Prevalence and risk factors of diabetes mellitus and hypertension in North East Tunisia calling for efficient and effective actions.

Diabetes and hypertension are a serious public health problem worldwide. In the last decades, prevalence of these two metabolic diseases has dramatically increased in the Middle East and North Africa region, especially in Tunisia. This study aimed to determine the prevalence of type 2 diabetes (T2D) and High Blood Pressure (HBP) in Zaghouan, a North-East region of Tunisia. To this end, an exploratory study with stratified random sampling of 420 participants has been carried out. Various data were collected. Blood samples and urine were drawn for biochemical assay. Then, all data were analyzed using the statistical R software. Results showed an alarming situation with an inter-regional difference in prevalence of obesity (50.0%, CI 95.0%), HBP (39.0%, CI 95.0%) and T2D (32.0%, CI 95.0%). This study allowed the discovery of 24, 17 and 2 new cases of T2D, HBP and T2D&HBP respectively. The association of some socio-economic factors and biochemical parameters with these chronic diseases has been highlighted. To conclude, the health situation in the governorate of Zaghouan requires urgent interventions to better manage the growing epidemic of non-communicable diseases (NCD) in the region. This study demonstrated the importance of engaging health policy makers in road mapping and implementing national NCD prevention programs.

The first exome wide association study in Tunisia: identification of candidate loci and pathways with biological relevance for type 2 diabetes.

Introduction: Type 2 diabetes (T2D) is a multifactorial disease involving genetic and environmental components. Several genome-wide association studies (GWAS) have been conducted to decipher potential genetic aberrations promoting the onset of this metabolic disorder. These GWAS have identified over 400 associated variants, mostly in the intronic or intergenic regions. Recently, a growing number of exome genotyping or exome sequencing experiments have identified coding variants associated with T2D. Such studies were mainly conducted in European populations, and the few candidate-gene replication studies in North African populations revealed inconsistent results. In the present study, we aimed to discover the coding genetic etiology of T2D in the Tunisian population. Methods: We carried out a pilot Exome Wide Association Study (EWAS) on 50 Tunisian individuals. Single variant analysis was performed as implemented in PLINK on potentially deleterious coding variants. Subsequently, we applied gene-based and gene-set analyses using MAGMA software to identify genes and pathways associated with T2D. Potential signals were further replicated in an existing large in-silico dataset, involving up to 177116 European individuals. Results: Our analysis revealed, for the first time, promising associations between T2D and variations in MYORG gene, implicated in the skeletal muscle fiber development. Gene-set analysis identified two candidate pathways having nominal associations with T2D in our study samples, namely the positive regulation of neuron apoptotic process and the regulation of mucus secretion. These two pathways are implicated in the neurogenerative alterations and in the inflammatory mechanisms of metabolic diseases. In addition, replication analysis revealed nominal associations of the regulation of beta-cell development and the regulation of peptidase activity pathways with T2D, both in the Tunisian subjects and in the European in-silico dataset. Conclusions: The present study is the first EWAS to investigate the impact of single genetic variants and their aggregate effects on T2D risk in Africa. The promising disease markers, revealed by our pilot EWAS, will promote the understanding of the T2D pathophysiology in North Africa as well as the discovery of potential treatments.

Whole-exome sequencing reveals novel variants of monogenic diabetes in Tunisia: impact on diagnosis and healthcare management.

Introduction: Monogenic diabetes (MD) accounts for 3%-6% of all cases of diabetes. This prevalence is underestimated due to its overlapping clinical features with type 1 and type 2 diabetes. Hence, genetic testing is the most appropriate tool for obtaining an accurate diagnosis. In Tunisia, few cohorts of MD have been investigated until now. The aim of this study is to search for pathogenic variants among 11 patients suspected of having MD in Tunisia using whole-exome sequencing (WES). Materials and methods: WES was performed in 11 diabetic patients recruited from a collaborating medical center. The pathogenicity of genetic variation was assessed using combined filtering and bioinformatics prediction tools. The online ORVAL tool was used to predict the likelihood of combinations of pathogenic variations. Then, Sanger sequencing was carried out to confirm likely pathogenic predicted variants among patients and to check for familial segregation. Finally, for some variants, we performed structural modeling to study their impact on protein function. Results: We identified novel variants related to MD in Tunisia. Pathogenic variants are located in several MODY and non-MODY genes. We highlighted the presence of syndromic forms of diabetes, including the Bardet-Biedl syndrome, Alström syndrome, and severe insulin resistance, as well as the presence of isolated diabetes with significantly reduced penetrance for Wolfram syndrome-related features. Idiopathic type 1 diabetes was also identified in one patient. Conclusion: In this study, we emphasized the importance of genetic screening for MD in patients with a familial history of diabetes, mainly among admixed and under-represented populations living in low- and middle-income countries. An accurate diagnosis with molecular investigation of MD may improve the therapeutic choice for better management of patients and their families. Additional research and rigorous investigations are required to better understand the physiopathological mechanisms of MD and implement efficient therapies that take into account genomic context and other related factors.

