Phenotype and molecular characterization of Wilson's disease in Morocco.

BACKGROUND AND STUDY AIMS: In Morocco the prevalence of Wilson disease (WD) and the spectrum of mutations are not known. The aim of the present study was to estimate the prevalence of WD in Morocco, to evaluate the phenotype among a large cohort of WD patients, and to characterize ATP7B variants in a subgroup of WD patients. PATIENTS AND METHODS: We collected data from 226 patients admitted to five university hospital centers in Morocco between 2008 and 2020. The diagnosis was based on clinical manifestations, function tests and biochemical parameters. The genotype was characterized in 18 families diagnosed at the University Hospital Center of Marrakesh, by next generation sequencing. RESULTS: The mean annual prevalence in Morocco was 3.88 per 100,000 and the allele frequency was 0.15 %. Among the 226 patients included (121 males and 105 females), 196 were referred for a hepatic or neurological involvement and 30 were asymptomatic. The mean age at diagnosis was 13 ± 5.1 years (range: 5 - 42 years). Consanguinity was found in 63.3 % of patients. The mean duration of illness was 2.8 ± 1.9 years. Kayser-Fleischer rings were found in 131 (67.9 %) of 193 patients. Among the 196 symptomatic patients, 141/159 (88.7 %) had low serum ceruloplasmin (<0.2 g/L) and a high 24-hours urinary copper (>100 µg/day) was found in 173/182 (95.1 %) patients. The initial treatment was D-penicillamine in 207 patients, zinc acetate in five, zinc sulfate in five, and nine patients were not treated; 60/207 (29 %) patients have stopped treatment. A total of 72 patients died; the mortality rate was 31.9 %. Eight different ATP7B variants were identified among the 18 patients studied, of which two were novel (p.Cys1104Arg and p.Gln1277Hisfs*52), and six previously published (p.Gln289Ter, p.Cys305Ter, p.Thr1232Pro, p.Lys1020Arg, p.Glu583ArgfsTer25 and c.51+4A>T). All informative patients were homozygous for the disease-causing mutation. CONCLUSION: In Morocco, a high prevalence due to consanguinity and a high mortality rate due to the difficulty of diagnosis and lack of treatment were observed in WD patients. NGS sequencing identified new ATP7B variants in WD patients from Morocco.

Microbiological quality of gluten-free meals, naturally gluten free foods, and gluten free-labelled products.

Background: The rising prevalence of gluten-related disorders such as celiac disease explains the increased consumption of gluten-free foods (GFF). However, these foods must be safe in terms of both gluten content and contamination by pathogenic microorganisms in order to avoid food poisoning. Objective: The objective of this study was to assess the microbiological quality of gluten-free meals, naturally gluten free foods, and gluten free-labelled products. Material and Methods: We collected 62 GFF samples including 20 meals (M-GF), 22 naturally gluten free (N-GFF) and 20 labelled (L-GFF) products, which were investigated for microbiological contamination according to Moroccan regulations guidelines, issued by the International Organization for Standardization (ISO). The analysis consisted of the detection of Salmonella and Listeria monocytogenes in each sample, and the quantification of the microbial load of the following six micro-organisms: total aerobic mesophilic flora, total coliforms, fecal coliforms, Staphylococcus aureus, Sulphite-Reducing Anaerobic, and yeasts and molds. Results: A total of 372 analyses were carried out, showing a microbiological contamination rate of 5.1%. This contamination concerned N-GFF in 8.3% (predominantly with yeasts and molds), and meals prepared at home in 11.7 (predominantly with Staphylococcus aureus and coliforms). Only one case (0.8%) of contamination was observed in products labelled gluten-free and no contamination was noticed in meals prepared in food services. Listeria monocytgenes and Salmonella were not detected in any samples of food analyzed. These results indicate a good compliance of L-GFP and M-GF prepared in food services, while unsatisfactory quality was observed in N-GFF and M-GF prepared at home. Conclusion: Therefore, rigorous hygienic practices and adequate corrective measures should be considered by celiac patients, especially regarding the N-GFF and M-GF prepared at home.

Immunoglobulin E-Mediated Food Sensitization in a Moroccan Pediatric Population with Celiac Disease.

