Genetic Association of LEP Gene Polymorphisms with Obesity in Moroccan Individuals: Case-Control Study and Updated Meta-analysis.

Obesity is a global epidemic disease representing the fifth leading cause of death in the world. It was shown that it is caused by the interaction between environmental factors and genes including leptin gene (LEP). This paper aimed to analyze the association between the LEP gene polymorphisms rs7799039 and rs11761556 with obesity in Moroccan individuals as well as to perform an update meta-analysis of this genetic association. Both polymorphisms were genotyped in 146 obesity patients and 104 controls using real-time PCR technique. The genetic association analysis and the comparison of quantitative parameters were carried out using the R language. Moreover, a meta-analysis including 20 genetic association studies was performed using Review Manager 5.3 software. No significant association was found between the polymorphisms rs7799039 and rs11761556 and the risk of obesity. The comparison of biochemical and clinical parameters between the genotypes of the rs7799039 polymorphism, showed a significant increased triglycerides levels in carriers of AA or GA genotypes (P value = 0.040). The meta-analysis showed no significant association between the rs7799039 polymorphism and obesity under all genetic models. In conclusion, the case-control study and meta-analysis demonstrated that the LEP gene polymorphisms rs7799039 and rs11761556 cannot be considered as genetic risk factors for obesity.

First characterization of congenital myasthenic syndrome type 5 in North Africa.

BACKGROUND: Congenital myasthenic syndromes (CMS) are associated with defects in the structure and the function of neuromuscular junctions. These rare disorders can result from mutations in the collagenic tail of endplate acetylcholinesterase (COLQ) essentially associated with autosomal recessive inheritance. With the lowered cost of genetic testing and increased access to next-generation sequencing, many mutations have been reported to date. METHODS AND RESULTS: In this study we identified the first COLQ homozygous mutation c.1193T>A in the North African population. This study outlines the genetic and phenotypic features of a CMS patient in a Moroccan family. It also describes a novel COLQ missense mutation associated with CMS-5. CONCLUSION: COLQ mutations are probably underdiagnosed in these North African populations, this is an issue as CMS-5 may be treated with ephedrine, and albuterol. Indeed, patients can seriously benefit and even recover after the treatment that should be planned according to genetic tests and clinical findings.

Vitreoscilla massiliensis sp. nov., Isolated From the Stool of an Amazonian Patient.

Strain SN6T is a non-motile and non-spore-forming gram-negative bacterium which was isolated from the stool sample of an Amazonian patient. The optimum growth was observed at 37 °C, pH 7, and 0-5 g/l of NaCl. Based on the 16S rRNA gene sequence similarity, the strain SN6T exhibited 97.5% identity with Vitreoscilla stercoraria strain ATCC_15218 (L06174), the phylogenetically closest species with standing in nomenclature. The predominant fatty acid was hexadecenoic acid (31%). The genomic DNA G + C content of the strain SN6T was 49.4 mol %. After analysis of taxonogenomic data, phenotypic and biochemical characteristics, we concluded that strain SN6T represents a new species of the genus Vitreoscilla for which the name Vitreoscilla massiliensis sp.nov is proposed. The type strain is SN6T (=CSUR P2036 = LN870312 = DSM 100958).

X-Linked Agammagobulinemia in a Large Series of North African Patients: Frequency, Clinical Features and Novel BTK Mutations.

PURPOSE: X-linked agammagobulinemia (XLA) is a primary immunodeficiency caused by Bruton's tyrosine kinase (BTK) gene defect. XLA patients have absent or reduced number of peripheral B cells and a profound deficiency in all immunoglobulin isotypes. This multicenter study reports the clinical, immunological and molecular features of Bruton's disease in 40 North African male patients. METHODS: Fifty male out of 63 (male and female) patients diagnosed with serum agammaglobulinemia and non detectable to less than 2% peripheral B cells were enrolled. The search for BTK gene mutations was performed for all of them by genomic DNA amplification and Sanger sequencing. RESULTS: We identified 33 different mutations in the BTK gene in 40 patients including 12 missense mutations, 6 nonsense mutations, 6 splice-site mutations, 5 frameshift, 2 large deletions, one complex mutation and one in-frame deletion. Seventeen of these mutations are novel. This large series shows a lower frequency of XLA among male patients from North Africa with agammaglobulinemia and absent to low B cells compared with other international studies (63.5% vs. 85%). No strong evidence for genotype-phenotype correlation was observed. CONCLUSIONS: This study adds to other reports from highly consanguineous North African populations, showing lower frequency of X-linked forms as compared to AR forms of the same primary immunodeficiency. Furthermore, a large number of novel BTK mutations were identified and could further help identify carriers for genetic counseling.