SARS-CoV-2 Serology: Utility and Limits of Different Antigen-Based Tests through the Evaluation and the Comparison of Four Commercial Tests.

Introduction: SARS-CoV-2 serology have several indications. Currently, as there are various types available, it is important to master their performance in order to choose the best test for the indication. We evaluated and compared four different commercial serology tests, three of them had the Food and Drug Administration Emergency Use Authorization (FDA-EUA). Our goal was to provide new data to help guide the interpretation and the choice of the serological tests. Methods: Four commercial tests were studied: Elecsys® Roche® on Cobas® (total anti-nucleocapsid (N) antibodies), VIDAS® Biomerieux® (IgM and IgG anti- receptor binding domain (RBD) antibodies), Mindray® (IgM and IgG anti-N and anti-RBD antibodies) and Access® Beckman Coulter® (IgG anti-RBD antibodies). Two panels were tested: a positive panel (n = 72 sera) obtained from COVID-19-confirmed patients with no vaccination history and a negative panel (n = 119) of pre-pandemic sera. The analytical performances were evaluated and the ROC curve was drawn to assess the manufacturer's cut-off for each test. Results: A large range of variability between the tests was found. The Mindray®IgG and Cobas® tests showed the best overall sensitivity, which was equal to 79.2% CI 95% (67.9−87.8). The Cobas® test showed the best sensitivity after 14 days of COVID-19 molecular confirmation; which was equal to 85.4% CI 95% (72.2−93.9). The Access® test had a lower sensitivity, even after day 14 (55.5% CI 95% (43.4−67.3)). The best specificity was noted for the Cobas®, VIDAS®IgG and Access® IgG tests (100% CI 95% (96.9−100)). The IgM tests, VIDAS®IgM and Mindray®IgM, showed the lowest specificity and sensitivity rates. Overall, only 43 out of 72 sera (59.7%) showed concordant results by all tests. Retained cut-offs for a significantly better sensitivity and accuracy, without significant change in the specificity, were: 0.87 for Vidas®IgM (p = 0.01) and 0.14 for Access® (p < 10−4). The combination of Cobas® with Vidas® IgM and IgG offered the best accuracy in comparison with all other tests combinations. Conclusion: Although using an FDA-EUA approved serology test, each laboratory should carry out its own evaluation. Tests variability may raise some concerns that seroprevalence studies may vary significantly based on the used serology test.

Presumed Protective Role for Anti-Hepatitis B Virus Antibodies Against COVID-19 Severe Cases: A Clinical Study Confirming in silico Hypothesis.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID-19 disease which is known to have a broad clinical spectrum, from asymptomatic to critical presentation leading to death. Many researchers have investigated the factors impacting the course of the disease. Our previous in silico study suggested a possible protective effect of Hepatitis B, Tetanus and Measles vaccines against COVID-19. In continuity, we conducted a cross-sectional clinical study in order to confirm our in silico assumptions regarding the HBs-Ag antibodies. Methods: A representative sex- and age-matched sample of patients with confirmed COVID-19 was selected (n = 340). All clinical presentations were equally represented. Using an ELISA test, each patient benefited of a serology for the detection and measurement of the anti-HBs specific IgG antibodies. The obtained results allowed determining the different correlations between these antibody titers and the disease severity. The R® software and the MedCalc® software served to calculate the Spearman's coefficient of rank correlation (rho) for the obtained titers per severity group as well as the different other calculations and figure representations. Results: A significant positive correlation was found with the anti-HBs titers (rho = 0.107; p = 0.04). High anti-HBs titers were significantly associated with the mild presentation of COVID-19. A significant difference was found between the obtained titers per severity class (chi-2 test, p = 0.03). Discussion/Conclusion: Our findings demonstrated that anti-HBs titers were significantly higher for patients having mild COVID-19 presentations. We presume that being immunized against the HB may play a protective role in the course of the disease. Our study provided more key elements in understanding the disparity of the clinical spectrum among regions.

