9.23%) in December. However, there was no relationship between the period of recruitment of cases and controls (p=0.5009)  years were recruited in this study0.5009) 11.Thick blood count was positive in 87.80% (CI 95%=81.8-92.39%) of cases and 2.74% (CI 95%=1.45-5.135%) of controls. There was an association between the result of the thick blood test and whether the child was a case or a control (chi-square-corrected (Yates)=365.23 and p<0.0001). Plasmodium falciparum was the only species found on positive slides for both cases and controls. The mean parasite density was 13820.18 (+/-19393.43) in cases versus 4119.66 (+/- 3827.74) in controls and this difference was statistically significant .Mosquito net. Net ownership was much higher among controls  than among cases  and this difference was not statistically significant (chi-square-corrected (Yates)=2.22 and p=0.13). The rate of net use was higher among controls who had slept under a net the day before the survey  compared to cases . This difference was statistically significant (chi-square-corrected (Yates)=5.23 and p=0.007). Comparing the possession or not of a long-lasting impregnated mosquito net (LLIN) in cases and controls, an odds ratio of 0.61 (CI 95%=0.34-1.10) was found. This gives an effectiveness of the LLIN of 39% in this study.Exposure to SMC. This study has also assessed the use of SMC among case and controls. It was reported that the controls  had taken more SMC than the cases  and this difference of proportion was statistically significant (chi-square-corrected (Yates)=27.15 and p<0.0001). Comparing the use or not of SMC between cases and controls, an odds ratio of 0.12 (CI 95%=0.04-0.28) was found. This gives an effectiveness of 88% to this strategy. Of the cases who received SMC, 68.38% (CI 95%=59.86-76.08%) were recruited after less than 28 days from the last time they took SMC, 27.94% (CI 95%=20.59-36.28%) between 29 and 42 days, and 3.68% (CI 95%=1.20-8.37%) more than 43 days. For controls who received the drug, 79.10% (CI 95%=74.24-83.25%) were recruited before 28 days, 18.65% (CI 95%=14.71-23.35%) between 29 and 42 days, and 2.25% (CI 95%=1.09-4.57%) after more than 43 days..There was no statistically significant difference between cases and controls, regardless of the time period between the date of the last administration of SMC and the date of recruitment . Among the cases, 26.99% (CI 95%=20.35-34.5%) had not received any SMC cycle, 23.93% (CI 95%=17.60-31.22%) received one cycle; 21.47% (CI 95%=15.44-28.58%) two cycles; 16.56% (CI 95%=11.21-23.18%) three and 11.04% (CI 95%=6.68-16.89%) four. For controls, the proportions of children also varied according to the number of cycles received. Indeed, 4.27% (CI 95%=2.56-7.04%) had not received any; 22.56% (CI 95%=16.41-29.73%) one; 25.30% (CI 95%=20.9-30.28%) two; 24.09% (CI 95%=19.77-29%) three and 17.07% (CI 95%=13.39-21.52%) four cycles. These differences in proportions between these two groups of children according to the number of cycles received, were statistically significant (Chi-square=54.88 and p=0.0001). In the case group, 82.55% (CI 95%=75.49-88.27%) of the children reported that the community health worker had left the doses of day 2 (D2) and day 3 (D3), compared to 98.45% (CI 95%=96.42-99.33%) of the controls. This difference was statistically significant (p<0.0001). Compliance with these doses was more observed in the controls with 98.43% (CI 95%=96.38-99.33%) having taken the dose on D2 and 96.24% (CI 95%=93.54-97.84%) on D3. In the cases, the compliance was 86.51% (CI 95%=79.28-91.94%) for D2 and 73.02% (CI 95%=64.38-80.53%) for D3. There was a statistically significant difference between cases and controls for both D2 (Chi-square=27.89 and p=0.0001) and D3 (Chi-square=54.43 and p=0.0001). The number of controls who used both net and SMC (84.45%) was higher than that of cases (60.97%). This difference in proportion was statistically significant (p=0.032) 11.et al., and related to the rigor required for this type of study, were encountered. The usefulness of case-control studies for determining the efficacy of SMC as well as that of a vaccine has been reported. Indeed, this type of study would allow a better understanding of many parameters that could have an impact on it. Home visits to collect information were not facilitated by the rainy season, which sometimes made access to the villages difficult, but which was also linked to farming activities. This case-control study has resulted in a protective efficacy of 89% of the SMC not exceeding 28 days. Similar results, with an efficacy of 88% in the first 28 days. The same effectiveness were also almost obtained in a study that evaluated SMC in 5 West African countries where SMC was also implemented. The Access SMC consortium, which supervised the scaling up of SMC in West and Central Africa, also found during its evaluation that this strategy, similar to ours, was protective. This very good efficiency of the SMC around 90%, had already been demonstrated in many studies conducted in the research context. This observation shows that the transition from research to scale-up of this strategy does not affect its effectiveness. However, it is strongly related to the complete treatment as demonstrated by this and several other studies.In this case-control study chosen to evaluate the effectiveness of SMC administered in a mass campaign in Senegal, the same difficulties as those described by Cairnset al., in 2015 in Gambia (85% vs. 49.9% in 2015). The efficacy of the net around 50% found in this study had also been showed in other studies that sought to evaluate. On the other hand, efficiencies higher than ours