Chronic granulomatous disease in Morocco: genetic, immunological, and clinical features of 12 patients from 10 kindreds.

PURPOSE: Chronic granulomatous disease (CGD) is characterized by an inability of phagocytes to produce reactive oxygen species (ROS), which are required to kill some microorganisms. CGD patients are known to suffer from recurrent bacterial and/or fungal infections from the first year of life onwards. From 2009 to 2013, 12 cases of CGD were diagnosed in Morocco. We describe here these Moroccan cases of CGD. METHODS: We investigated the genetic, immunological and clinical features of 12 Moroccan patients with CGD from 10 unrelated kindreds. RESULTS: All patients were children suffering from recurrent bacterial and/or fungal infections. All cases displayed impaired NADPH oxidase activity in nitroblue tetrazolium (NBT), dihydrorhodamine (DHR) or 2',7' dichlorofluorescein diacetate (DCFH-DA) assays. Mutation analysis revealed the presence of four different mutations of CYBB in four kindreds, a recurrent mutation of NCF1 in three kindreds, and a new mutation of NCF2 in three patients from a single kindred. A large deletion of CYBB gene has detected in a patient. The causal mutation in the remaining one kindred was not identified. CONCLUSION: The clinical features and infectious agents found in these patients were similar to those in CGD patients from elsewhere. The results of mutation analysis differed between kindreds, revealing a high level of genetic and allelic heterogeneity among Moroccan CGD patients. The small number of patients in our cohort probably reflects a lack of awareness of physicians. Further studies on a large cohort are required to determine the incidence and prevalence of the disease, and to improve the description of the genetic and clinical features of CGD patients in Morocco.

Usefulness of lipid ratios and atherogenic index of plasma in obese Moroccan women with or without metabolic syndrome.

OBJECTIVES: Our study aimed to study the impact of obesity and metabolic syndrome (MetS) on lipoprotein profiles and cardiovascular risk through lipid ratios and atherogenic index of plasma (AIP) in Moroccan women. METHODS: Our study included 240 Moroccan women, aged 53.31 +/- 8.51 years, divided into three groups: controls (group 1, n = 80), obese without MetS (group 2, n = 80) and obese with MetS (group 3, n = 80). Anthropometric and lipid measurements were taken and specific lipid ratios assessed, as well as Non-HDL cholesterol (Non-HDL-C) and atherogenic index of plasma (ALP). RESULTS: Group 2 presented similar lipoprotein profiles compared with group 1. Group 3 had higher triglyceride (TG) levels than group 1, which, in turn, increased HDL and AIP values. Dyslipidemia in group 3 was demonstrated by higher TG levels, lipid ratios and AIP and lower HDL-C levels compared with group 2. All of these abnormalities are responsible for elevations of risks of cardiovascular diseases. Closer associations were found between cardiovascular risk and lipid ratios and AIP than lipids alone. CONCLUSION: Our study confirms that MetS affects the serum lipoprotein profile of obese women. Lipid ratios, non HDL-C and AIP remain useful tools for the diagnosis and prognosis of cardiovascular disease by their associations with lipid parameters and their high predictive values.

Inflammation and cardiovascular risk assessment in Moroccan obese patients with and without metabolic syndrome: importance of lipoproteins ratios.