Vitamin D in healthy Tunisian population: Preliminary results.

Background: Vitamin D deficiency is one of the most common medical conditions worldwide. In Tunisia, several studies evaluated Vitamin D status, but this was concerning specific populations (pregnant women, obese or diabetic patients and children with asthma). The only study that evaluated Vitamin D status in a healthy Tunisian population was conducted by Meddeb and associeties in 2002. The update of data available, based on the currently recommended limits, is necessary. This study aimed to estimate the prevalence of hypovitaminosis D in a healthy Tunisian population, and correlate the values with potential risk factors. Methods: It was conducted on 209 Tunisian healthy subjects. Data collected included clinical characteristics and dietary intakes. We measured 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), glycemia, creatinine, calcium, phosphorus, and alkaline phosphatase concentrations. Hypovitaminosis D was retained for 25(OH)D concentrations <75 nmol/L. Vitamin D deficiency was defined by 25(OH)D concentrations <25 nmol/L. Results: The prevalence of hypovitaminosis D and vitamin D deficiency were respectively 92.3% and 47.6%. The main factors that were significantly associated with low vitamin D levels in our multivariate analysis were veiling, living in rural areas and sunscreen use. However, sex, age, socioeconomic level, phototype, solar exposure score, smoking and bone mass index, were not statistically associated with hypovitaminosis D. The study of relationship between vitamin D status and serum PTH levels showed a significative and negative correlation (P < 0.005). Conclusions: Given the high prevalence of vitamin D, an adapted health policy is essential. A widespread vitamin D supplementation and food fortification seems to be necessary in Tunisia.

The Role of Dietary Intake in Type 2 Diabetes Mellitus: Importance of Macro and Micronutrients in Glucose Homeostasis.

The prevalence of Type 2 diabetes (T2D) is increasing worldwide. Genetics and lifestyle, especially diet, are contributing factors. Analyses of macro- and micronutrient intake across global populations may help to explain their impact on glucose homeostasis and disease development. To this end, 420 Tunisians were enrolled in a prospective cross-sectional study of daily food consumption. Various data were collected and blood samples were drawn for biochemical assay. A 24-h recall questionnaire was obtained from participants to evaluate dietary intake. Statistical analyses were conducted using Nutrilog and R software. Biochemical analyses stratified the studied population (n = 371) into three groups: diabetics (n = 106), prediabetics (n = 192) and controls (n = 73); 49 subjects were excluded. Our results showed that Tunisians had hypercaloric diets high in carbohydrates and fat with variability in the levels of some vitamins and minerals, including riboflavin and niacin, that were statistically different among groups. The lower intake of vitamin D was associated with a greater risk of T2D. Higher vitamin A and sodium intake were associated with poor glucose homeostasis, although protein intake may improve it. In perspective, nutrigenomic studies can provide insight into problematic diets and poor eating habits and offer opportunities to analyze the effects of behavioral changes that can mitigate T2D development and progression.

SARS-CoV2 RT-PCR assays: In vitro comparison of 4 WHO approved protocols on clinical specimens and its implications for real laboratory practice through variant emergence.

