Young children in Sub-Saharan Africa (SSA), particularly those from resource-limited settings, are heavily burdened by anemia and malaria. While malaria infected children frequently become anemic (hemoglobin < 110 g/L), anemia is a strongly multifactorial disease with many other risk factors than malaria. Due to the complex and often overlapping contributors to anemia, it remains challenging to isolate the true impact of malaria on population level hemoglobin concentrations.We quantified the malaria-induced effect on hemoglobin levels in children under 5 years of age, leveraging data from 7,384 twins and other multiples, aged 6 to 59 months, from 57 nationally representative Demographic and Health Surveys (DHSs) from 23 SSA countries from 2006 to 2019. The quasi-experimental twin fixed-effect design let us minimize the impact of potential confounders that do not vary between twins.p<0.001). The relative risk of severe anemia was higher  among malaria positive children, compared to malaria negative children. Conversely, malaria positive children are only half as likely to be non-anemic .Our analyses of twins revealed a malaria-induced hemoglobin decrease in infected twins of 9 g/L (95% CI -10; -7, Even after rigorous control for confounding through a twin fixed-effects study design, malaria substantially decreased hemoglobin levels among SSA twins, rendering them much more susceptible to severe anemia. This effect reflects the population-level effect of malaria on anemia. Young children from low- and middle-income countries frequently suffer from a wide range of health burdens specific to their under-resourced settings. In Sub-Saharan Africa (SSA) these often include anemia of multifactorial origin and endemic infectious diseases such as malaria, where recent estimates have suggested a prevalence of up to 60%  1–4). C–4. C1–4)Plasmodium parasite genus whose species differ in both geographic distribution and severity of the resulting disease . During the surveys, trained field workers conducted extensive interviews, collected biomarkers, and gathered anthropometric data from a representative cluster-based sample of households in the respective survey country  and Malaria Indicator Surveys (MISs)  R package and extracted the variabes sex, hemoglobin and RDT result for analysis. Furthermore, we extracted the survey identifier, the household identifier, the date of birth and the mother identifier to identify the twin pairs. The datasets were restricted to children with complete RDT and hemoglobin measurements.All surveys from the SSA region that contained hemoglobin measurements and rapid diagnostic tests (RDT)-based malaria measurements were eligible for analysis. We downloaded the survey data using the P. falciparum  available in the given data. RDTs are frequently used in remote clinical settings and surveys because they work well-with minimal laboratory equipment while providing solid and immediate results on site. The tests detect  malaria , 35.Unfortunately, the data did not contain reliable information on potentially confounding causes of anemia such as iron status, coinfections or genetic disorders.The flowchart illustrates the process of data preparation and eligibility assessment . The eliSince there was no predefined twin-identifier in the given data, we manually constructed a twin-identifier variable from the country code, survey year, cluster identifier, household identifier, mother identifier and date of birth (century month code) of the child, thus ensuring that only twins and higher order multiples shared a common indicator. The resulting main dataset contained 7,384 monozygotic and dizygotic twins with complete data and was used in the analysis of the main model.With the given data it is not possible to exclusively identify monozygotic twins. Instead, we approached a selection of monozygotic twins by creating a subset excluding twin pairs with differing sex and all multiples , to maximize the probability of genetic similarity . This suWe first described the overall patterns in the population structure of all children and of the twin subsets to enable easier comparison between the groups and assess the representativeness of the twins.main, same-sex, different-sex) we constructed a linear twin-fixed effect regression model. Each of the models estimated the expected difference in hemoglobin of twins with parasitemia compared to those without, with a separate intercept for each pair of twins. In more descriptive terms, the model considers a twin pair as a single entity, where the individual siblings within a twin pair represent two simultaneous measurements of the same entity in two different exposure-states , moderate or worse (< 90 g/L) and severe anemia (< 70 g/L). These analyses illustrate in a more tangible way the consequences that are implied in the malaria-induced hemoglobin reductions. All analyses were performed using R version 4.0.2. We extracted, cleaned and analyzed the data with the help of the following r packages .plasmodium positive children than in negative ones (82.4 vs. 56.2%) (T-test: p<0.001). The pooled prevalence of plasmodium parasitemia also remained largely constant between survey years . The distribution of hemoglobin in each survey is shown in T-test .An overview of the demographic composition of the datasets used for analysis is provided in . 56.2%) . The avemain model, adjusted for sex and the twin fixed effect, showed a 9 g/L  hemoglobin decrease in children with positive RDT compared to their RDT-negative twins. Female sex increased hemoglobin by 2 g/L . The main model outcome and the outcomes for each separate country analysis are illustrated in The same-sex model, for the twin fixed effect only and found a malaria-induced hemoglobin decrease of 9 g/L . Based on the Weinberg rule we estimated that a third of the same-sex data set are monozygotic twins  hemoglobin reduction induced by plasmodium presence among the different-sex twins. The effect of parasitemia in the different-sex model is thus marginally smaller than the effect of the main and same-sex model , , moderate anemia or worse (Hb < 90 g/L),  and severe anemia (Hb < 70 g/L), . At the same time, their “risk” of being healthy (Hb≥110) was reduced . The outcomes of the quasi-poisson regression are illustrated in Based on the main model we also conducted a quasi-poisson regression with anemia severity as binary outcomes. In this model series plasmodium-infected children to their healthy twin counterparts and confirmed that malaria substantially impacts population-level hemoglobin. Plasmodium presence was associated with a 9 g/L hemoglobin decrease among all twins and among the same sex twins. Among the different-sex twins the parasitemia-associated hemoglobin decrease was marginally smaller. Since the anemia severity groups are distinguished by steps of 20 g/L hemoglobin, the effect sizes are large enough to be clinically relevant, particularly if considering that our calculations are based on cross-sectional data. This is also illustrated in the poisson regression, where children with parasitemia are only about half as likely to be non-anemic and three times more likely to be severely anemic than their plasmodium-negative peers. The slightly larger effect of the same-sex model in comparison to the different-sex model might point toward a relevant genetic predisposition, e.g., sickle cell anemia, G6PD-deficiency or thalaessaemias , to a marginal -1 g/L among adults in a regional setting in south-east Asia , 39. In f -5 g/L . Korenrof -5 g/L . Our poif -5 g/L , 43. Thef -5 g/L , 45.Our study benefits from several strong innovations. It draws data from a large, representative sample of children in SSA, the region where the greatest gains against malaria have been made and yet much remains to be done. Additionally, from this representative sample we identified a very large number of twins as a basis for our fixed effect statistical model, controlling for most relevant confounding that is shared between twins , 28.Our study has several limitations. Firstly, the nature of cross-sectional data is vulnerable to certain types of bias. This includes survival bias, where children with severe anemia are likely treated in clinics to receive intensified medical care and therefore cannot be included in the surveys . We expeSecondly, we decided to use RDT result as our indicator for malaria status, rather than microscopy although microscopy is considered the gold standard. We made this choice because the RDTs most effectively reflect the diagnostic situation in SSA. RDTs are used in most DHS surveys, are a staple in clinical settings of rural Africa where microscopy is frequently not feasible and have very similar sensitivity and specificity , 47. Thein-utero and perinatal exposures, the mother's education, location of birth, age of the child, early childhood exposures etc .Publicly available datasets were analyzed in this study. This data can be found at: TS and TB designed the study. TS performed the data analysis and drafted the manuscript with support of CB. All co-authors contributed significantly to the revision of the manuscript and provided scientific guidance. All authors contributed to the article and approved the submitted version.For the publication fee we acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publikationskosten as well as by Heidelberg University and the Heidelberg Institute of Global Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown.Polymorphisms in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygotes , heterozygote T/C and wildtype C/C ) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank.Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ½ ± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p < 0.0001). Growth of a Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p < 0.01). Genotype group did not affect parasite adhesion in vitro or var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50 = 122uM CI: 110–134) confirming its sensitivity to calcium channel blockade.Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression  and slower rates of calcium expulsion (calcium tATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.The data support the hypothesis that this  Plasmodium falciparum malaria remains one of the most common infectious diseases, infecting about 241 million people worldwide in 2020  of the ATP2B4 gene]. Previous analysis of four of these SNPs  demonstrated that they were in complete LD .Taken together, these data are consistent with the conclusion that polymorphisms in ATP2B4 haplotype will likely diminish each of these risks and thus explain its association with severe malaria.Although cytoadherence is critical to some of these complications, host immune response to the presence of the malaria parasite also plays an important role. For example, the reaction against the parasite pigment released during RBC lysis is the cause of the cyclic febrile episodes in malaria disease . Additio


This remained significant after adjusting for CRP and PCT . Area under the ROC curve for CRP and PCT were 0.754 and 0.779, respectively, but increased to 0.803 and 0.832, respectively, when combined with viremia.Most children with fever without source (FWS) require diagnostic laboratory tests to exclude a serious bacterial infection (SBI), often followed by admission and empirical antibiotics. As febrile children with a viral infection are less likely to have a SBI, identifying patients with systemic viral infection could contribute to exclude SBI. We evaluated whether the presence of virus in the blood could be used as a biomarker to rule out SBI. Children < 3 years old with FWS were prospectively enrolled and had real-time (reverse-transcription) PCR performed on the blood for adenovirus, enterovirus, parechovirus, and HHV6. 20/135 patients had SBI, and in 47/135, at least one virus was detected in the blood. Viremia had a higher sensitivity and negative predictive value (90% and 96%) to rule out SBI compared to CRP (65% and 93%) and PCT (55% and 90%). The odds ratio (OR) for the presence of SBI among non-viremic patients was 5.8  PCR in the plasma in management algorithms of children with FWS.The online version contains supplementary material available at 10.1007/s00431-022-04690-7. Fever without source (FWS), defined as temperature ≥ 38.0 °C with no cause identified after a thorough medical history and clinical examination , is one Even though most children with FWS have a self-limited viral infection, laboratory investigations performed in this context are meant to rule in or rule out SBI. For example, biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP), have been shown to be good predictors of SBI –11. SimiThis is a substudy of a prospective, single-center, epidemiological diagnostic study . PatientStudy patients were divided into two groups based on the diagnosis of SBI. SBIs consisted of documented bacteremia requiring antibiotic treatment (blood cultures interpreted as contaminant were not considered as SBI), bacterial meningitis, osteomyelitis, pneumonia, or urinary tract infection (UTI). UTIs were defined as a positive urinalysis and ≥ 10e5 CFU/ml of a single uropathogenic organism in appropriately collected specimens, as per the American Academy of Pediatrics . More deThis study was approved by Geneva’s Ethics Committee (CCER #15-082) and registered under Clinicaltrials.gov (NCT03224026). No investigation was performed before signature of the informed consent.p values < 0.05 were considered significant. Statistics were calculated using SPSS software, version 23.0 , and all statistical tests were two-sided.Continuous variables were compared using the Mann–Whitney test. Dichotomous variables were compared using Chi-squared or Fisher exact test. Proportions are reported with 95% confidence intervals (Clopper-Pearson exact method). Univariate and bivariate logistic regression was used to evaluate biomarkers performance individually and in combination. The combination CRP + viremia was obtained with a multivariable logistic regression model: CRP and viremia were linearly combined using the regression coefficients. The same was true for the combination PCT + viremia. Odds ratios assessed with logistic regression models are reported with 95% confidence intervals. Receiver operating characteristic (ROC) curves with area under the curve (AUC) were also used to evaluate biomarkers performance (non-parametric approach). n = 18), followed by H. influenzae (type f) bacteremia with concomitant meningitis (n = 1) and P. aeruginosa meningitis (no known comorbidities). The demographics of study patients are detailed in Table One hundred and thirty-five patients enrolled between November 1, 2015, and December 31, 2017, had blood tested for HEV, HPeV, AdV, and HHV6. Among those, 20 were diagnosed with SBI. Most SBIs consisted of UTIs  polymerase chain reaction (PCR) in the plasma. Namely, HEV was detected in 19 patients, HHV-6 in 15, HPeV in 8, and AdV in 7 . Within the dataset, two patients had a concomitant SBIs and viral systemic infection .p = 0.011)  (Table There were significantly less viremic patients in patients with SBI than in those without SBI (1) Table . Similary) Table .Viremia had a higher sensitivity and negative predictive value  to rule out SBI when compared to CRP < 40 mg/l  and PCT < 0.5 ng/ml  Table . Taking p = 0.0225), compared to 5.51 for CRP ≥ 40 mg/l  and 3.67 for PCT ≥ 0.5 ng/ml   (Table p = 0.03 for both)   for having SBI among non-viremic patients was 5.79  Table . Similary) Table . Using bh) Table . After ay) Table .The AUC for CRP and PCT were 0.754 and 0.779, respectively Fig. . When viThis study evaluated the prognostic accuracy of the absence of viremia as a predictor of SBI in children with FWS. The main finding was that the sensitivity and negative predictive value of viremia compared to commonly used biomarkers such as CRP and PCT allowed to better rule out an SBI. This remained true whether viruses were analyzed together or individually. The superiority of viremia when compared to CRP or PCT was particularly evident in children < 3 months, which is important because most of them will require blood testing given their higher likelihood of SBI. Similarly, the likelihood of SBI was approximately six times lower among viremic patients, even after adjusting for CRP and PCT. Using ROC curves, CRP and PCT displayed better prognostic accuracies when combined to viremia, confirming the independent prognostic value of the absence of viremia and its potential interest when used in conjunction with CRP and/or PCT. These findings are in line with previous evidence showing that febrile children with a documented viral infection are less likely to have a concomitant SBI , 14–21. With the SARS-CoV-2 pandemic, there has been a dramatic development of point-of-care testing (POCT) RT-PCR testing, with turnaround times as fast as 20 min , 33. In This study has some limitations. First, only four viruses were tested. However, those viruses were selected because of (1) their frequent epidemiology in FWS as shown by other groups  and (2) In conclusion, we showed that in FWS, viremia displayed a better independent prognostic accuracy to discriminate children with or without SBI when compared to commonly used biomarkers and could potentially be used in conjunction with CRP and/or PCT in the evaluation of children with FWS. Larger studies should evaluate the role and cost-effectiveness of point-of-care testing of viruses by (RT)-PCR in the plasma or whole blood in management algorithms of children with FWS.Supplementary file1 (XLSX 12 KB)Supplementary file2 (XLSX 11 KB)Supplementary file3 (XLSX 10 KB)Supplementary file4 (XLSX 10 KB)Supplementary file5 (DOCX 78 KB)Below is the link to the electronic supplementary material.