BACKGROUND: Obesity predisposes an individual to numerous risk factors for cardiovascular diseases. Inflammation, reported as a link between obesity and cardiovascular disease, contributes to the development of atherosclerosis. OBJECTIVES: The aim of our study was to assess the relationship between lipid parameters, low grade inflammation and metabolic syndrome in a sample of obese Moroccan adults with or without metabolic syndrome (MetS). PATIENTS & METHODS: Our study included 235 obese patients, mean aged 53.30 ± 9.73 years, with or without MetS. Our data included anthropometric measurements, lipoprotein and apolipoproteins profiles and several lipid ratios. RESULTS: In patients with MetS, lipoprotein profile alterations and low-grade inflammation were observed. Lipid ratios were better predictors of cardiovascular risk than lipids alone because of their relative associations with lipoproteins and apolipoproteins. CONCLUSION: Our study shows that Moroccan obese adults with MetS have altered lipoproteins profiles and suffer from low-grade inflammation. Indeed, we have detected a high level of small dense LDL particles and HDL particles defectiveness. Hence, we propose that risk management of cardiovascular events should be based on lipoprotein ratios rather than lipids alone. Treatments should also take into account inflammatory markers and LDL heterogeneity.

Association of apolipoprotein E gene polymorphism with end-stage renal disease and hyperlipidemia in patients on long-term hemodialysis.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death of patients with chronic renal failure. Apolipoprotein E (apoE) plays an important role in the homeostasis of cholesterol and triglycerides. OBJECTIVE: We aimed to investigate the possible link(s) between apoE gene polymorphism, inflammation and lipoproteins in hemodialysis patients. METHODS: We studied 109 end-stage renal disease (ESRD) patients and 97 controls. The serum lipids, apolipoproteins, lipoprotein particles, high-sensitivity C-reactive protein (hs-CRP) and total homocysteine (t-Hcy) levels and paraoxonase (PON) activity were determined in our patients. We also analyzed apoE gene polymorphism in the patients and controls. RESULTS: The analysis of the apoE gene demonstrated a predominance of the e3 allele in both the patients and controls, followed by the e4 and then the e2 alleles. The analysis of the apoE genotype and allele frequencies showed significantly higher e4 allele and E3E4 genotype frequencies and decreased e3 allele and E3E3 genotype frequencies in the patients compared with the controls. The e2, e4 and E3E4 carriers within the ESRD patient population presented an atherogenic lipid profile. However, there were no significant variations in the serum PON activity and the hs-CRP and t-Hcy levels between individuals with different apoE polymorphisms. CONCLUSIONS: Our findings suggest an association between the e4 allele, E3E4 genotype and ESRD. The apoE polymorphism affects the serum lipoprotein levels, and the ESRD patients who are e4 and e2 allele carriers are more likely to present an atherogenic lipoprotein profile that may be a major factor associated with increased risk of CVD.

An overview of a preliminary multicenter retrospective study on food and drug allergies in Moroccan pediatric population.

Introduction: this study aimed to investigate the prevalence and management of food allergies (FA) and drug allergies (DA) in Morocco. Sparse and conflicting epidemiological data exist on the exact prevalence of allergies in the country. The rise in allergies can be attributed to various factors. Methods: the study analyzed data from patients with suspected FA and DA who sought medical attention. Statistical tests were used to analyze the data, percentages were computed for qualitative variables, and for quantitative variables, medians or means accompanied by standard deviations (SD) were calculated. The Chi-square test was employed to assess categorical variables. A p-value < 0.05 was considered statistically significant. Results: Cow's milk was the most reported food allergen (58.2%), followed by egg and nuts (23.4% and 12.1%, respectively). The most affected age group was children under 5 years. Antibiotics were the leading cause of reported drug allergies (44.8%), particularly Beta-lactams. Immediate reactions were commonly associated with antibiotics and nonsteroidal anti-inflammatory drugs (NSAIDs). Symptoms of FA included acute urticaria, vomiting, anaphylactic shock, and facial edema. Urticaria was the most frequent symptom of DA. Antihistamines and corticosteroids were the main treatments used for both FA and DA. Conclusion: the prevalence of FA and DA in Morocco remains uncertain due to limited data. There is a need for centralized data collection and awareness among clinicians and the general population regarding allergies. The study highlights the importance of proper diagnosis and management of allergies to ensure patient safety. The findings emphasize the necessity of establishing a mandatory center for allergy care in Morocco to improve the understanding and management of allergic conditions.