INTRODUCTION: RT-PCR testing on nasopharyngeal swabs is a key component in the COVID-19 fighting, provided to use sensitive and specific SARS-CoV2 genome targets. In this study, we aimed to evaluate and to compare 4 widely used WHO approved RT-PCR protocols on real clinical specimens, to decrypt the reasons of the diverging results and to propose recommendations for the choice of the genome targets. METHODS: 260 nasopharyngeal samples were randomly selected among the samples tested between Week-16, 2020 and week-16 2021, in the Institut Pasteur de Tunis, Tunisia, one of the referent laboratories of COVID-19 in Tunisia. All samples were tested by Charité, Berlin protocol (singleplex envelop (E) and singleplex RNA-dependent RNA polymerase (RdRp)), Hong Kong Universiy, China protocol (singleplex nucleoprotein (N) and singleplex Open reading frame Orf1b), commercial test DAAN Gene® (using the CDC China protocol), (triplex N, Orf1ab with internal control) and Institut Pasteur Paris protocol (IPP) (triplex IP2(nsp9) and IP4 (nsp12) with internal control). For IPP, a selection from samples positive by IP2 but negative with IP4 was re-tested by exactly the same protocol but this time in singleplex. New results were described and analyzed. RESULTS: In vitro analysis showed discordant results in 29.2% of cases (76 out of 260). The most discordant protocol is DAAN Gene® due to the false positive late signals with N target. Discordant results between the two protocol's targets are more frequent when viral load are low (high Ct values). Our results demonstrated that the multiplexing has worsened the sensitivity of the IP4 target. CONCLUSION: We provide concise recommendations for the choice of the genome targets, the interpretation of the results and the alarm signals which makes suspect a gene mutation.

Multiallelic Rare Variants in BBS Genes Support an Oligogenic Ciliopathy in a Non-obese Juvenile-Onset Syndromic Diabetic Patient: A Case Report.

Juvenile-onset diabetes may occur in the context of a rare syndromic presentation, suggesting a monogenic etiology rather than a common multifactorial diabetes. In the present study, we report the case of a young diabetic Tunisian patient presenting learning problems, speech deficits, short stature, brachydactyly, and a normal weight. Whole exome sequencing analysis revealed five heterozygous genetic variants in BBS1, BBS4, BBS8, MKS1, and CEP290. These genes are involved in the regulation of cilium biogenesis and function. We analyzed variant combinations pathogenicity using the recently developed ORVAL tool, and we hypothesized that cumulative synergetic effects of these variants could explain the syndromic phenotype observed in our patient. Therefore, our investigation suggested a genetic diagnosis of Bardet-Biedl syndrome with an oligogenic inheritance pattern rather than a monogenic diabetes. Although there is no curative therapy for this ciliopathy at the moment, a genetic diagnosis may offer other supportive care options, including the prevention of other possible clinical manifestations of this syndrome, mainly renal abnormalities, obesity, liver fibrosis, and hypertension, as well as the genetic counseling for family members.

Effects of Zinc supplementation on serum copper to Zinc and CRP to albumin ratios in hemodialysis patients.

BACKGROUND: Zinc (Zn) deficiency is a common condition and could contribute to poor outcomes in hemodialysis (HD) patients. The aim of this study was to evaluate the effects of Zn supplementation on serum copper (Cu) to Zn and C-reactive protein (CRP) to albumin ratios (CAR) in HD patients. METHODS: Seventy-seven HD patients were enrolled in a multicentre simple-blind randomized clinical trial. Only 37 HD patients completed the study; they were randomly divided into two groups and supplemented with zinc sulphate (n=17) or placebo (n=20) for two months. Serum Zn and Cu were measured by atomic absorption spectrophotometry. Serum albumin and hypersensitive-CRP were assessed by colorimetric and immunoturbidimetric method, respectively. Determinations were performed before and after supplementation. RESULTS: After two months of supplementation, serum Zn significantly increased, and Cu to Zn ratio decreased in Zn supplemented group, but remained unchanged in the placebo group. In parallel, serum albumin concentrations significantly increased, and CAR decreased in Zn supplemented group only. CONCLUSIONS: Zn supplementation reduces Cu to Zn and CRP to albumin ratios in HD patients. These changes point towards an improvement in nutritional, oxidative and inflammatory status. The study findings suggest that correcting Zn deficiency reduces poor outcomes in HD patients.

Correlation of Fgf23 and Balp with Bone Mineral Density in Hemodialysis Patients.

