Développement et évaluation de méthodes pour le diagnostic prénatal non-invasif à partir des cellules fœtales circulant dans le sang maternel

Abstract : Current prenatal diagnosis depends on invasive procedures and is thus offered only to high-risk pregnancies. Development of non-invasive prenatal diagnosis (NIPD) would change the risk-benefit ratio and make it likely that more women would benefit from prenatal testing. Scientists have documented the presence of rare fetal cells in maternal blood and envisioned targeting them with specific markers and their use in NIPD. Considering their extremely low frequency in maternal blood, fetal cells have been difficult to retrieve and use in clinical practice. Therefore, there is a pressing need for systematic sequential studies to evaluate their feasibility in NIPD. Generally, detection of rare cells within a large cell population carries great potentialities for the prospects of cancer management and NIPD. Manual scanning is very cumbersome and time-consuming Therefore; the first part of our project was, dedicated to the optimization of an effective strategy to evaluate retrieval of rare cells. We have developed a way of distributing a controlled number of target cells among hundreds of thousands of other cells on microscope slides. This strategy allows the precise evaluation of the retrieval of rare events and the comparizon of the efficacy of different techniques and enrichment approaches by knowing the definite number and locations of target cells on the slides. Furthermore, it allows the evaluation of hybridization of missed events. We have also developed a robust custom-made detection algorithm for rare cells using the MetaSystems automated platform and have used this strategy in the validation of manual and automatic scanning of 60 slides with a pre-defined number of rare male cells among a pure population of female cells using XY-FISH. Consequently, we tested the developed classifier for the detection of real fetal cells from maternal blood in both normal and aneuploid pregnancies with Down syndrome. We further evaluated the number of fetal cells with different methods of enrichments in the first and second trimesters. The data collected confirmed the early presence of fetal cells in all of the pregnancies tested and their frequencies were higher in cases of aneuploidies. Fetal cells are in a state of dynamic change throughout the pregnancy. Higher numbers of these cells can be obtained by optimizing the harvest time and methods of enrichment. We found that automatic scanning is more sensitive and reliable than manual detection. Furthermore, it alleviates the burden of scanning large numbers of cells and thus is more suitable for clinical application. We also demonstrated the feasibility of using rare cells in NIPD. Five microdissected amniotic fetal cells from 26 cases of normal and aneuploid pregnancies were quite enough to provide accurate NIPD through using whole genome amplification coupled with QF-PCR. Our findings laid the ground for the use of rare fetal cells in maternal blood for NIPD. // Résumé : Le diagnostic prénatal résulte encore aujourd’hui de procédures invasives, qui présentent des risques pour la grossesse. Le développement du diagnostic prénatal non-invasif (DPNI) changerait le rapport risque : bénéfice, rendant le diagnostic prénatal plus intéressant pour les femmes enceintes. Plusieurs chercheurs ont montré la présence de cellules fœtales dans le sang maternel et des travaux ont été entrepris afin de les cibler et de les utiliser éventuellement en DPNI. Toutefois, la faible concentration des cellules fœtales dans le sang maternel réduit les possibilités d’isolement ainsi que celles de leur utilisation en clinique. Un autre aspect technique du DPNI, le balayage manuel, est très laborieux, surtout en terme de temps technique. Il y a donc un besoin certain pour des études approfondies afin d’évaluer et d’améliorer la faisabilité du DPNI. La détection d’évènements rares dans une grande population cellulaire offre un potentiel pour le diagnostic en oncologie mais aussi en diagnostic prénatal. Dans cette thèse, la première étude était dédiée à l’optimisation d’une stratégie pour détecter les cellules rares. Nous avons développé une méthode d’étalement sur lame d’un nombre précis de cellules cibles parmi des centaines de milliers de cellules. Cette stratégie a permis d’évaluer le taux de détection d’évènements rares et de comparer l’efficacité des techniques d’enrichissement en connaissant le nombre exact et la localisation de cellules cibles sur les lames. De plus, il a été possible d’évaluer les problèmes d’hybridation des évènements manqués. Nous avons, par la suite, développé un algorithme robuste pour la détection de cellules rares en utilisant la plateforme de microscopie automatisée MetaSystems et utilisé cette approche dans la validation des balayages manuel et automatique d’un nombre précis de cellules mâles parmi une large population de cellules femelles marquées avec la technique FISH. Nous avons testé ce classificateur avec des échantillons de sang de femmes enceintes de grossesses normales et aneuploïdes et évalué la fréquence de cellules fœtales isolées par différentes méthodes d’enrichissement au cours des premier et second trimestres de grossesse. Les données accumulées ont confirmé la présence de cellules fœtales chez toutes les grossesses et leur fréquence plus élevée dans les grossesses aneuploïdes. Le nombre de cellules fœtales est dynamique tout au long de la grossesse. De plus, un nombre plus élevé de cellules fœtales peut être obtenu en optimisant le moment du prélèvement et les méthodes d’enrichissement. De plus, le balayage automatique s’est avéré plus sensible et constant que le balayage manuel, ce qui permet de balayer un grand nombre de cellules et devient plus approprié pour une application clinique. Nous avons aussi montré la faisabilité d’utiliser des cellules fœtales dans le cadre du DPNI. Cinq cellules amniotiques microdisséquées, provenant de grossesses normales et aneuploïdes, ont suffi pour poser un diagnostic prénatal par une combinaison de l’amplification du génome complet et de la technique QF-PCR (réaction quantitative en fluorescence d’amplification entraînée par une polymérase) permettant la détection d’anomalies chromosomiques. Nos résultats ouvrent la voie à l’utilisation de cellules fœtales dans le sang maternel pour le DPNI