Undiagnosed malarial infectionis associated with significant mortality and morbidity. Laboratory investigations leading to rapid, accurate and timely diagnosis of malaria is still a challenge. This study was done to assess the utility of abnormal White blood cell differential fluorescence (WDF) scattergram for diagnosis of malaria. Our aim was to study the utility of WDF scattergram for early detection of malarial parasite.All EDTA anti-coagulated blood samples received in laboratory during a period from Dec 2019 to May 2020 were analyzed on anautomated hematology analyzer, Sysmex XN 1000. All abnormal WDF scattergrams pertaining to plasmodium specie were critically evaluated and recorded. Review of Leishman-stained peripheral smears as well as immune-chromatographic assay by rapid test devices (RTD) was done. Accordingly, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of malaria by abnormal scattergram were calculated.Out of total 1, 26,000of samples analyzed, abnormal WDF scattergrams were detected in 96 cases. Amongst these, 95.8% (92) were positive for MP on Leishman-stained peripheral smear as well as on ICT with a p-value of 0.05. WDF scattergram abnormalities typical of malaria showed a sensitivity of 80% and specificity of 93.26%. Positive predictive value of 95.8% whereas negative predictive value of 99.9% was detected. Significant findings of hemolysis, platelet clumps, nucleated RBCS (NRBCs) and RBC agglutination were noted in cases (n=4) with abnormal WDF scattergram negative for malaria on peripheral smear.Interpretation of abnormal WDF scattergram not only increases the early detection rate for malarial parasite but isa strong indicator for presence of hemolysis, RBC agglutination, platelet clumps and leucoerythroblastic blood picture as well. P.vivax is the most commonly found species in Afghanistan, Pakistan, Iran and Iraq. In Pakistan, 1.5 million cases per annum have been estimated. In different provinces of Pakistan, the prevalence of P.vivax found to be 2.4% in Punjab, 10.8% in Sindh whereas prevalence of P.falciparum ranged from 0.1% in Islamabad and 3.8% in Balochistan.1Despite advances in diagnosis and management of malaria over the past decade, it remains the most common and important human vector-borne disease worldwide. Amongst the four known species of plasmodium (P) globally, the dominant species is P. falciparum however Microscopy of Giemsa-stained peripheral smears of patients with suspected malaria are still gold standard for its diagnosis. The liability of this method is based on the professional expertise in staining technique and microscopy. Moreover, it is also time consuming in setups with heavy workload and limited resources. Keeping these limitations in view, several new techniques for detection of malarial parasite were offered. These include antigen-coated dipstick tests, quantitative buffy coat examination using fluorescent dyes and polymerase chain reaction.5Several latest automated haematology analyzers indicate flagging or graphical representation of malaria as a new emerging technique for its early detection. Automated haematology analyzers e.g., Sysmex XN-1000 perform a complete blood count and detect the presence of plasmodium species by showing an abnormality of the WDF, WNR, and RET scattergram. WDF scattergrams are graphical representation of the findings of the WDF channel. Different types of white blood cells are counted in WDF channel in XN series.Various studies have been conducted worldwide based on results of the automated hematology analyzers like, GEN S andLH750, XE2100 and XS100i7 for malarial detection.It was a prospective study conducted at Fauji Foundation Hospital Rawalpindi over a period of six months duration from December 2019 to May 2020. After taking ethical approval from ethical review board , all the complete blood counts (CBC) samples received in pathology laboratory from both outpatient departments (OPD) as well as wards were processed on automated Haematology analyzer Sysmex XN-1000. All the CBCs that were requested specifically for malaria and ICT MP were excluded from the studies. The CBCs showing abnormal scattergram pattern pertaining to malaria were selected and Leishman-stained peripheral smear stained were prepared and examined. ICT MP was also performed for all these selected cases. The abnormalities in the scattergram included double neutrophil line, multiple neutrophil lines, pseudo eosinophilia, right ward shift of ghost area and greying of the neutrophil/eosinophil zone. The peripheral smear was reviewed by post graduate resident and verified by consultant haematologist. All cases found to be positive for plasmodium species were documented in the software system and notified to the respective wards. The walk-in patients and patients from OPD were contacted on cell numbers to report to hospital immediately. Data analysis was done through SPSS version 17. All quantitative variables like age and gender were noted. Frequency and positive predictive and negative predictive value of malaria positive cases with abnormal scattergram was recorded. Sensitivity and specificity were also calculated for these selected cases.A total of 1,26,000 CBCs received in hematology laboratory during the period of six months (Dec 2019-May 2020) were analyzed along with their respective WDF scattergrams. Of the total CBC, 75,600 received were of admitted cases in various wards, 44,100 cases were booked from outpatient department and6,300 CBC were booked directly in the laboratory as walk-in patients. Amongst the total cases, 96 CBC cases showed abnormal WDF scattergram pertaining to malaria. Of these 96 cases, 92 cases were positive for malarial parasite on peripheral smear and ICT with a p-value of 0.05. 79.3% (73) were females and 20.6% (19) were males with mean age of 40 years.Amongst these, 93.7% (90) were found to be positive for MP on Leishman-stained peripheral smear as well as on ICT MP. While 02 cases with no evidence of malaria on peripheral smear tested positive on ICT.Out of the total 96 cases showing abnormal scatter gram, 4.1% (4) cases tested negative for malaria species on both peripheral smear as well as on ICT showed other findings such as RBC agglutination, platelet clumps, leucoerythroblastic blood picture and hemolysis on peripheral film examination .Amongst these cases only one case had plasmodium falciparum whereas mixed infection with plasmodium vivax and falciparum was detected in two cases. The rest of the 89 cases showed plasmodium vivax. No Ovale, Malariae species were reported . SensitiThe detection of clinically unsuspected malaria is of significant importance in order to prevent the mortality and morbidity of the disease.3 the GEN.S and LH-750 ,4 and the XE-2100 and XS-1000i .11In various studies Haematology analyzers evaluated for malaria detection include the Cell-Dyn ,The present study showed abnormalities in hematological parameters like anemia, leukopenia and thrombocytopenia which were also reported in previous studies.A study conducted in India by Ningombam A et al showed 99.2% parasite detection by abnormal scattergram on similar haematology analyzer used in present study thus showing comparable results.13P.vivax is the most common plasmodium infections in Pakistan but P. falciparum and mixed species infections are also prevalent.16In present study 89 cases were positive for plasmodium vivax, two for falciparum and one was mixed infection. It is in accordance with most of the studies reported plasmodium vivax as the most common infection globally. A study conducted by Zuluaga GC performed on XE-2100 indicated that malaria alarm produced by abnormal WDF scattergram can be helpful for early malaria microscopic diagnosis.17According to the study conducted by Buoro et al. nine patients were positive to Plasmodium falciparum, one to Plasmodium ovale, and one to Plasmodium malaria and three to Plasmodium vivax. Significant abnormalities were observed in both white blood cell differential (WDF) and white cell nucleated (WNR) scatter grams (sensitivity 0.64 and specificity 1.0) in nine samples with parasites at gametocyte or schizonts stage, while characteristic scattergram abnormalities were not detected in the 5 samples containing only parasites at the trophozoites stage. The specific variations of some cell population data (CPD) could be recorded (sensitivity 1.00 and specificity 0.91) in these samples.18P.vivax infected samples, however in contrast to our study an increase in highly fluorescent lymphocyte-coded along with elevated reticulocyte/IRF ratio events in the DIFF scatterplot in a P. falciparum-infected patients were also detected.21In another study of the 80 patients who presented with fever and suspicion of malaria, 29 patients were positive for malaria and 10 cases were diagnosed incidentally by the findings on the cell counter and were confirmed by Giemsa-stained blood smears. The sensitivity and specificity of the abnormalities detected in the WBC-Diff channel in detecting malaria is 82% and 100% respectively. Using WBC-BASO channel abnormality for initial diagnosis the sensitivity and specificity is 50% and 92.5% respectively.It is a single center study. This study should be extended on multicenter to better enhance and validate the research findings. Furthermore, this idea can also be replicated for different CBC analyzers to assess their utility in this regard as well.Timely detection of malaria is life-saving. Knowledge about interpretation of abnormal WDF scattergram generated by automated haematology analyzers and its application can be utilized as an adjunctive diagnostic tool for early diagnosis of malaria especially in unsuspected cases where diagnosis can be overlooked. It has the potential to facilitate the diagnostic approaches, and significantly impact therapeutic monitoring and efficacy of anti-malarial and even clinical trials. It can also serve as the ideal blood donor screening tool for presence of malaria especially in malaria-endemic regions.MR: Literature search, manuscript writing, data acquisition, manuscript drafting and editing and critical review for intellectual content and final approval of the version to be published. Corresponding author and accountable for accuracy and integrity of work.AK: Concept and design of the study, data acquisition and statistical analysis, critical review.FN: Data collection, Data analysis & Data interpretation, critical review.