INTRODUCTION: Celiac disease is a chronic autoimmune disorder that occurs following the ingestion of gluten, in genetically predisposed individuals. Patients with celiac disease, especially children, are likely prone to develop allergic reactions to different food allergens. However, the relationship between food allergy and celiac disease remains not elucidated. The aim of this pioneering study was to evaluate the prevalence of allergic food sensitization in children with celiac disease in Morocco. METHODS: A total of 57 children with confirmed celiac disease, including 25 males and 32 females with a mean age of 8.6 ± 4.4 years, underwent a food allergen-specific immunoglobulin E (IgE) screening. This screening was conducted using a multiparametric immunodot assay (Euroline Food "Maghreb," Euroimmun). Statistical analysis was performed using R software. RESULTS: Among the 57 cases tested, the overall rate of IgE-mediated sensitization to food allergens was found to be 48% (27/57), dominated by chicken, with 51.9% (14/27), followed by almond, 40.7% (11/27), sesame, 40.7% (11/27), potato 33.3% (9/27), and apple 18.5% (5/27). Of the s-IgE positive cases, 74% were sensitized at least to one allergen, 37% (10/27) were sensitized to both chicken and almond allergens. A significant correlation was observed between almond, sesame, chicken, and potato. CONCLUSION: The current study highlighted a high prevalence of food allergen sensitization in children with celiac disease. This underlines the potential benefit in screening for food allergy in celiac patients.

Clinical, biochemical and molecular characterization of Wilson's disease in Moroccan patients.

Background: Wilson Disease (WD) is an autosomal recessive inherited metabolic disease caused by mutations in the ATP7B gene. WD is characterized by heterogeneous clinical presentations expressed by hepatic and neuropsychiatric phenotypes. The disease is difficult to diagnose, and misdiagnosed cases are commonly seen. Methods: In this study, the presented symptoms of WD, the biochemical parameters as well as its natural history are described based on cases collected in Mohammed VI Hospital University of Marrakech (Morocco). We screened and sequenced 21 exons of ATP7B gene from 12 WD patients that confirmed through biochemical diagnosis. Results: Mutational assessment of the ATP7B gene showed six homozygous mutations in 12 individuals however, 2 patients had no evidence of any mutation in promoter and exonic regions. All mutations are pathogenic and most were missense mutations. c.2507G > A (p.G836E), c.3694A > C (p.T1232P) and c.3310 T > C (p.C1104R) that were identified in 4 patients. The other mutations were a non-sense mutation (c.865C > T (p.C1104R)) detected in 2 patients, a splice mutation (c.51 + 4A > T) detected in 2 patients and a frameshift mutation (c.1746 dup (p.E583Rfs*25) detected in 2 patients. Conclusion: Our study is the first molecular analysis in Moroccan patients with Wilson's disease, the ATP7B mutational spectrum in the Moroccan population is diverse and still unexplored.

Digestive manifestations of Covid-19 in children: a retrospective study.

Background: The world is currently facing a pandemic due to a new species of the Coronaviridae family called SARS-CoV-2, discovered in the city of Wuhan in China in December 2019. The WHO has named the resulting disease COVID-19 (Coronavirus Disease 2019). It has been a global health problem due to its major socio-economic damage. The aim of this study was to show the prevalence of gastrointestinal and hepatic manifestations in symptomatic children with COVID-19. Methods: We performed a retrospective study, including 36 symptomatic children infected by SARS-CoV-2 hospitalized at the mother and child hospital of university hospital of Mohammed VI, Marrakech in Morocco, over a period of 7 months. Clinical and biological manifestations of the digestive system were evaluated for all patients. Results: The digestive symptomatology came in second place after the respiratory manifestations. 14 patients (38.89 % of symptomatic patients) in our study had digestive symptoms on admission: 12 (33.33%) presented with diarrhea, 4 (11.11%) had abdominal pain and only one child (2.78%) had vomiting. Aspartate aminotransferase (AST) was elevated in one patient, while alanine transaminase (ALT) was elevated in 6 patients. The prothrombin level was normal in all patients. All patients were discharged with good general condition without morbidity and mortality. Conclusion: This study concludes with the high prevalence of digestive manifestations of COVID-19 in symptomatic children. There were no severe clinical or biological abnormalities in our study. Digestive manifestations during COVID-19 in children are frequent, which requires the awareness of health professionals.

Quality of life of Moroccan patients with celiac disease: Arabic translation, cross-cultural adaptation, and validation of the celiac disease questionnaire.