can also be noted.In this study, the evaluation of the efficacy of the net was also conducted at the same time as the SMC. It was found that SMC was more effective than the net (89% vs 45%). The same observation was also made by CairnsNMCP. Epidemiological report, 2019.), . Although the combination of malaria control strategies is a strong recommendation from WHO and many health authorities, the use of new approaches in their deployment would have a greater impact . The strengthening of awareness and education strategies for health promotion for and by the populations themselves could be reinvigorated. A generalization of SMC administration to the whole population with mass drug administration campaigns could be a good approach as adults constitute an important parasite reservoir.In this study, controls had higher use of both SMC and nets. This indicates the need to strengthen advocacy for the integrated use of all malaria prevention strategies to have a greater impact on malaria indices  or came from smaller families, had more conscientious parents, presence of a grandmother in the house older mothers vs. younger mothers better housing etc? It was also interesting that 68.38 % of cases had received some SMC and got malaria within 28 days. What were the associated factors for this? Even more interesting was that 11.04 % of cases had received 4 doses of SMC and 4.27% of controls had not received any SMC, were they older children who might have some immunity already? These are all future research questions perhaps but could be described as being limitations of this study. The conclusion might therefore add some opinion on the unknown. We have one of the know facts seen in this paper is that malaria prevalence is still high in the general population even if slightly lower in children under five years and that this will continue indefinitely unless resources are directed to driving down transmission as well as preventing infection for a short period every year in a defined age group.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Malaria and childhood diseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I am ok.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:EpidemiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. It should be made clear when the scaling up to aged 10 years was done.This study was done in 2016 why is it only being published now?The odds ratios presented for both SMC and bed net usage efficacy are crude estimates in that they are not adjusted for confounding by each other. Adjusted (for confounding) odds ratios can be obtained by fitting a multivariable logistic regression model with both explanatory variables. Alternatively adjusted odds ratios could be estimated using stratification.Be consistent with presentation of P values - give them to 4 decimal points only. If smaller than that then present as e.g. P < 0.0001.NMCP. Epidemiological report, 2019,6)""Malaria in Kédougou is a real public health problem because, in 2019 for example, the proportional malaria morbidity was 27%, the rate of test positivity (RDT and blood smear) in the general population was 51%, in children under 5 years of age this rate was 26%. The proportional malaria mortality in this region was 27% and 50% in children under 5 years of age  However has child mortality reduced significantly so the numbers of children dying from malaria may have been reduced even if proportionate mortality is still 50% I am not sure what this study has to add to the evidence provided by several other case control studies that have been done to show the effectiveness / efficacy of SMC in reducing malaria cases in the Sahel. Its main difference is that it is conducted in children 3 months to 10 years (number of months) and that the average age of cases in this study was in slightly older children than would be expected if looking at traditional SMC in children from 3 - 59 months. New information of value in this study could have been if SMC was as effective in older children (those over five years) as in the younger age group which might have led to more programmes adopting the strategy of including older children which might in turn have a greater effect in the long run of reducing the malaria burden and leading down the road to actually eliminating malaria which from their data on malaria morbidity and mortality in 2019 was still very high in both the general population and in children under five. SMC it seems reduces morbidity in children especially if all four cycles are completed with 3 days of medication and the protective effect is best in the 28 days following the dose. If in spite of many years of implementation malaria transmission remains high, as it seems to here, we need to develop other strategies if we really want to have a serious effect on morbidity and morality over the long term as nets and SMC have not led to a reduction in transmission - perhaps this could be mentioned in the discussion even when extended to older children?This is an interesting study with interesting results although the results only confirm numerous other cases control studies that have been done across the SahelIs the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Malaria and childhood diseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 1. In Senegal the target of SMC, since the beginning of its implementation and scaling up in 2013 the southern regions of the country, has been children aged 3 to 120 months. The expansion of the SMC target in Senegal was decided at the time the strategy was adopted by the country's health authorities. This choice was reinforced by the numerous studies on intermittent preventive treatment (IPT) of malaria in children, which had shown that children aged 5 to 10 years were just as vulnerable as those under 5 years. This idea was submitted to WHO, which endorsed it, thus enabling the country, with the support of its technical and financial partners, to implement SMC for this group age 2. This study is part of a project to evaluate the impact of scaling up SMC in Senegal. This evaluation was requested from our organisation by the NMCP and was to be an ongoing process. However, it has not been carried out since 2016, although we had hoped to continue it in order to try to determine the dynamics of the effectiveness of the scaled-up SMC over time. This situation explains why it's took time before to write this article. 