Metabolic syndrome (MS) is not a single disease but a cluster of metabolic disorders associated with increased risk for development of diabetes mellitus and its complications. Currently, the definition of MS published in 2009 is widely used, but there are more versions of the diagnostic criteria, making it difficult to conduct scientific discourse in this area. Increased arterial stiffness (AS) can predict the development of cardiovascular disease both in the general population and in patients with MS. Pulse wave velocity (PWV), as a standard method to assess AS, may point out subclinical organ damage in patients with hypertension. The decrease in PWV level during antihypertensive therapy can identify a group of patients with better outcomes independently of their reduction in blood pressure. The adverse effect of metabolic disturbances on arterial function can be offset by an adequate program of exercises, which includes mainly aerobic physical training. Non-insulin-based insulin resistance index can predict AS due to a strong positive correlation with PWV. The purpose of this paper is to present the results of the review of the literature concerning the relationship between MS and its components, and AS assessed by PWV, including clinical usefulness of PWV measurement in patients with MS and its components. Metabolic syndrome (MS) is not a single disease but a cluster of metabolic disorders such as abdominal obesity, atherogenic dyslipidemia, elevated blood pressure (BP), insulin resistance (IR), and higher glucose levels . For decMS is linked to an increased risk of developing type 2 diabetes mellitus (T2DM) and its complications. Cardiovascular diseases (CVDs) are one of the most important causes of morbidity and mortality in the population of patients living with T2DM ,13,14. MMS was shown to be associated with increased oxidative stress. Obesity and IR are the MS component that contributes the most to this relationship . On the Therefore, it is important to identify subclinical vascular damage in patients with MS and its components who should be taken into careful consideration for appropriate treatment. Increased arterial stiffness (AS) can be considered to be a good predictor of the development of subclinical cardiovascular dysfunction, so the assessment of AS can be useful in the identification of patients with increased cardiovascular risk ,21.According to the American Heart Association as well as European experts’ consensus, the measurement of pulse wave velocity (PWV) remains a standard method to assess AS ,23. TherHowever, there are some limitations to the measurement of PWV. Specialized equipment and experienced personnel are required to perform the measurement. In addition, care should be taken to ensure that the measurement is carried out in appropriate conditions, such as, among others: a quiet, dry room, ensuring thermal comfort; moreover, the measurement should be carried out after a ten-minute rest in a lying position. The BP value and heart rate (HR) should also be taken into account during the interpretation of the PWV value, as these parameters can influence the PWV measurement result .The purpose of this paper is to present the results of the review of the literature concerning the relationship between MS, its components, and AS, with the greatest emphasis on AS assessed by PWV, including the clinical usefulness of PWV measurement in patients with MS and its components.Increased systolic blood pressure (SBP) or diastolic blood pressure (DBP) are both MS components. In patients with high cardiovascular risk such as patients with MS, it is crucial to assess subclinical dysfunction of the cardiovascular system in order to initiate preventive measures, adequate life changes, and treatment early ,26. TherIncreasing AS defines the loss of the arterial wall’s resistance to expansion by an increment in volume; as a result, it is linked to increased BP ,32. AS ip = 0.003) [In a cohort study in which 54,849 middle-aged patients participated, it has been shown that both SBP and DBP depend on AS. Compared to patients with normal PWV values, moderate, moderate–high, and high PWV values were responsible for a 3.03-, 5.44-, and 7.87-times higher incidence of hypertension, respectively . Hyperte= 0.003) .p < 0.001) and HR (p = 0.07) levels between visits during the period [However, in a four-week observational study performed by Kubalski et al., it was found that the PWV value changes with BP and HR. PWV was related to BP , an analysis of 8450 patients demonstrated a 42% lower risk of death associated with a decrease in the PWV value observed after one year of antihypertensive therapy compared to patients in whom the PWV value remained not reduced. Furthermore, the results of the SPRINT study were independent of BP reduction and Framingham risk score [In the management of arterial hypertension, it is crucial to pay attention to a low sodium diet, as well as exercise training. High sodium intake is related to water retention, an increase in systemic peripheral resistance, and changes in the structure and function of large elastic arteries . The impWhen considering abdominal obesity, it has been shown that in obese patients the PWV is significantly higher compared to normal healthy individuals, proving that abdominal obesity is a significant factor in the development of AS ,49,50. BIn a prospective study performed by Kae-Woei Liang, it has been proved that after a three-month weight reduction program, brachial–ankle PWV in patients with obesity and MS, compared to the healthy control group, decreased slightly. However, in those in whom weight regaining was observed in a sixty-month follow-up period, compared to the healthy control group that also experienced weight regaining, the brachial–ankle PWV increased by a significantly higher amount. Interestingly, brachial–ankle PWV at the 60th month after weight regaining in obese patients with MS was even worse than the baseline values, was associated only with SBP or DBP increments, and when compared to healthy individuals proves to be beyond the aging process. In addition, brachial–ankle PWV values after weight regaining were independent of body weight, high-sensitivity C-reactive protein (hs-CRP), or insulin resistance changes . HoweverThe weight loss program in obese patients is based on diet changes and regular exercise ,60,61. IDyslipidemia is a metabolic disturbance associated with an impaired concentration of basic lipid parameters, such as blood level of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and/or the presence of dysfunctional lipoproteins in the blood .The whole spectrum of possible underlying pathophysiological mechanisms of the influence of lipid profile on atherosclerosis has not yet been well established. Lipid disorder may lead to cardiovascular disease in different ways . Elevatep = 0.024). Moreover, the results of this study were independent of other MS components, age, gender, smoking status, LDL-C, and statin treatment [In a prospective study involving 1934 patients, the correlation between elevated TG and AS was analyzed. The authors of this study define AS by cardio–ankle vascular index (CAVI) as independent of BP compared to brachial–ankle PWV. The elevated level of TG was shown to be associated with higher CAVI values , BMI , TC level , LDL-C level , TG level , non-HDL level , and TG/HDL-C ratio  was shown to have a significant impact on PWV values [In the retrospective study conducted by Alparslan Kilic, in which data from 1584 patients were analyzed, age  is defined as a logarithm from the TG/HDL-C ratio . In a reAS increases in patients with MS and IR ,79. Predp < 0.001). In addition, they proved METS-IR to have the strongest correlation with AS over other non-insulin indexes, including TG/HDL-C ratio. When including METS-IR in the Framingham Hypertension Risk Prediction Model, a more precise hypertension incident predicament was observed [In a prospective study published in 2019, researchers proved the correlation between METS-IR and PWV, SBP, and DBP independently of age, sex, smoking status, and hypertensive therapy. METS-IR in 34% predicts PWV changes  calculated from waist circumference, BMI, and body height has been confirmed to identify people with MS and increased AS . Nedogodp < 0.001) and to have higher PWV  than those without MS [p < 0.05 for all variables) [p = 0.005) or increased all-cause mortality (p = 0.002) [Patients diagnosed with arterial hypertension have a high prevalence of metabolic syndrome regardless of controlled or resistant hypertension . Hypertethout MS . Predictriables) . Chen et= 0.002) .The conclusions from selected studies on vascular stiffness in patients with MS are presented in As shown in our analysis, there is no single pattern for elevated risk of CVD in patients with MS. MS, as a group of metabolic disorders, is strongly associated with subclinical vascular damage, marked by increased PWV value ,100. As Abdominal obesity is an important component of MS. The ambulatory AS index of the obese subjects is significantly higher than in the healthy controls ,105,106.It is important to measure PWV under stabilized BP and HR values, particularly in patients with newly diagnosed hypertension in whom detection of organ damage mediated by asymptomatic hypertension has an impact on risk stratification ,112. PWVThe risk of increased AS depends on hyperlipidemia at young ages . DyslipiAs shown in a study performed by Gong et al., AS became significant with increasing numbers of the metabolic components . HoweverDue to the strong correlation between PWV and METS-IR that extends beyond sex, age, smoking status, and hypertensive treatment, as shown in the analyzed studies, METS-IR could be treated as a subrogating of AS in routine primary care . FurtherMS and its components are a significant problem for healthcare systems worldwide. CVD is the leading cause of morbidity and mortality, and, on the other hand, MS predisposes the development of CVD. The identification of patients with features of subclinical dysfunction of the cardiovascular system in the population of patients with MS and its components is also very important in clinical practice, and PWV measurement is a valuable tool for this purpose.AS defined by PWV is positively correlated with both SBP and DBP. It may indicate subclinical organ damage in patients with hypertension. In addition, the decrease in PWV level during hypertensive therapy can identify a group of patients with better outcomes independently from BP reduction.Obese patients with MS who have lost and regained weight are predisposed to have a significant increase in PWV, above their baseline levels. The adverse effect of metabolic disturbances on arterial function can be offset by an adequate program of exercises, which includes mainly aerobic physical training.The TG/HDL-C ratio demonstrates the ratio between the atherogenic and preventive lipoproteins. The increased TG/HDL-C levels ratio is positively correlated with increased PWV levels and is an independent risk factor for cardiometabolic disease, with a consecutive correlation that was not proved separately for TG and HDL-C levels.IR affects AS and accelerates arterial aging. Non-insulin-based IR index, such as METS-IR, can predict arterial stiffens due to a strong positive correlation with PWV.We have presented a heterogeneous group of studies focused on PWV in patients with MS and its components. It should be highlighted that it is important to take into account the level of cardiovascular risk in the population with MS. In addition, several modalities are available in the assessment of AS including recording the pulse waves by a tonometer transducer, standard BP cuff, doppler ultrasound, and magnetic resonance imaging (MRI). Most of the studies presented have used the tonometry transducer method. However, different points of measurement were taken into consideration, such as carotid, femoral, brachial, and ankle. In addition, the distance between the two surface sites and the time delay between the waveform is used to determine PWV, and it may be impacted by the obesity in measurements.Although this subject is of great interest and the knowledge of the association between AS and MS and its components has increased significantly, further research is necessary, which perhaps will allow the use of the measurement of PWV rroutinely in the treatment of patients with MS. A novel approach and applications of existing drugs to specifically target pathways involved in modulating AS may provide further support to its broader assessment and treatment to improve cardiovascular outcomes.