Prevalence of extensively drug-resistant gram negative bacilli in surgical intensive care in Egypt

Introduction: the prevalence of extensively drug resistant gram negative bacilli (XDR-GNB) is rapidly progressing; however in Egypt data are sparse. We conducted the present study to  quantify the incidence, risk factors and outcome of patients harboring XDR-GNB. Methods: a one year prospective study was done by collecting all the bacteriological reports  for cultures sent from the surgical intensive care unit, Cairo university teaching hospital.  XDR-GNB were defined as any gram negative bacilli resistant to three or more classes of  antimicrobial agents .Patients with XDR-GNB compared with those sustaining non extensively drug-resistant infection. A multivariate logistic regression model was created to identify independent predictors of multi-resistance. Results: during one-year study period, a total of 152 samples (65%) out of 234 gram negative  bacilli samples developed extensively drug resistant infection. XDR strains were significantly  higher in Acinetobacterspp (86%), followed by Pseudomonas (63%), then Proteus (61%),  Klebsiella (52%), and E coli (47%). Fourth generation cephalosporine (Cefipime) had the lowest susceptibility (10%) followed by third generation cephalosporines (11%), Quinolones (31%), Amikacin (42%), Tazobactam (52%), Carbapinems (52%), and colistin (90%).Relaparotomy was the only significant risk factor for acquisition of XDR infection. Conclusion: extensively drug-resistant gram negative infections are frequent in our ICU. This  is an alarming health care issue in Egypt which emphasizes the need to rigorously implement  infection control practices

Pulse Pressure Variation-Guided Fluid Therapy during Supratentorial Brain Tumour Excision: A Randomized Controlled Trial