3. We accept your recommendation to adjust the odd ratio before the efficiency calculation. We will do this and include it in the new version we submit to the journal. 4.We have standardized the presentation of the p-value as you suggested. The changes have been made in the new version submitted to the journal. 5. We have reworded the sentence because, as noted, it is confusing. We also think that the part about the burden of malaria in the proposed new version helps to present it better. Our study was conducted in children aged 3 to 120 years, unlike those conducted in the Sahel to determine the effectiveness of SMC, all of which, as you rightly pointed out, were targeted at children under the age of 5. We believe that even though we did not carry out an analysis to determine the effectiveness of this strategy in children under and over 5 years of age, this constitutes a strong point for our study. In addition, we believe that the other strength of this study is that the data collection  was done by the trained community level and not by the research team. We have added a recommendation at the end of our discussion to suggest additional strategies that could further reduce malaria-related morbidity Specify SMC campaign rather than mass. I approve this version with one comment:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:EpidemiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Introduction (needs more detail):Reference: harmonize the reference citations in the main document.The author should give more details on study justification because the effectiveness of SMC has already been proven: more cases of malaria are screened despite the SMC?Please specify: SMC campaign or mass campaign? If different to SMC campaign,  what approaches of mass campaign was used to administer antimalarial drug.SMC is recommended in children up to 10 in Senegal?Methods (need to clarify some points):Study Site The rate of test positivity in the general population was 51%: please specify the test (RDT or blood smear).Study type, time-period, and populationControls were recruited at concession level based on an apparent good health and a negative RDT.Good health: any malaria symptom or any symptom?Data management and analysis:2 Mann, Kruskall.The author just needs to specify which tests used and for what comparison: ANOVA, T-test ChiWhat part of the result section were these test were used?Results (the interpretation of some sentences needs to be clarified):All cases in our study were recruited on the basis of a positive rapid diagnostic test (RDT) and the controls a negative RDT. Thick blood count was positive in 87.80% (CI 95%=81.8-92.39%) of cases and 2.74% (CI 95%=1.45-5.135%) of controls.-9). Make this part clearerSpecify the test used for malaria diagnosis: There was an association between the result of the thick blood test and whether the child was a case or a control  than among cases  and this difference was not statistically significant (p=0.053).Reword this sentence: Net ownership was much higher among controls because it’s not significant.(p=0.0000004): also harmonizes p-values.Conclusion (without aim)It is not necessary to give against the objective of the study.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:EpidemiologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reference: We have respect the guidelines given by the journal for this part. However if you can give me an example of that I will take in account to improve the presentation of references.Justification of the study: You right when you say that the effectiveness of SMC has already shown, but most often it is in the context of operations research, which we have tried to demonstrate by posing the problem. The justification for this study is that the effectiveness of this strategy in mass campaigns has not been evaluated in Senegal. We think we have done this, but remain open to improving this part.You right when you say SMC campaign and I'll delete mass campaign in the new versionSMC is not recommended for children up to 10 years but for those aged 3 months to 10 years. We have therefore amended the sentence in the new version to make it clearer. Method Study site: The rate of test positivity is for RDT and blood smear and we add it in the new version of this article.Study type, time-period and population: an apparent good health means for us without any clinical symptom. We put it in brackets in the new version for more clarifications.Data management and analysis: We take in account your suggestions for the test used and we add in the new version in brackets the test and the p-value for each comparaison. ResultFor the results of the slide reading we have in the new version removed the first sentence. This sentence can be confusing. We have also added for each comparison the statistical test used. ConclusionWe have deleted the objective in this part as you have suggested Introduction