BACKGROUND AND STUDY AIMS: Celiac disease (CD) management is based on a lifelong gluten-free diet (GFD) that affects the quality of life (QoL) of patients with CD. Specific instruments have been used to evaluate this QoL, such as the CD-Questionnaire (CD-Q). This study aimed to translate, validate, and cross-culturally adapt the CD-Q in an Arabic version and then apply it to evaluate the QoL of Moroccan adult patients with CD. PATIENTS AND METHODS: The Moroccan version of the CD-Q (M-CD-Q) was administered to 150 patients with CD, and 112 of them completed it. The reproducibility and reliability of the M-CD-Q were studied by the intraclass coefficient (ICC) and Cronbach's α, respectively. Parametric and nonparametric tests, confirmatory factor analysis, and Spearman correlation were used for the statistical analysis performed by SPSS, and the goodness-of-fit test was determined using SPSS AMOS. RESULTS: No difficulties were found during the translation and cultural adaptation of the CD-Q. Cronbach's α showed good internal consistency. The retest showed excellent reproducibility (ICC > 0.4). The study of the psychometric properties of the M-CD-Q showed good acceptance, zero ceiling effect, and floor effect. The model fit was good [(root mean square error of approximation = 0.075 (<0.08) and χ2 = 509.04, p < 0.001]. The total scores showed a neutral QoL. This QoL was worse in the worries subscale, which is related to gluten-free products. The GFD did not improve the QoL of the examined samples. CONCLUSION: The M-CD-Q is the first reliable and adapted instrument in an Arab country for the evaluation of QoL in patients with CD. CD negatively influences this QoL, especially items related to gluten-free products.

Acute Vision Loss and Conjunctival Hemorrhage as Telltale Symptoms of PIMS-TS.

There is a global concern about children presenting with inflammatory syndrome with variable clinical features during the ongoing COVID-19 pandemic. This paper reports the first pediatric case of bilateral serous retinal detachment and conjunctival hemorrhage as a revealing pattern of the pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS). Despite the severity of multisystemic involvement, the management with steroids was very successful. Complete remission was obtained within 3 months of acute onset.

Challenges in the Diagnosis and Management of Nephrotic Syndrome in a Child With Multisystemic Tuberculosis.

We report the case of a child who had tuberculosis associated with nephrotic syndrome. In this case, it was difficult to identify if the renal involvement was due to renal infection with mycobacterium tuberculosis, the consequence of nephrotoxicity of anti-bacillary drugs, or due to new onset of nephrosis. Management was complex as the use of high-dose steroids can disseminate the infection.

Update of a colorimetric method for quantitative determination of galactose in blood samples: A simple and rapid method for the early detection of inherited metabolic diseases.

BACKGROUND: A colorimetric microassay for the quantitative determination of galactose in the blood was taken and updated. This method helps in diagnosis and follow-up of several inherited metabolic diseases connected to galactose metabolism deficiency such as galactosemia, glycogenosis, glycosylation, tyrosinemia and citrin deficiency. Galactose assay in the blood presents difficulties due to interference with glucose. In this study, we update a method to get around these difficulties. METHOD: This procedure was based on the incubation of whole blood with orcinol in a strongly acidic solution to form a galactose and glucose complexes able to absorb at two different wavelengths. RESULTS: The standard curve analysis for the individual solutions of these two sugars showed a wide range of linearity from 0 to 200 mg / l. Under optimal experimental conditions, the stirring time of the orcinol is 3 minutes, the heating time of the reaction is 20 minutes at 56 ° C, and the duration of the incubation in the dark is 40 minutes. The analysis is carried out on fresh blood. The maximum absorbance of galactose and glucose is respectively 569 nm and 421 nm. An adapted diagnosis algorithm was developed based on our results. CONCLUSION: this method could help in screening and identifying patients with hypergalactosemia that need further investigations. It could represent a promising method for neonatal screening in countries with limited resources.

Laboratory abnormalities in children with novel Coronavirus Disease 2019.

The novel Coronavirus disease 2019 continues to be a worldwide pandemic. Yet, little is still known about the biological features of this emergent infection in children. In this prospective study, we collected 68 children infected with SARS-COV-2 from March 2020 to May 2020, in Marrakesh, Morocco. No severe cases were observed in this cohort, and 66% of the patients were asymptomatic. The main laboratory abnormalities were hematological, as we found Leucopoenia in 4.4% of the cases, hyperleukocytosis in 1.6%. Neutropenia was found in 5 patients (7%) and only 2 cases (3%) had Lymphopenia. The inflammation and coagulation biomarkers were normal in the majority of the cases, as for liver and kidney function. Lactate dehydrogenase (LDH) serum levels were elevated in 8 cases (11.67%). The COVID-19 in children seems to have mild course and better outcome than in adults, which impacts the laboratory findings in this category. More studies must be conducted to learn more about the laboratory abnormalities in pediatric COVID-19.