BACKGROUND: Goal-directed fluid therapy (GDFT) improved patient outcomes in various surgical procedures; however, its role during mass brain resection was not well investigated. AIM: In this study, we evaluated a simple protocol based on intermittent evaluation of pulse pressure variation for guiding fluid therapy during brain tumour resection. METHODS: Sixty-one adult patients scheduled for supratentorial brain mass excision were randomized into either GDFT group (received intraoperative fluids guided by pulse pressure variation) and control group (received standard care). Both groups were compared according to the following: brain relaxation scale (BRS), mean arterial pressure, heart rate, urine output, intraoperative fluid intake, postoperative serum lactate, and length of hospital stay. RESULTS: Demographic data, cardiovascular data (mean arterial pressure and heart rate), and BRS were comparable between both groups. GDFT group received more intraoperative fluids {3155 (452) mL vs 2790 (443) mL, P = 0.002}, had higher urine output {2019 (449) mL vs 1410 (382) mL, P < 0.001}, and had lower serum lactate {0.9 (1) mmol versus 2.5 (1.1) mmol, P = 0.03} compared to control group. CONCLUSION: In conclusion, PPV-guided fluid therapy during supratentorial mass excision, increased intraoperative fluids, and improved peripheral perfusion without increasing brain swelling

The Impact of Goal-Directed Fluid Therapy in Prolonged Major Abdominal Surgery on Extravascular Lung Water and Oxygenation: A Randomized Controlled Trial

BACKGROUND: A growing interest had been paid to goal-directed fluid therapy (GDT) in abdominal surgery; however, its impact on the respiratory profile was not well investigated. AIM: We evaluated the impact of GDT on postoperative extravascular lung water and oxygenation after prolonged major abdominal surgery. METHODS: A randomised, controlled study was conducted in Kasr Alainy hospital from April 2016 till December 2017 including 120 adult patients scheduled for prolonged major abdominal surgery. Patients were randomised into either GDT group (n = 60) who received baseline restricted fluid therapy (2 mL/Kg/hour) which is guided by stroke volume variation, or control group (n = 60) who received standard care. Both study groups were compared according to hemodynamic data, fluid requirements, lung ultrasound score, and PaO2/fraction of inspired oxygen ratio (P/F ratio), RESULTS: Intraoperatively, GDT group received less volume of fluids and showed higher intraoperative mean arterial pressure compared to the control group. Postoperatively, lung ultrasound score was lower, and P/F ratio was higher in the GDT group compared to the control group. The number of patients who showed a significant postoperative increase in LUS was higher in the control group 44 (73%) patients versus 14 (23%) patients, P < 0.001). CONCLUSIONS: Using stroke volume variation for guiding fluid therapy in prolonged, major abdominal operations were associated with better hemodynamic profile, less intraoperative fluid administration, lower extravascular lung water and better oxygenation compared to standard care

Technical considerations for Electrorestoration in historical metal products

Abstract:Electrochemical restoration is considered a technique dealing with the treating of historical metal products and the repairing of the distortion occurred in them as a result of multiple factors and to achieve this, electrochemical formation processes are used with different metals, whether they are precious such as silver and gold and their alloys or non-precious as copper and its alloys by taking advantage of the unique properties of these metals.Hence, the corrosion process has many problems and defects affect on historical metal products, that may cause visible distortion in the product.Therefore, there are technical considerations affecting the quality of the electrochemical restoration process, which are represented in several factors related to precise control during the procedure as controlling the temperature of the solution, current density, and the concentration of metal ions in the solution.............. and other factors.Where the failure to pay attention to the control factors will lead to undesirable results in the part properties that is being restored, especially the mechanical and chemical properties.So, the success of the electrochemical restoration process depends on the accuracy of attention to these technical considerations, and then obtaining the required technical specifications in the places that are being restored.Therefore, the success of the electrochemical restoration process depends on the accuracy of attention to these technical considerations, and then obtaining the required technical specifications in the places that are being restored.Through the foregoing, the research problem is determined by the need to identify the most important different technical factors affecting the electrochemical restoration process of historical metal products.The aim of the research is to develop a scientific methodology to determine the most important factors affecting the cathodic electrochemical systems for the restoration and treatment of historical mineral products by conducting the analytical study and knowing its methods and the most important solutions used in it and standing on the aesthetic, economic and environmental values added to the product. And this will be done under the premise that careful control of the various factors of the electrochemical restoration process achieves a great deal of accuracy in treating the deformations caused by multiple corrosion factors in historical metal products.Based on the descriptive analytical method