Biochemical, Cellular, and Proteomic Characterization of Hereditary Spherocytosis Among Tunisians.

BACKGROUND/AIMS: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. METHODS: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5'-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. RESULTS: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. CONCLUSION: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

Hb Moscva [ß24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A]: An Unstable Hemoglobin Newly Detected as a De Novo Mutation in a Mauritanian Patient.

Unstable hemoglobins (Hbs) are a group of Hb disorders that could be the origin of chronic hemolytic anemia. Most of these disorders are caused by point mutations taking place in the globin genes and affecting the stability of the Hb molecule. They are inherited as autosomal dominant diseases and described worldwide. Herein we report a new observation of an unstable variant in the Mauritanian population. The patient was a young girl of Mauritanian origin. She presented with chronic hemolytic anemia with an unknown etiology after being referred to several medical centers. Laboratory investigations based on routine analyses, capillary electrophoresis (CE), cation exchange high performance liquid chromatography (HPLC) and DNA sequencing revealed an abnormal unstable Hb known as Hb Moscva [ß24(B6)Gly→Asp (GGT>GAT), HBB: c.74G>A] that occurred as a de novo mutation newly detected in an African girl of Mauritanian origin.

Homozygous Mutation on the ß-Globin Polyadenylation Signal in a Tunisian Patient with ß-Thalassemia Intermedia and Coinheritance of Gilbert's Syndrome.

We report here the clinical, hematological and molecular data in a 50-year-old patient with ß-thalassemia intermedia (ß-TI) caused by a homozygous ß+ mutation on the ß-globin gene polyadenylation (polyA) signal (AATAAA>AAAAAA). ß Haplotype analysis was accomplished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype and framework analysis showed that this mutation is associated with the [- - - - + + +] ß haplotype and framework 1 (CCGCT) (FW1). This mutation was previously reported in the heterozygous state in association with the codon 9 (+TA) mutation in a ß-TI patient originating from Tunisia. To the best of our knowledge, this is the first report describing this mutation in the homozygous state. The case reported here, coinherited Gilbert's syndrome, which is characterized by hyperbilirubinemia. This conclusion was reached by the investigation of the promoter region [A(TA)nTAA] motif of the UGT1A1 gene, showing the (TA)6/(TA)7 genotype.

Prediction of intensive care unit mortality: Interest of serum lactates.

INTRODUCTION: Many prognostic indices have been developed to assess clinical status and predict the probability of death in the intensive care unit (ICU) but none have perfect sensitivity or specificity. AIM: To evaluate the prognostic value of admission lactate in patients admitted to ICU. METHODS: A cohort, observational, prospective study was carried out in the intensive care unit (ICU) of Mongi Slim Hospital, la Marsa, over 12 months period. Arterial blood lactate (ABL) was measured in ICU admission (H0), then 6 hours (H6), 12 hours (H12), 24 hours (H24) and 48hours (H48) after admission. Prognostic scores were calculated 24 hours after the admission. We also recorded biological data, hemodynamic parameters, and the evolution during the stay in intensive care. Primary endpoint was ICU mortality. RESULTS: We included 135 patients. The average age was 47.22 ± 16.88 years with a sex-ratio of 1.75. ICU mortality was 48%. The mean ABL at admission was 3.05 ± 2.63 mmol/l, higher in the dead group with a statistically significant difference. Prognostic value of lactate at admission was less powerful than severity indices in this study but remains excellent with an AUC >0, 7 defining « cut-off ¼ values with a good sensitivity and specificity. In multivariate analysis, initial lactate > 2 mmol/l was found to be an independent predictive factor of ICU mortality with an Odd Ratio [IC 95%] =1.16 [1.07 - 3.6]; p=0.04. CONCLUSIONS: Monitoring lactatemia in ICU could allow better identification of patients at high risk of death and the reassessment of therapeutic efficacy.

Novel mutations in Uridyl-diphosphate-glucuronosyl-transferase 1A1 (UGT1A1) gene in Tunisian patients with unconjugated hyperbilirubinemia.

INTRODUCTION: Unconjugated hyperbilirubinemia (UCB) is a feature of Gilbert's syndrome (GS) and Crigler-Najjar's syndrome (CNS), which are two hereditary defects in bilirubin metabolism. Both syndromes are linked to mutations in the UGT1A1 gene, which cause either the decrease or the absence of the UGT1A1 enzymatic activity. Here, we investigated the molecular basis of the UGT1A1 gene in Tunisian patients presenting with unconjugated hyperbilirubinemia. METHODS: Twenty-four patients with UCB were investigated. The screening protocol for hemoglobinopathies, enzymopathies, and membrane defects was executed in all patients. Afterward, the molecular analysis of the entire UGT1A1 gene was performed by DNA Sanger sequencing. Several bioinformatic tools were used to explore the effects of novel mutations. RESULTS: Fifteen different UGT1A1 variations were identified, among which four are described here for the first time. In exon 5, the c.1412C > G; p.(Ala471Gly) and c.1589C > T; p.(Ser530Phe) mutations were detected in patients presenting with CNS type I and GS, respectively. In the 3'UTR region of UGT1A1, the c.*90C > T mutation was detected in 3 patients with CNS type I. In the same region, the c.*388C > T defect was found in a GS patient. A deleterious and damaging effect on the UGT1A1 protein were predicted for both exonic mutations. Furthermore, novel microRNAs were identified as targetting the mutated sequences for the 3'UTR mutations. CONCLUSION: Our study provides novel data on UCB among Tunisians. Furthermore, we report four novel mutations associated with both GS and CNS. The identification of these mutations increases the spectrum of the UGT1A1 mutations and contributes to an understanding of the molecular abnormalities associated with unconjugated hyperbilirubinemia.