Obesity and its related factors among women from popular neighborhoods in Casablanca, Morocco.

OBJECTIVES: Study of overweight and obesity among women from modest neighborhoods in Casablanca and the related factors influencing this phenomenon. DESIGN: A survey was conducted in 6 neighborhoods of Casablanca using multistage cluster sampling. Anthropometric parameters and body composition were measured, and information about food habits, sociodemographic situation and body image perception were collected using a questionnaire. PARTICIPANTS: 425 adult women from popular neighborhoods in Casablanca. RESULTS: We found a prevalence of 47% obesity and 36% overweight. Obesity and overweight exceeded 80% in all age categories except among women < 25 years, where we found a prevalence of 44% overweight. Furthermore, abdominal obesity prevalence was high in all age categories with an overall prevalence of 67%. Body image perception data showed that 62% women perceived their weight as being either normal or insufficient, 46% of those women were, in fact, either overweight or obese. Fattening products were used by 12% of the population at least once, especially by women < 35 years, 68% of these women were overweight or obese at the time of the study. CONCLUSIONS: The problem of obesity in this society is amplified by its perception of a regular weight and by the use of fattening substances even among young and educated women.

Molecular docking analysis of PPARγ with phytochemicals from Moroccan medicinal plants.

PPARγ agonists play a crucial role in regulating metabolic homeostasis for treating type-2 diabetes (T2D). Due to the adverse side effects associated with thiazolidinediones, a class of PPARγ agonists, there is a growing interest in identifying natural compounds from medicinal plants that have the potential to bind PPARγ. In this study, we extensively investigated Moroccan phytochemicals using computational structure-based screening with the crystal structure of the PPARγ ligand-binding domain (PDB ID: 7awc) to discover novel phytochemicals targeting PPARγ. The docking results of 540 Moroccan phytochemicals were integrated into online databases for further exploitation through in-depth studies. Drug-likeness analysis was performed to assess the phytochemicals drug-like properties. Two promising phytochemicals, 3,4-dicaffeoylquinic acid and Chlorogenic acid, were identified, both exhibiting high docking affinity and unique binding site interactions compared to the established PPARγ full agonist, rosiglitazone. Molecular dynamics simulations of 100 ns were conducted to examine the stability of the complexes formed by both compounds within the PPARγ active site, and their dynamic behavior was compared to the reference structure of PPARγ alone and with rosiglitazone. Binding free energy calculations demonstrated that 3,4-dicaffeoylquinic acid and Chlorogenic acid exhibited higher binding free energy than the reference agonist, suggesting their potential as candidates for experimental validation in future drug discovery efforts targeting PPARγ for the treatment of T2D and metabolic syndrome.

Identification of Zika virus NS2B-NS3 protease and NS5 polymerase inhibitors by structure-based virtual screening of FDA-approved drugs.

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (-8.27 kJ/mol), the highest binding affinity (-5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of -150.624 kJ/mol, a binding affinity of -5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro, in vivo, and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates.Communicated by Ramaswamy H. Sarma.

Identification of novel T-cell epitopes on monkeypox virus and development of multi-epitopes vaccine using immunoinformatics approaches.