BACKGROUND: Chronic kidney disease (CKD) is associated with numerous complications such as bone mineral disorder. The aim of our study was to analyze the correlation of bone turnover markers with Bone Mineral Density (BMD) measurements in Tunisian end stage renal diseases (ESRD) patients. METHODS: This study included 100 ESRD Tunisian patients. Their estimated glomerular filtration rate (eGFR) was < 15 mL × min-1 × (1.73 m2)-1, which requires hemodialysis. Bone-specific alkaline phosphatase (BALP) serum concentration was determined with a chemiluminescence immunoassay. Fibroblast Growth Factor 23 (FGF23) serum was assessed by Enzyme-Linked Immunosorbent Assay method. The serum levels of 25-Hydroxyvitamin D (25(OH)D), intact parathyroid hormone (iPTH) and Beta cross-laps (CTX) was measured by Electrochemiluminescence Technology. DEXA (dual-energy x-ray absorptiometry) technique was used to evaluate BMD. RESULTS: We observed a statistically significant negative correlation between BALP levels and total body BMD (r = -0.268; P = 0.015) particularly in femoral neck (FN) (r = -0.219; P = 0.037). BALP concentrations were negatively associated with total BMD especially in FN for patients with BMI < 30, FGF23 concentrations were also negatively correlated with BMD in lumbar spine site (LS) (r = -0.209; P = 0.046). For osteopenic patients we found an inverse correlation between 25(OH)D concentrations and BMD in LS position (r = -0.336; P = 0.038). In men group, we have also found a negative correlation between iPTH and total BMD (r = -0.326; P = 0.015). However we found a positive correlation between calcium expression and BMD in LS site (r = 0.270; P = 0.031). CONCLUSIONS: FGF23 and BALP can predict bone loss in ESRD through their strong correlation with BMD in LS and FN sites respectively.

FGF 23, PTH and vitamin D status in end stage renal disease patients affected by VDR FokI and BsmI variants.

OBJECTIVES: The aim of this study was to evaluate the association between two VDR SNPs FokI and BsmI and mineral status in ESRD patients. DESIGN AND METHODS: Our case-control study included 100 patients with chronic renal failure in ESRD and 149 healthy subjects. We measured the serum Vitamin D levels and the serum intact PTH level by Electrochemiluminescence Technology (cobas E411 analyzer). We evaluated the serum FGF23 levels by indirect ELISA method. The genotyping of two VDR gene variants FokI and BsmI was carried out by PCR-RFLP technique. RESULTS: In our study, the FokI TT genotype was associated with lower risk of ESRD development (OR = 0.176, Padj = 0.039). The difference in PTH and FGF23 levels between cases and controls was statistically significant. The expression of FokI CT genotype in subjects with diabetic nephropathy was associated with a negative correlation between VD and PTH levels (r = -0.620, P = 0.032) and a positive correlation between VD and FGF23 levels (r = 0.967, P = 0.012). A significant differences in VD levels between patients and controls was observed in the presence of FokI TT (P = 0.044) and CT (P = 0.036) genotypes. The expression of FGF23 serum level was significantly elevated in patients than in controls in the presence of the FokI CC and BsmI AG genotypes. CONCLUSIONS: In conclusion, our study shows the existence of an association between VDR FokI, BsmI polymorphisms and mineral status in ESRD patients. The presence of VDR variants affect the protein expression of VD, phosphorus, FGF23 and PTH.

Jumping improves lower limbs bone mass and lean mass in elite jumpers.

BACKGROUND: The present study was designed to examine the influence of extreme impact loading induced by jump training on bone mineral density (BMD) and bone mineral content (BMC) in high level jumpers. METHODS: Forty boys volunteered for the study aged 20 to 21 years. They were 22 high level jumpers and 18 controls. Bone mass and body composition measurements were performed by dual-energy X-ray absorptiometry, in the total body and at different sites. RESULTS: The results showed that BMC, BMD, bone area, and lean mass (P<0.0001) were significantly higher in the jumpers compared to the controls. A strong positive correlation was found between lean mass and bone parameters (BMC at the right femur, r=0.80; P<0.01). The values of the same correlation were weak in the control group. In addition, the effects of the regular practice of jumping on the BMD, BMC, and bone area were more pronounced in the lower limbs (P<0.01). These adaptations were site-specific, with increased bone mass at the lower limbs (P<0.01), especially at the legs, right and left leg (LRL) (P<0.05). It appears that the time dedicated to this activity may be account for the difference between jumpers and controls. CONCLUSIONS: The practice of high level jump starting at pubertal age generates an increase and an acquisition of the bone mass in males. This adaptation is further enhanced by the times dedicated for this activity. Therefore, it would be interesting to program jumping activities daily to conserve bone mineral and to prevent osteopenia.