[The rate of development of myopia during hyperbaric oxygen therapy].

BACKGROUND: Hyperbaric oxygenation (HBO) is a common treatment both for emergency medicine as well as for chronic treatments. One of the most common indications for treatment is a non-healing wound due to diabetes. It is known that prolonged HBO treatments cause a myopic change in refraction. From the literature we know that the myopic shift is usually temporary, reversing back to basic refraction within a few weeks of cessation of the HBO course. There is controversy in the literature regarding the cause of refraction change, but research with animals reinforces the view that the source is the lens nucleus. PURPOSE: To measure the refractive change following 30 HBO treatments, and to quantify the rate of change. METHODS: A prospective study that included 44 eyes of diabetic patients treated with HBO in Rambam and Elisha Hyperbaric Center. Follow-up examinations were carried out every 10 treatments for spherical equivalents, sphere components, cylinder powers and axes (SPHER, SE, CYLINDER and AXIS respectively). The measurement device was an autorefractometer. A single examiner performed all the measurements; the examiner was blinded to the previous results. For each parameter, a regression equation was calculated after plotting the myopic change over time. RESULTS AND CONCLUSIONS: A positive correlation was found between HBO treatments and a myopic shift in the refraction. The correlation was statistically significant for: SPHER, SE. (0.58D, 0.61D) respectively. The change accrued from the first examination and remained throughout the follow-ups at a steady rate. Both eyes behaved similarly. The authors did not find a correlation between the myopic shift and gender, age or basic refraction. This information is vital both to clinicians, to understand the physiologic changes occurring during chronic HBO treatments, and especially to our patients, who are about to receive HBO treatments.

Are our stethoscopes contaminated ?

BACKGROUND: Health care-associated infections are a real public health problem. Contaminated medical equipments such as stethoscopes are often an overlooked vector. In our study, we were interested in proving our doctors stethoscopes contamination and in studying the microbiological profile of isolated germs. METHODS: This was about a cross-sectional study that lasted 2 months (May and June 2014) including 39 personal stethoscopes of all grade doctors working in 8 different departments in Habib Thameur Hospital. The swabs were taken from the membranes of the stethoscopes and sent quickly to our bacteriology service. Then, the bacteriological samples were subcultivated on blood agar. The reading tooks place 24 hours later. Were considered positive the cultures that contained more than 104 colony forming units/ml. Then we proceeded to the identification of the germ. RESULTS: Fifteen samples from 39 were positive that was 38 %. The coagulase- negative Staphylococcus was the predominant germ. The pathogenic germs were found in 5 positive samples that was 12 % from all the samples. They were distributed as follows: 2 Methicillin-susceptible Staphylococci Aureus, 2 methicillin-resistant coagulase- negative Staphylococci and one Pseudomonas aerogenusa. The pathogenic germs were found in stethoscopes taken from cardiology and intensive care unit (ICU). In cardiology, 7/9 stethoscopes were positive (5 coagulase- negative Staphylococcus among them 2 resistant methicillin and 2 Staphylococcus aureus.) In ICU, 6/13 stethoscopes were positive among them one Pseudomonas aerogenusa. CONCLUSION: The stethoscopes constitute bacterial reservoirs. It is necessary to perform a procedure for the good practices of disinfection.

Parecoxib in laparoscopic surgery for simple vesicular lithiasis.

AIM: To evaluate the contribution of parecoxib to the protocol of multimodal analgesia for simple vesicular lithiasis by laparoscopy. METHODS: A prospective, randomized, double-blind study was carried out at Habib Thameur Hospital (Tunis). We included 60 patients, ASA I or II, scheduled for cholecystectomy by laparoscopy. The patients were randomized to 2 groups. The parecoxib group (PG) receiving parecoxib 40 mg 30 minutes before the induction and the control group (CG) receiving physiological saline. Data were collected during hospitalization and a follow-up was done one year after the operation by a questionnaire. RESULTS: The pain scores at rest and at cough were significantly lower in the PG than in the CG during the first postoperative day (p < 10-3). Ten percent of the patients of the CG and no patient of the GP required Morphine in the recovery room (p = 0,07). The requirement of Tramadol was significantly less frequent in the PG (70 % of the PG, 16,6 % of the CG and p < 10-3). A chronic pain was found in 37,5 % and 8 %, respectively, in the GC and GP (p = 0,013). This pain was intense in 2 GC patients requiring analgesics and a work stoppage. CONCLUSIONS: The results of our study are in favor of the use of Parecoxib 40 mg 30 minutes before laparoscopic cholecystectomy for its effects on acute pain, opioid sparing and chronic pain.

Rapid and inexpensive detection of common HBB gene mutations in Tunisian population by high-resolution melting analysis: implication for molecular diagnosis.

In Tunisia, ß-thalassemia is a common hereditary disease with a carrying rate of 2.21%. Up to now, detection of responsible mutations was made by laborious, expensive, and/or time consuming methods. The aim of this study is to develop and validate a specific assay for detection of the two most frequent mutations in Tunisian population, the IVS-I-110 (G â†’ A) and Cd39 (C â†’ T) mutations. In this study, we optimize high resolution melting analysis (HRMA) conditions for these mutations, using control DNAs. Then, we evaluate the strength of this methodology by screening a cohort of patients with ß-thalassemia. All examined reference DNA samples were unambiguously distinguished from each other. For the blinded test, the results were completely compatible with direct sequencing, performed after the HRMA. As HRMA represents a highly sensitive and high-throughput gene scanning method, it can provide timely diagnosis at low cost for effective clinical management of ß-thalassemia.