Monkeypox virus (MPV) is closely related to the smallpox virus, and previous data from Africa suggest that the smallpox vaccine (VARV) is at least 85% effective in preventing MPV. No multi-epitope vaccine has yet been developed to prevent MPV infection. In this work, we used in silico structural biology and advanced immunoinformatic strategies to design a multi-epitope subunit vaccine against MPV infection. The designed vaccine sequence is adjuvanted with CpG-ODN and includes HTL/CTL epitopes for similar proteins between vaccinia virus (VACV) that induced T-cell production in vaccinated volunteers and the first draft sequence of the MPV genome associated with the suspected outbreak in several countries, May 2022. In addition, the specific binding of the modified vaccine and the immune Toll-like receptor 9 (TLR9) was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores confirmed the stringency of the modified vaccine. The stability of the interaction was confirmed by a classical molecular dynamics simulation and normal mode analysis. Then, the immune simulation also indicated the ability of this vaccine to induce an effective immune response against MPV. Codon optimization and in silico cloning of the vaccine into the pET-28a (+) vector also showed its expression potential in the E. coli K12 system. The promising data obtained from the various in silico studies indicate that this vaccine is effective against MPV. However, additional in vitro and in vivo studies are still needed to confirm its efficacy.Communicated by Ramaswamy H. Sarma.

Association between nutritional status, body composition, and fitness level of adolescents in physical education in Casablanca, Morocco.

Aim: This study aims to analyze and compare dietary intake, as well as to examine the associations between energy intake in terms of macronutrients, body composition, and physical fitness (PF) specifically cardiorespiratory endurance (CE) among a sample of young adolescents aged 15 to 18 years, who participate in physical education and sports sessions in public schools in Casablanca, Morocco. Materials and methods: A total of 311 participants, including 156 girls and 154 boys, were included in the study. Each participant maintained a food diary for 3 days during the same study week. Additionally, body composition measurements were taken using bioelectrical impedance analysis (BIA). The PF was assessed using the validated mini-Cooper test (6 min). Results: The results show that the participants had an average total energy intake of 2386.7 ± 492.7 kcal. A significant difference was observed between boys and girls, with average energy intakes of 2468.8 ± 531.1 kcal and 2304.0 ± 437.0 kcal, respectively. These dietary intakes were significantly lower than their needs and nutritional recommendations. The associations of nutritional status, sex, body mass index (BMI) and physical fitness (PF) were tested and a positive correlation was observed following an adequate intake of carbohydrates (CHO) and proteins on Vo2max, while a negative association was observed with regard to Body fat for both sexes. Boys exhibit significantly better PF than girls (p < 0.01). Obese participants had the lowest PF and an unbalanced nutritional status, the adolescents with a normal weight p < 0.01 displayed a high level of PF compared to individuals in other weight categories. Conclusion: The PF is significantly associated with macronutrient intake status and body composition, especially BMI and BF. The Underweight, overweight, and obese students demonstrated poorer performance in physical fitness indices compared to normal-weight. Adolescents adhering to recommended CHO and protein intake levels tend to exhibit enhanced physical fitness. Implementing strategies to encourage students to maintain a balanced diet and engage in regular physical exercise is essential.

Design of a multi-epitope Zika virus vaccine candidate - an in-silico study.

Zika virus (ZIKV), an RNA virus, rapidly spreads Aedes mosquito-borne sickness. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. In this study, to address these unmet medical needs, we aimed to design B- and T-cell candidate multi-epitope-based subunit against ZIKV using an in silico approach. In this study we applied immunoinformatics, molecular docking, and dynamic simulation assessments targeting the most immunogenic proteins; the capsid (C), envelope (E) proteins and the non-stuctural protein (NS1), described in our previous study, and which predicted immunodominant B and T cell epitopes. The final non-allergenic and highly antigenic multi-epitope was constituted of immunogenic screened-epitopes (3 CTL and 3 HTL) and the ß-defensin as an adjuvant that have been linked using EAAAK, AAY, and GPGPG linkers, respectively. The final construct containing 143 amino acids was characterized for its allergenicity, antigenicity, and physiochemical properties; and found to be safe and immunogenic with a good prediction of solubility. The existence of IFN-γ epitopes asserts the capacity to trigger strong immune responses. Subsequently, the molecular docking among vaccine and immune receptors (TLR2/TLR4) was revealed with a good binding affinity with and stable molecular interactions. Molecular dynamics simulation confirmed the stability of the complexes. Finally, the construct was subjected to in silico cloning demonstrating the efficiently of its expression in E.coli. However, this study needs the experimental validation to demonstrate vaccine safety and efficacy.Communicated by Ramaswamy H. Sarma.