Short height can bias the diagnosis of wasting when using the weight-for-height indicator. The aim of this study was to evaluate the diagnostic concordance of the anthropometric indicators of wasting and the relationship between wasting and stunting in children from highly vulnerable communities in Guatemala. (2) Methods: The sample consisted of 13,031 anthropometric records of children under five years of age , including weight, height, and mid-upper arm circumference (MUAC), collected in March–August 2019. The proportions of stunting, underweight, and wasting, assessed by three different indicators, as well as their concurrence through the Composite Index of Anthropometric Failure were calculated. (3) Results: Stunting affected 73% of the sample, and 74.2% showed anthropometric failure. Wasting varied by indicator . Concordance between MUAC and weight-for-height was very low . MUAC identified more wasted children in the stunted group (53.6% vs. 26.5%), while the opposite occurred in the non-stunted group (34.8% vs. 46.7%). (4) Conclusion: The presence of stunting affected the diagnosis of wasting, and both indicators should be included as diagnostic criteria for screening campaigns and in the treatment of moderate to acute wasting in vulnerable populations affected by multiple forms of undernutrition. The Central American Dry Corridor region has been the most affected historically, with a continuously high degree of food insecurity due to cyclical drought processes . It is eIn 2019, Guatemala was the second poorest country in Latin America, with 61.1% of its inhabitants living in multidimensional poverty . The couStunting is closely related to delayed neuromotor development. As stated by Guatemala’s government, children with stunted growth: learn to sit, stand, and walk later; have lower cognitive capacity; perform worse in school and are more likely to repeat academic years; miss more days of school due to illness; and are more likely to drop out than children with good nutritional status. It is estimated that, if trends do not change, by 2026, Guatemalan children will have missed almost 33 million equivalent years of learning in school and the country will lose Q146,207 million due to causes directly related to chronic malnutrition [Wasting affects around 0.7% of the child population in Guatemala, but depending on the age group and geographical location, figures above 2% can be registered . CurrentResearch studies have repeatedly shown that both anthropometric indicators identify different population groups, and that the presence of short height could condition the diagnosis of acute malnutrition if the WHZ indicator is used . AccordiThe REDAC study (from the Spanish “Relación entre Desnutrición Aguda y Crónica”) is a cross-sectional observational study funded by the European Civil Protection and Humanitarian Aid Operations Commission (ECHO) as part of a humanitarian food assistance intervention in highly food-insecure communities of Guatemala. The study involved eight humanitarian organizations performing anthropometric monitoring of vulnerable communities from March to August 2019 in 14 municipalities of four departments  selected for the impact of droughts in recent years on rural households’ food insecurity . The parAll of the anthropometrists attended a workshop on harmonization in measurement. The weight (kg), height (cm), and MUAC (mm) of children from 0–59 months were measured following the SMART guidelines for humanitarian interventions  with theFrom these direct measurements, the nutritional status of the participants was calculated based on various anthropometric indicators: stunting or chronic malnutrition was assessed through the height-for-age z-score (HAZ), underweight or global malnutrition through the weight-for-age z-score (WAZ), and wasting or acute malnutrition through the presence of edema or three different anthropometric indicators , and MUAC) as an absolute value with fixed cut-off points (moderate wasting < 125 mm and severe wasting < 115 mm). For HAZ, WAZ, WHZ, and MUACAZ, deviation values were calculated with respect to the World Health Organization Child Growth Standards  (z-scoreThe Composite Index of Anthropometric Failure (CIAF)  was applThe consolidated database initially included 13,124 records. A total of 91 records of children over 59 months and another without sex were eliminated, leaving 13,031 records in the final database. Of those, 49.5% (6447) were girls, and the average age was 27.9 (14.5–42.5) months. A strict data cleaning protocol was applied based on the WHO–UNICEF recommendations for anthropometric surveys : (a) fixp < 0.05. For continuous variables, the normality was assessed using the Kolmogorov–Smirnoff test with Lilliefors correction. Accordingly, Student’s t-test or the Mann–Whitney test was used to compare central parameters. Contingency tables were used to compare proportions, and the chi-square test with Yates correction was applied. The analysis was performed using IBM SPSS v.25 software . All tests were carried out under a bilateral hypothesis, considering a significance level of Univariate logistic regression analysis was applied to quantify the association between the undernutrition categories through the odds ratio (OR). Cohen’s Kappa test was used to analyze the agreement between anthropometric indicators by considering the concordance thresholds proposed by Landis and Koch . Using Wp < 0.001), with WHZ revealing the lowest figures. Regarding the detection of severe wasting cases, WHZ and MUAC detected the same proportion of cases, while MUACAZ identified more than twice as many children.p < 0.001). Analyzing the diagnostic efficacy of MUAC, the results were 40.9% sensitivity, 96.9% specificity, 95.2% accuracy, and 61.0% likelihood ratio. The concordance analysis  showed tp < 0.001) because, in boys, WHZ and MUAC diagnosed a similar proportion of cases , while in the female series, MUAC identified many more cases of wasting .The results confirm the nutritional vulnerability of this sample by finding the prevalence of undernutrition to be markedly higher than the national figures reported in the last National Survey of Maternal and Child Health : 47% of Likewise, 74.2% of the children showed some anthropometric failure. This value is markedly higher than the 51.3% reported for an international study with more than 100,000 children from 14 countries . AnotherAnthropometric failure was significantly higher in boys, coinciding with the results of the meta-analysis published by Thurstants et al.  This pheIn the present study, 1% of cases with the three types of simultaneous undernutrition were recorded (67 boys and 53 girls). Another study with anthropometric surveys from 51 countries showed that the concurrence of all types of undernutrition implies a 12.3-fold increased risk of death versus a normal nourished state, whereas that risk value is 2.3 for those children with wasting only and 1.5 for those with stunting only . Based oThe present study confirmed the low concordance between WHZ and MUAC, which shows a prevalence of wasting of 2.8% and 4.4%, respectively. In another Guatemalan survey with 906 vulnerable children from Zona Reina (Quiché), a lower prevalence of wasting by WHZ (1.8% vs. 2.5%) was also found . ResearcTo explain the population differences in the discrepancy between MUAC and WHZ, it can be thought that MUAC could better reflect wasting in populations with a high prevalence of stunting, which is an indicator that is independent of height. Our study showed that MUAC and MUACAZ identify a higher proportion of children with wasting in the stunted group than WHZ . In contrast, these differences are not as evident in children without stunting . In addition, of all wasting cases identified in the stunted group, the majority were based on MUAC alone (53.6%), while in the non-stunted group, the highest proportion was identified by WHZ alone (46.7%). A similar trend was found in the aforementioned international study (20), where the prevalence of wasting was markedly higher in the group with stunting using MUAC (15.2% vs. 5.9% without stunting), and only slight differences were found using WHZ (13.3% vs. 11.5%). That study reported an overall MUAC–WHZ concordance (K: 0.358) similar to that of the present study (0.310). However, they found that the agreement was higher in the stunted group (K: 0.486 vs. 0.292 without stunting). However, the highest prevalence of acute malnutrition was obtained with MUACAZ, which would reach figures within the high emergency threshold according to the current classification of the WHO (10–15%) . This isThere are pilot experiences in Guatemala with children over five years of age testing the use of MUAC bracelets adapted to introduce age adjustment for the classification of nutritional status . Their qA limitation of the present study is that it included a convenience sample not representative of the territory or the general population, given that the participating communities were selected based on vulnerability criteria. On the contrary, it was an adequate sample for the study’s objective, given that the undernutrition risk, specifically wasting risk, was higher than in the general Guatemalan child population.The study results highlight the need to include MUAC and WHZ as diagnostic criteria for wasting in vulnerable populations with a high prevalence of stunting. This should be considered for epidemiological surveillance, community screening campaigns, and access to therapeutic or nutritional supplementation programs for moderate and acute wasting. Currently, MUAC is not included as a valid criterion in the national surveillance protocols. In the present study, 358 cases with acute malnutrition by MUAC were recorded, which would not have been identified by WHZ and would not have access to the necessary treatment. Additionally, MUAC is accepted for screening in the community, but if a child is identified with moderate wasting by MUAC, it should be confirmed by WHZ at the treatment site. The present study confirmed the low concordance between those indicators and, therefore, a child identified in the community may not be admitted to outpatient treatment. This situation endangers the children’s lives and can cause community disaffection with the nutrition programs committing their attendance in the future. However, the inclusion of MUAC as a diagnostic criterion may lead to an increase in the prevalence of acute malnutrition among the most vulnerable populations and, therefore, may require more resources and personnel for managing cases at the community level.


GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed antibody responses and the association of naturally acquired immunity with incidence of malaria in one of the trial sites, Banfora in Burkina Faso. The analysis included 453  children aged 12-60 months old. Children were followed-up for clinical malaria episodes for 12 months after final vaccine administration. Antibody levels against GMZ2 and eleven non-GMZ2 antigens were measured on days 0 and 84 . Vaccine efficacy (VE) differed by age group , p = 0.0615). During the twelve months of follow-up, VE was 1%  and 23%  in the 12 - 35 and 36 – 60 months old children, respectively. In the GMZ2 group, day 84 anti-GMZ2 IgG levels were associated with reduced incidence of febrile malaria during the follow up periods of 1-6 months (hazard ratio (HR) = 0.87, 95%CI = ) and 7-12 months ) in the 36-60 months old but not in 12-35 months old children. Multivariate analysis involving day 84 IgG levels to eleven non-vaccine antigens, identified MSP3-K1 and GLURP-R2 to be associated with reduced incidence of malaria during the 12 months of follow up. The inclusion of these antigens might improve GMZ2 vaccine efficacy. Plasmodium falciparum candidate vaccine, and is designed with the aim to emulate naturally acquired anti-malarial immunity  and merozoite surface protein (MSP) 3 which are major epitopes for antibodies  involved in NAI remains elusive, we and others have shown that monocyte mediated opsonic phagocytosis (OP) of rozoites , 21, 22 rozoites  are elicrozoites .® . GMZ2/alum was well tolerated and immunogenic in three phase 1 studies . The local Ethics Committees and regulatory authorities for each site and country approved the clinical trial protocol before the start of the trial. Signed informed consent was obtained from parent/guardian of children before their inclusion in the study. The protocol was registered with the Pan African clinical trial registry with registration number ATMR2010060002033537.P. falciparum is responsible for nearly 100% of all clinical malaria cases and children under five years and pregnant women are the populations at highest risk. Study design and details were previously described  children’s specimen collected from Banfora, Burkina Faso in the GMZ2/alum phase 2b clinical trial. Malaria is endemic in Burkina Faso and occurs throughout the year, with seasonal peak between June and October, a period when rainfall is highest. escribed . BrieflyThin and thick blood films were prepared from a finger prick. The thin film was fixed with methanol for a few seconds. Both blood films were then stained with 10% Giemsa stain for 15 minutes for malaria parasite identification and quantification. The stained blood smears were rinsed with running tap water for about 10 seconds and allowed to air dry. Malaria parasites were counted (trophozoites) against 200 white blood cells (WBCs) on the thick film by two independent experienced microscopist using a light microscope under oil immersion at 100x magnification. Negative result was assigned after examining 200 high power fields of the thick film at x100 magnification. Parasite counts were converted to parasites density/μL of blood assuming 8000 WBCs/μL of blood. Malaria species identification was done using thin blood smears.Pf38, Pf12 and MSP1-19) and GMZ2 in a multiplex assay as described elsewhere  -labelled goat antihuman IgG (Jackson Immuno Research) was added at 1:3500 for the detection of IgG bound antibodies and incubated for 1 hour. For the quantification of IgG subclasses, mouse antihuman IgG1 or IgG3 diluted 1:5000 were added followed by a PE-labelled goat antimouse IgG diluted 1:200. Between steps, plates were washed 3 times each with assay buffer E . Mean fluorescent intensity (MFI) was measured with Luminex 200 Bio-Plex analyser .IgG antibody levels were determined against a panel of 11 antigens  and GraphPad Prism version 8. Differences in geometric mean antibody levels were compared using a t-test after log transforming antibody data to base 10. Cox regression was used to estimate vaccine efficacy and to determine the association of antibody levels with incidence of clinical malaria, using a robust standard error to allow for repeated events in the same child. To compare effects by age group and time period, Wald tests were used to assess interactions. To standardise the antibody levels to the 12 antigens, levels were transformed to logarithms and the logged values, x, then transformed to z-scores The phase 2b efficacy trial of GMZ2/alum was conducted at 5 sites in East- West- and Central-Africa . The preP. falciparum mono-infections. The remaining episodes were mixed infections of P. falciparum and P. ovale or P. malariae. Children from the study cohort were stratified into younger (12-35 month) and older (36-60 month) age groups  (During the 12-months of follow-up (after dose 3 of vaccine was administered), 98.9% of the malaria episodes were b trial) . The incWe have previously observed a significant vaccine efficacy (VE) in the ATP analysis . In BanfGMZ2 IgG levels were first compared between vaccine groups at days 0 and 84 , respectively. Levels of GMZ2 IgG were similar between the vaccine groups at day 0, however, at day 84, the GMZ2 group had significantly higher levels than the rabies group  . In the rabies group, there was no association between GMZ2 IgG levels and incidence of malaria in any of the age groups at any of the defined follow up periods  (Recently we developed a bead-based phagocytosis assay (BPA) to measure the functional activity of antibodies against distinct merozoite surface antigens . The GMZFirst, we used a flow cytometry-based immunofluorescence assay (FC-IFA) to quantify anti-merozoite antibodies . In eachCollectively, these findings are consistent with the notion that anti-merozoite immunity develops in the present study population irrespective of vaccine group.Having shown through the FC-IFA that natural exposure boosted anti-merozoite IgG responses in the study population, we next sought to delineate the potential specific merozoite antigens involved. Levels of merozoite specific antibodies were measured against a panel of 11 merozoite antigens not present in GMZ2 Table 2.In summary, we showed VE in Banfora increased with increasing age of the children at enrolment, and older children (36-60 months) benefitted most from GMZ2/alum vaccination in the 12 months of follow-up. We further showed that naturally acquired antibodies to MSP3-K1 and GLURP-R2 measured at day 84 , were associated with reduced malaria incidence in the study population during the trial period. Incorporation of these antigens in some form into future GMZ2 designs may help improve VE.Considering all sites in the GMZ2 efficacy study, we observed that children in the GMZ2/alum group with high levels of GMZ2 IgG had a lower incidence of clinical malaria, after adjusting for age, compared with children with low levels . Here, wWe found that increasing anti-GMZ2 IgG levels were associated with reduced incidence of febrile malaria in older children 36-60 months of age during the first 12 months of follow-up. However, these analyses did not establish anti-GMZ2-IgG as the sole correlate of vaccine protection as they did not exclude potential confounders such as acquisition of antibodies to other blood stage antigens and age-dependent maturation of cell mediated immunity . AlthougWe further observed that older children had lower parasite densities during febrile malaria attacks than the younger ones and that this difference was most pronounced in the GMZ2 vaccine group. This finding is consistent with observations that the parasite threshold at which fever is triggered depends on the age of the affected child. Older individuals were found to have a much lower pyrogenic threshold compared to younger ones , 39. WheIt has previously been proposed that multiple anti-merozoite antibody specificities act in concert to provide protection against clinical malaria , 20 afteP. falciparum parasites with different distributions among different regions and the finding that parasites may evolve over time possibly as a result of immune selection [reviewed in , the German Federal Ministry of Education and Research  and Ministry of Foreign Affairs of Denmark (DFC file no.14-P01-GHA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Opportunistic infections remain a significant cause of morbidity and mortality after kidney transplantation. This retrospective cohort study aimed to assess the incidence and predictors of post-transplant DNA virus infections  in kidney transplant recipients (KTR) at a single tertiary centre and evaluate their impact on graft outcomes. Methods: KTR transplanted between 2000 and 2021 were evaluated. Multivariate logistic regression analysis and Cox proportional hazard analyses were used to identify factors associated with DNA virus infections and their impact on allograft outcomes respectively. A sub-analysis of individual viral infections was also conducted to describe the pattern, timing, interventions, and outcomes of individual infections. Results: Data from 962 recipients were evaluated . 30% of recipients (288/962) had infection(s) by one or more of the DNA viruses. Individually, CMV, EBV, BKV and JCV viruses were diagnosed in 13.8%. 