Implementation of an Affordable Method for MPS Diagnosis from Urine Screening to Enzymatic Confirmation: Results of a Pilot Study in Morocco.

BACKGROUND: Rapid and accurate diagnosis of mucopolysaccharidoses (MPS) is still a challenge due to poor access to screening and diagnostic methods and to their extensive clinical heterogeneity. The aim of this work is to perform laboratory biochemical testing for confirming the diagnosis of mucopolysaccharidosis (MPS) for the first time in Morocco. METHODS: Over a period of twelve months, 88 patients suspected of having Mucopolysaccharidosis (MPS) were referred to our laboratory. Quantitative and qualitative urine glycosaminoglycan (GAG) analyses were performed, and enzyme activity was assayed on dried blood spots (DBS) using fluorogenic substrates. Enzyme activity was measured as normal, low, or undetectable. RESULTS: Of the 88 patients studied, 26 were confirmed to have MPS; 19 MPS I (Hurler syndrome; OMIM #607014/Hurler-Scheie syndrome; OMIM #607015), 2 MPS II (Hunter syndrome; OMIM #309900), 2 MPS IIIA (Sanfilippo syndrome; OMIM #252900), 1 MPS IIIB (Sanfilippo syndrome; OMIM #252920) and 2 MPS VI (Maroteaux-Lamy syndrome; OMIM #253200). Parental consanguinity was present in 80.76% of cases. Qualitative urinary glycosaminoglycan (uGAGs) assays showed abnormal profiles in 31 cases, and further quantitative urinary GAG evaluation and Thin Layer Chromatography (TLC) provided important additional information about the likely MPS diagnosis. The final diagnosis was confirmed by specific enzyme activity analysis in the DBS samples. CONCLUSIONS: The present study shows that the adoption of combined urinary substrate analysis and enzyme assays using dried blood spots can facilitate such diagnosis, offer an important tool for an appropriate supporting care, and a specific therapy, when available.

[Cyclophosphamide in idiopathic nephrotic syndrome: Outcome and outlook]. / Le cyclophosphamide dans le syndrome néphrotique idiopathique : résultats et perspectives.

INTRODUCTION: Cyclophosphamide (CYP) has been used for over 40 years in patients with steroid-sensitive nephrotic syndrome (NSSS) presenting frequent relapses (NSRF) or steroid dependence (NSSD). However, the long-term success of treatment with cyclophosphamide is difficult to predict. The objectives of this study are to determine long-term outcomes of cyclophosphamide and identify the factors associated with sustained remission. METHODS: We retrospectively studied the data from 50 patients with idiopathic nephrotic syndrome, treated by oral cyclophosphamide and followed at service of pediatric for more than 8 years for idiopathic nephrotic syndrome and related factors for survival without relapse were evaluated by univariate analysis. RESULTS: The median age at the time of diagnosis was 4.3 years, and median follow-up time was 1.7 years with the median of 8 years at the first use of CYC. Patients had received a median cumulative dose of 168mg/kg. At the end of follow-up, 38% of patients entered into remission after using CYC while 62% failed to respond and further relapses then occur. The median time of stopping corticosteroid therapy was three month. The survival without relapse was respectively 56% (28 patients), 52% (26 patients), 48% (24 patients), and 38% (19 patients), at 6 months, one year, two years and more than two years. In univariate analysis, the survival without relapse was related to the age at the moment of starting the therapy par CYC (the median was 5 months for an age < 8 years and 41 months for an age≥8 years; P=0.049), the type of nephrotic syndrome [36 months for SNRF, 4 months for NSSD and nephrothic syndrome steroid resistant (NSSR); P=0.068], and the histological lesion (6 months for diffuse mesangial proliferation, 2 months for segmental glomerulosclerosis; P=0.009). The age at the moment of diagnosis, the sex and the cumulative dose of CYC did not have significant influence. CONCLUSION: The results presented in this study suggest the use of oral cyclophosphamide for short period remain second line effective therapy. Further well-designed trials are required to evaluate the efficacy of other steroid-sparing agents.