Reverse vaccinology-based prediction of a multi-epitope SARS-CoV-2 vaccine and its tailoring to new coronavirus variants.

The genome feature of SARS-CoV-2 leads the virus to mutate and creates new variants of concern. Tackling viral mutations is also an important challenge for the development of a new vaccine. Accordingly, in the present study, we undertook to identify B- and T-cell epitopes with immunogenic potential for eliciting responses to SARS-CoV-2, using computational approaches and its tailoring to coronavirus variants. A total of 47 novel epitopes were identified as immunogenic triggering immune responses and no toxic after investigation with in silico tools. Furthermore, we found these peptide vaccine candidates showed a significant binding affinity for MHC I and MHC II alleles in molecular docking investigations. We consider them to be promising targets for developing peptide-based vaccines against SARS-CoV-2. Subsequently, we designed two efficient multi-epitopes vaccines against the SARS-CoV-2, the first one based on potent MHC class I and class II T-cell epitopes of S (FPNITNLCPF-NYNYLYRLFR-MFVFLVLLPLVSSQC), M (MWLSYFIASF-GLMWLSYFIASFRLF), E (LTALRLCAY-LLFLAFVVFLLVTLA), and N (SPRWYFYYL-AQFAPSASAFFGMSR). The second candidate is the result of the tailoring of the first designed vaccine according to three classes of SARS-CoV-2 variants. Molecular docking showed that the protein-protein binding interactions between the vaccines construct and TLR2-TLR4 immune receptors are stable complexes. These findings confirmed that the final multi-epitope vaccine could be easily adapted to new viral variants. Our study offers a shortlist of promising epitopes that can accelerate the development of an effective and safe vaccine against the virus and its adaptation to new variants.Communicated by Ramaswamy H. Sarma.

Data insights from a Moroccan phytochemical database (MPDB) derived from aromatic & medicinal plants.

The geographical location of Morocco and the diversity of its topography ensure a high variability of climate conditions, ranging from humid to Saharan, and extending through subhumid, arid, and semi-arid stages. This variability offers a high floristic diversity, while the medical use of these phytochemicals has not been fully explored. Advanced computer-aided drug discovery utilizes chemical biology to accelerate the study of phytochemicals at the molecular level and discover novel therapeutic pathways. Currently, there is no online resource for phytochemicals in Morocco. Therefore, it is of interest to describe the Moroccan Phytochemicals Database (MPDB), accessible, featuring over 600 phytochemicals derived from journal articles and other reports. The web interface of the database, which is simple and easy to use, provides each phytochemical's reference, plant sources, 3D structures, and all related information. Furthermore, we provide direct links to commercially available analogs from Mcule. In addition, we provide the results of the first virtual screening against cardiovascular targets. We present these data to facilitate further exploration and exploitation of Morocco's rich phytochemical resources, and to contribute to the global understanding and application of these compounds in the medical and scientific communities.

MBL2 gene polymorphisms related to HIV-1 infection susceptibility and treatment response.

Human Mannose-binding lectin (MBL) is a protein encoded by MBL2 gene involved in the activation of the lectin-complement pathway. Several studies emphasized the role of MBL2 gene in several infectious diseases' susceptibility, including HIV-1 infection. We aim to investigate the impact of 10 MBL2 gene polymorphisms located in the promoter, 5'UTR and exon 1 regions on HIV-1 physiopathology. The polymorphisms genotyping of 400 individuals, which 200 were HIV-1 positive patients and 200 were controls, was performed by PCR-sequencing. Our results showed that rs503037 and rs1800451 polymorphisms are associated with a high risk of HIV-1 infection susceptibility while rs7096206 and rs11003123 showed a protective effect. A significant association between haplotype CGA and HIV-1 infection susceptibility was also found in the exon 1 region. Moreover, rs11003124, rs7084554, rs36014597 and rs11003123 polymorphisms revealed an association with treatment response outcome as measured by RNA viral load. This study highlights the importance of MBL2 polymorphisms in the modulation of HIV-1 infection susceptibility and the contribution to treatment response outcomes among Moroccan subjects.