11.3%, 8.9% and 4.4% of recipients respectively. Factors associated with increased risk of post-transplant DNA virus infection included recipient female gender, higher number of HLA mismatch, lower baseline estimated glomerular filtration rate (eGFR), CMV seropositive donor, maintenance with cyclosporin (rather than tacrolimus) and higher number of maintenance immunosuppressive medications. The slope of eGFR decline was steeper in recipients with a history of DNA virus infection irrespective of the virus type. Further, GFR declined faster with an increasing number of different viral infections. Death-censored graft loss adjusted for age, gender, total HLA mismatch, baseline eGFR and acute rejection was significantly higher in recipients with a history of DNA virus infection than those without infection ). In contrast, dialysis-free survival did not differ between the two groups of recipients . Conclusion: Post-transplant DNA viral infection is associated with a higher risk of allograft loss. Careful management of immunosuppression and close surveillance of at-risk recipients may improve graft outcomes. Patients who undergo solid organ transplantation have a significantly higher risk of developing infections and infection-related complications than the general population . InfectiPathogens that commonly infect transplant recipients include viruses, bacteria, and protozoa. Of these, viruses pose the biggest threat . Viruses+ myeloid progenitor cells as well as in CD14+ lymphocytes, monocytes, dendritic cells, and megakaryocytes [The most common of these virus infections is CMV, a double-stranded DNA virus of the Herpesviridae family with a seroprevalence rate of 70–90% in the general adult population . The dorryocytes . Reactivryocytes . Charactryocytes . In addiryocytes . The incEBV, a double-stranded DNA gamma herpesvirus, is another ubiquitous pathogen seroprevalent in more than 90% of the adult population. It persists within B lymphocytes without active infection in the majority of hosts. In transplant recipients, however, newly acquired or reactivated latent infections can cause invasive diseases. The spectrum of such diseases range from non-neoplastic asymptomatic viral replication through EBV end-organ diseases  to EBV-mediated post-transplant lymphoproliferative disorder (PTLD) . There iUntil recently, polyomavirus infections were not recognized as common sequelae of transplantation. However, rates of infection in renal transplant patients have increased in recent decades with the advent of newer and more potent immunosuppressive agents. Incidence rates now range from 10–60% . The twoRecent advances in immunosuppressive therapy have reduced the incidence of acute rejection, but unfortunately, have worsened post-transplant infection rates , hence tThis study therefore aims to assess the incidence and risk factors and outcome of CMV, EBV, BKV and JCV infection in a large cohort of kidney transplant recipients transplanted and managed over a 20-year period.In this retrospective cohort study, we collected data from 962 subjects who received kidney transplantation between 01 January 2000 and 31 December 2021 and who were followed at a tertiary nephrology centre, the Salford Royal Hospital, Northern Care Alliance NHS Foundation Trust . Subjects are usually repatriated to our hospital after a period of typically 3–4 months following transplantation at the regional transplant centre. Repatriation of some recipients might be delayed beyond the fourth month post-transplantation if there were early post-transplant complications that required close monitoring such as severe acute rejection, ureteric stenosis, or allograft vascular abnormalities. We excluded subjects who suffered graft loss within 3 months post-transplantation.Demographic, clinical and laboratory data were extracted from the electronic patient records (EPR). The data were collected until one of the following clinical endpoints occurred: graft loss, death, loss to follow-up, or the end of the study period (31 December 2021) which ever occurred first. Infections were identified from virology reports identifying patients who had developed a positive polymerase chain reaction (PCR) test for CMV, EBV, BKV or JCV virus since the date of their latest transplantation.Preimplantation transplant factors included preemptive transplantation, donor type, Human leucocyte antigen (HLA) mismatch and donor and recipient CMV status. perioperative factors included cold ischemia time and post-operative factors such as immunosuppression regimen, history of acute rejection, post-transplant cardiovascular events malignancy and baseline estimated glomerular filtration rate (eGFR) (measured at 3 months post-transplantation) were also recorded. eGFR was calculated using the Modification of Diet in Renal Disease (MDRD) Study equation.Post-transplant outcomes comprised of death censored graft survival (DCGS), dialysis-free survival (DFS) and death with a functioning allograft.The overall rate of change in eGFR (delta eGFR) was compared between the four viral infection groups as well as between patients with any post-transplant viral infection (infection group), and patients without any post-transplant viral infection (non-infection group).Death-censored graft loss (DCGL) was defined as a return to dialysis or re-transplantation. DFS was defined as a composite of DCGL and death with functioning graft.Cardiovascular disease was defined as a composite of myocardial infarction, coronary artery disease, stroke, heart failure or peripheral vascular disease. Heart failure was defined as the clinical symptoms of ventricular dysfunction such as dyspnea and raised jugular venous pressure and/or bibasilar crackles corroborated by elevated natriuretic peptide levels and clinical or radiological evidence of pulmonary or systemic congestion . PVD wasAcute and chronic rejections were confirmed by kidney transplant biopsy .All post-transplant malignancies were captured according to clinical history. Non-melanoma skin cancers were excluded from the study.All subjects were treated with CNI-based maintenance immunosuppression as either tacrolimus or cyclosporin. Those without contraindications also received anti-proliferative immunosuppression, mycophenolic acid (MPA) or azathioprine (AZA). Those at high immunologic risk were also maintained on glucocorticoids.We defined symptomatic infections as those that were associated with clinical signs and symptoms or laboratory evidence of organ dysfunction. In contrast, asymptomatic infections are those detected via routine viral screens without any associated clinical or other laboratory abnormalities.For the individual viral strains, symptomatic CMV was defined as those with clinical or laboratory evidence of localized CMV infection , and/or disseminated disease (affecting multiple organ systems).Symptomatic EBV was defined as the occurrence of mononucleosis or PTLD. Whereas symptomatic BK virus infection was defined as BK viremia associated with one or more of the following: haemorrhagic cystitis, ureteric stenosis or biopsy proven BK nephropathy, symptomatic JCV infection was defined as the occurrence of one or both of progressive multifocal leukoencephalopathy and JCV nephropathy.BKV and JCV nephropathy was diagnosed if there was histologic evidence of polyoma-associated nephropathy following a clinically indicated kidney biopsy.Monitoring for CMV, EBV, BK and JCV was usually undertaken monthly for the first 6 months or until repatriation from the regional transplant centre. Thereafter there was no routine policy for regular testing of patients during follow-up, but viral PCR testing was usually performed annually or if clinically indicated. More frequent testing to monitor viremia was conducted if an infection was confirmed.Viral PCR testing was undertaken in one central virology laboratory using a TaqMan PCR-based approach. The CMV PCR assay was conducted using the real-time Taqman PCR assay for CMV which was first introduced in 1999 . The CMV7 IU/mL [Similarly, EBV real-time TaqMan PCR, using 100µL of patient blood and able to detect viral DNA over a linear span of between 100 and 107 IU/mL . HoweverBK and JC polyomavirus assay was also carried out using the real-time TaqMan PCR which targeted the large T antigen (between 1999 and 2019). In 2019 it was redesigned to detect the VP2 gene of both BK and JC virus. The limit of sensitivity for BK and JC virus detection was 50 IU/mL.All samples were processed with Qiagen MDX using a bespoke extraction which is suitable for all sample types, and amplification was carried out using Thermo 7500 real-time instruments. Assays were considered positive if test results showed a viral DNA count greater than or equal to the limit of sensitivity for the virus assayed.t-test was used for parametric variables and Wilcoxon’s sign-rank test for non-parametric variables. Categorical variables were summarized as frequencies and percentages and compared using Pearson’s chi-squared or Fisher’s exact test.The cohort was first divided into 2 groups. The infection group consisted of subjects who had at least one episode of infection by any of the index viruses and the non-infection group consisted of subjects who experienced none of the evaluated infections. We also conducted a sub-analysis of individual DNA viral infections  to determine their incidence, time to infection, type of clinical disease, complications, treatment, and allograft outcomes. Continuous variables were presented as mean and standard deviation for normally distributed data or median and interquartile range for skewed data. For group comparison, the two-tailed p-value less than 0.20 remained in the model.For the evaluation of possible risk factors of DNA virus infection, multivariate logistic regression analysis was conducted. Covariates were chosen based on previous knowledge and if they satisfied the criteria for being a potential confounder . The varp-value < 0.05. All analysis were conducted using Stata version 14 statistical software package .Finally, two separate multivariate Cox-proportional hazards regression models were used to compare DCGS and DFS between recipients who developed viremia and those who did not and adjusted for the effects of confounders including age, gender, number of HLA mismatches, baseline eGFR, and history of biopsy-proven acute rejection (BPAR). Of those who experienced viremia, we estimated the probability of DCGL stratified by the virus subtypes using the Kaplan–Meier method. Statistical significance was defined as The study complies with the declaration of Helsinki and was registered with the Research and Innovation department of the Northern Care Alliance NHS Group (Ref: S21HIP03) who approved the methodological protocol as outlined above. As this was a retrospective observational study based on routinely collected and fully anonymised data, the need for individual patient consent was waived by the Research and Innovation review committee, who granted study approval.The demographic and clinical characteristics of the cohort are summarised in 2), and to have a higher average uPCR in the first year (20 vs. 15 mg/mmol). Furthermore, recipients with viremia had a significantly lower average haemoglobin concentration over the period of follow-up (123 vs. 126 g/L) and were more likely to be on long-term steroid therapy than those who did not experience viremia. However, we suspect that this association may have been a consequence of viremia rather than the cause, as viral infections are frequently managed by discontinuation of antimetabolites (MMF) and replaced with corticosteroids. The low haemoglobin and raised uPCR may have been consequences of viremia and viremia-induced loss of allograft function.30% of recipients (288) had at least one DNA virus infection (viremia group). There was no significant difference in age, sex, ethnicity, BMI or primary kidney disease between the viremia and non-viremia groups . However10 4.6 vs. 3.1 IU/mL; p < 0.001) whereas EBV, BKV and JCV viral counts were not different between symptomatic and asymptomatic infections. Furthermore, those with higher viremia log values were more likely to receive escalated interventions during follow-up, notably a combination of immunosuppression reduction and antiviral agents , and higher baseline eGFR,  per 10 mL/min increase. Conversely factors associated with increased odds of viremia include CMVD+R- serostatus , CMVD+R+ , Total HLA mismatch , cyclosporin as CNI rather than tacrolimus  and number of maintenance immunosuppressive medications  for each additional agent.Sixteen variables were included in the initial multivariate logistic regression model. By stepwise backward elimination of covariates with Over the follow-up period, 87 out of 962 recipients (9%) lost their graft and 161/962 (17%) died with functioning grafts. The most common causes of graft loss included chronic allograft injury (CAI) (38%), acute rejection (24%), and recurrent glomerulonephritis (14%).p = 0.042). Post-transplant malignancy was also more common in recipients who had viremia than in the non-viremia group (11% vs. 7% p = 0.14). Incidence of acute rejection, and new-onset diabetes mellitus after transplantation (NODAT) did not differ between the groups . Individually, recipients who had CMV, EBV, BKV and JCV viremia all showed a steeper rate of eGFR loss with JCV having the greatest decline (2.40 mL/min/1.73 m2/year), followed by CMV (1.50 mL/min/1.73 m2/year), EBV (1.36 mL/min/1.73 m2/year) and BKV (1.19 mL/min/1.73 m2/year), The eGFR slopes became steeper as the number of infection types increased shown in B suggestFor each of the virus strains, recipients with symptomatic infections showed a higher rate of eGFR decline compared to those with asymptomatic infections. Mean slope of eGFR decline in symptomatic vs. asymptomatic patients was −1.89 vs. −0.87 mL/min/year for CMV, −3.66 vs. −1.83 mL/min/year for EBV, −2.13 vs. −1.73 mL/min/year for BKV and −3.47 vs. −3.17 mL/min/year for JCV. Dialysis-free survival was also lower for recipients who experienced symptomatic viral infections compared to those who were asymptomatic. For instance, for recipients with symptomatic vs. asymptomatic CMV infections, this was 12.8 vs. 13.9 years. For recipients who experienced EBV infection, dialysis-free survival was 11 years for symptomatic and 15 years for asymptomatic infections. A similar result was noted for BKV (9.7 vs. 15.6 years) and JCV (2.6 vs. 14.7 years).p = 0.024) (p = 0.038)  a. Howeve= 0.038) b.p < 0.001 whereas EBV, BK and JCV viral counts did not differ between complicated and asymptomatic infections. Our study revealed a higher rate of eGFR decline in recipients with a history of viremia compared to those with no history of viremia. Recipients who experienced more than one type of viremia were also more likely to have a more rapid loss of eGFR, and a greater incidence of post-transplant cardiovascular disease and malignancy. Death-censored graft loss adjusted for confounders was significantly higher in recipients with a history of viremia than those without but there was no difference in dialysis-free survival between the 2 groups.In this retrospective single-center cohort study of 962 kidney transplant recipients, we explored the factors associated with post-transplant DNA viremia and the prognostic impact of experiencing these infections on allograft and recipient outcomes. 30% of kidney allograft recipients in our cohort experienced at least one episode of CMV, EBV, BKV or JCV infection over the period of follow-up. A quarter (71/288) of those with a history of viremia had more than one type of viral infection. Male gender and higher baseline creatinine were associated with a lower risk of viremia whereas pretransplant donor-positive CMV serology (irrespective of recipient CMV status), cyclosporin maintenance therapy and high immunosuppression burden were associated with the development of viremia. CMV viremia was the most commonly occurring of the four DNA viral infections, followed by EBV and polyomaviruses. The time to infection post-transplant was shortest with CMV infection (9 months) and longest with EBV (45 months). A quarter of patients with CMV infections experienced symptomatic organ dysfunction of which gastroenteritis was the most common feature. Less than 10% of patients with EBV (7%) or polyoma viremia (6%) suffered symptomatic disease. Symptomatic CMV viremia was associated with higher viral DNA count compared to asymptomatic infection (log 5.4 vs. 4.0) A similar study by McCaffrey et al. evaluated the same four viral infections in a small cohort (98 patients) of paediatric transplant patients and reported a two-fold higher overall incidence of viremia, 67.3% ,23. The The reported incidence of individual virus types differed amongst previous studies due to differing surveillance practices, duration of follow up and sensitivity of the assay. Furthermore, prospective studies tend to report a higher incidence of viremia than retrospective studies most likely due to a more proactive (research-oriented) surveillance procedure. Previous studies estimated the incidence of CMV in kidney transplant populations to be between 8% and 32%  which isAs has been reported previously , it was According to our results, recipients of CMV donor-positive allografts are at greater risk of contracting viremia than those who received CMV donor-negative allografts. This is consistent with findings of the study by McCaffrey et al.  and showp = 0.045 [There is a paucity of evidence on the impact of gender on the risk of viremia in transplant recipients. However, evidence from studies on immunocompetent populations suggests significant differences between the sexes in their immune responses to infections. This was attributed to the effects of sex hormones on females as well as the extra X chromosome in females ,28. Comp = 0.045  One explOur finding of decreased odds of infection with higher eGFR broadly supports the widely known fact that lower kidney function is associated with poorer immune response ,31. Low Unlike some previous studies ,34, we dOur study found that in all four DNA virus groups, viremia was associated with higher average annual decline in eGFR when compared to the non-viremia groups A. This iPre-transplant screening enables the risk stratification of patients especially as regards CMV and EBV viremia. Prophylactic antiviral agents  for CMV are commonly given to at risk individuals for the first 3 months after transplantation in most transplant centers. These are usually recipients with CMV D+R− serostatus and those who received a lymphocyte depleting agent at immunosuppression induction. Based on the result of this study, a case could be made for extending CMV prophylaxis to include all recipients of CMV donor positive allografts (CMV D+R− and CMV D+R+). The duration of antiviral prophylaxis aims to strike a balance between prevention of viremia and development of antiviral resistant strains. Most centers offer 3 months of prophylaxis. However, considering that the median time to CMV infection in our cohort was nine months, increasing the duration of prophylaxis is a worthy consideration.A significant finding from this study was that viremia adversely affected death-censored graft survival. Previous studies have reported conflicting results about the impact of viremia on graft outcomes. For instance, Blazquez-Navarro et al., noted that patients with viremia had significantly lower GFR in the first-year post-transplant compared to those with no viremia . On the A major strength of this study was the relatively large number of study subjects investigated, the detailed analysis of relevant baseline characteristics as well as post-transplant outcomes. Nevertheless, there are limitations that need to be acknowledged, first of which is the retrospective nature of the study. Secondly, viral surveillance beyond six months post-transplantation was inconsistent and depended on the supervising physician. This meant that asymptomatic and transient infections may have been undiagnosed and unrecorded. As a result, the true prevalence of viremia may have been under-represented by the observed prevalence. Thirdly, for some of the subjects, data from the regional transplant center where they were followed for the first 3–6 months post-transplantation were incomplete or unavailable. For these subjects, data on early post-transplant viremia was not available for analysis. Despite these limitations, our study evaluated the incidence of four key viral infections in an adult KTR population cohort. As far as we know, these infections have not been evaluated together previously.In conclusion, one third of our cohort of kidney transplant recipients had a history of post-transplant DNA viremia. Donor CMV positive serostatus, the type and burden of immunosuppression, female gender and low baseline allograft function were independent predictors of risk of viremia. Viremia was associated with a decline in allograft function and a higher risk of graft loss. In addition, GFR loss was faster when patients experienced multiple different types of viral infection. These findings highlight the need for adequate pre-transplant risk stratification, optimal prevention strategies, effective surveillance and appropriate intervention including those in recipients with low viral counts.