Characterization of fusidic acid-resistant Staphylococcus aureus isolates in the community of Casablanca (Morocco).

Resistance to fusidic acid in Staphylococcus aureus is caused by mutation of the elongation factor G (EF-G) encoded by fusA or by expression of a protein, encoded by fusB or fusC, that protects the drug target. Other mechanisms involved in this resistance are mutations in the riboprotein L6 operon within rplF. The aim of this study was to determine the prevalence and mechanisms of resistance to fusidic acid in clinical isolates of S. aureus in Casablanca (Morocco) and to define the phenotypic and genotypic traits of these isolates and their clonal relationship. All fusidic acid-resistant S. aureus (FAR-SA) isolates were tested for fusB and fusC genes and were evaluated for the detection of mutations in fusA and fusE (rplF). fusB-positive strains were tested for a cadDX operon, encoding cadmium resistance. The agr group and the presence of toxin genes were monitored to characterize all FAR-SA isolates which were typed by pulsed-field gel electrophoresis (PFGE) and spa typing. Among 140 clinical S. aureus isolates collected in 2007 and 2008, 18 (∼13%) exhibited resistance to fusidic acid. The most common resistance determinant was fusC, found in 16 isolates. Molecular typing showed that 14 of them harboured an agr group III and belonged to the same clonal complex (CC) spa type 127 and identical clonotype (cluster labelled A). These isolates also possessed the staphylococcal enterotoxin H gene. The second resistance determinant was fusB found in two isolates. These two isolates lacked cadDX gene and were found to belong to two unrelated clusters and spa types. While no isolate carrying mutations in rplF was found, 15 expressed a silent mutation in fusA (nucleotide 342). Only acquired fusidic acid resistance genes (mainly fusC) were prevalent among FAR-SA isolates with almost all of the clinical specimens belonging to CC-spa type 127. This study provides valuable data on the prevalence of fusidic acid-resistant S. aureus with the associated molecular mechanisms of resistance and the genetic background of the strains in Casablanca.

Comparability of CMV DNA Extraction Methods and Validation of Viral Load.

Human cytomegalovirus is a herpesvirus that has a worldwide seroprevalence of more than 60% of adults in developed countries and 90% in developing countries. Severe disabilities in newborns are characteristic of the human cytomegalovirus congenital infection, and this virus is implicated in graft rejection in transplant patients. To treat and follow-up the infection, the CMVPCR viral loads are required, and the DNA extraction step remains very important; however, the quantity, quality, and purity of extracted DNA from different biological fluids influence the results of PCR amplification, that is why for reliable results, the choice of nucleic acid extraction methods requires careful attention. Materials and methods: In this study, we compare 4 protocols, I (EZ1 DSP Virus kit), II (EZ1 Virus mini kit), III (QIAamp DSP virus kit), and IV (heating); the extractions are made from plasma collected on EDTA tubes, and the concentration of extracted DNA was measured on NanoDrop Lite followed by real-time CMVPCR using an Artus CMV QS-RGQ kit. All protocols are performed following the manufacturer's instructions. Results: This study is conducted on the samples of 135 transplant patients whose follow-up medical tests related to human cytomegalovirus infection; since most of the CMVPCR results are negative, we have chosen the 10 CMVPCR positive samples and 2 negative samples as controls to conduct this comparison study. By using NanoDrop Lite to evaluate the DNA concentration, the yield of extracted DNA is higher in our heating protocol than other protocols, the EZ1 DSP virus kit and EZ1 Virus mini kit show homogeneous quantities, and the QIAamp DSP virus kit shows very low DNA yields. Comparing cycle threshold and viral loads by real-time PCR, all these protocols identified negative samples (100%), and the previously positive samples used were as follows: protocol IV (90%), protocol II (60%), and protocol I (40%). QIAamp DSP virus kit results were not real-time PCR applicable and were non-conclusive because of the low DNA yields. Conclusion: Our developed heating method (protocol IV) is very effective, reliable, simple, fast, and cheap compared to the other protocols in our study.