A swift risk analysis for COVID-19 testing facilities using rapid tests

Introduction. COVID-19 is an infectious disease of International Concern, due to the wide-spread geographic impact and high transmissibility, causing severe illnesses. Many testing facilities were set-up for monitoring the spread of the SARS-CoV-2 virus, at the early days of the coronavirus pandemic. From Biosafety aspect this study investigates a reliable risk assessment method to identify and mitigate the risks of COVID-19 testing facilities using Rapid diagnostic tests (POCT), in order to protect the staff, the people who got tested, the community and the environment. Material and methods. Many techniques have been used so far for performing a risk assessment. In the present study, SWIFT analysis suitable for biosafety facilities and for risks of different magnitude, was used for identifying threats and hazards and to calculate the risks for COVID-19 testing facilities. Results. Our analysis showed several initial and potential risks, which could lead to unwanted exposure or release of the SARS-CoV-2, and/or unwanted infection of staff and patients. With minor adjustments of the testing facility, by creating standard operating procedures and awareness of the potential risks, most of the identified risks could be mitigated. Conclusions. Our study demonstrated that when setting up a COVID-19 testing facility, a proper risk assessment should be part of the process, in order to ensure the safety of staff, patients, and the environment. Additionally, we proposed a number of multiple mitigation measures and recommendations, with the goal to reduce the risks during the rapid testing diagnostic procedure.Introducere. COVID-19 este o boală infecțioasă cu un impact geografic larg răspândit și transmisibilitate ridicată, care poate provoacăboli grave. Încă de la debutul pandemiei de COVID-19 au fost înființate multe stații de testare pentru monitorizarea răspândirii virusu-lui SARS-CoV-2. Din punct de vedere al biosecurității acest studiu investighează o metodă de evaluare a riscurilor în vederea identificării și atenuării riscurilor stațiilor de testare COVID-19, care utilizează teste de diagnosticare rapidă (POCT) pentru a proteja personalul, pacienții, comunitatea și mediul. Material și metode. În prezentul studiu au fost aplicate diferite tehnici pentru realizarea unei evaluări a riscurilor. A fost utilizată analiza SWIFT pentru instalațiile de biosecuritate și pentru riscuri de diferită amploare pentru identificarea amenințărilor și pericolelor, și pentru a calcula riscurile pentru stațiile de testare COVID-19. Rezultate. Analiza noastră a identificat mai multe riscuri inițiale și potențiale, care ar putea duce la expunerea sau eliberarea nedorită a SARS-CoV-2 și/sau la infectarea nedorită a personalului și a pacienților. Cu ajustări minore ale stațiilor de testare, prin crearea de proceduri standard de operare și conștientizarea riscurilor potențiale, majoritatea riscurilor identificate ar putea fi atenuate. Concluzii. Prezentul studiu a demonstrat că atunci când se înființează o unitate de testare COVID-19, o evaluare adecvată a riscurilor ar trebui să facă parte din proces pentru a asigura siguranța personalului, a pacienților și a mediului. În plus, am propus o serie de măsuri și recomandări multiple de atenuare cu scopul de a reduce riscurile în timpul procedurii de diagnosticare a testării rapide

Expression, characterization and structural study through nuclear magnetic resonance spectroscopy of the catalytic center of the bacillus anthracis' metalloprotease lethal factor

Anthrax is an acute, infectious disease that became widely known as a biological weapon used in terrorist attacks after the events of 11th September 2001. The disease is caused by the toxic effect of Bacillus anthracis, a positive Gram bacterium. The difficulty of treating the disease, along with the resulting increased morbidity rate are due to the often delayed diagnosis and treatment, as the initial symptoms are non-specific. The bacterium’s toxicity is caused by the synergistic action of three bacterial proteins, namely the Protective Antigen, the Edema Factor and the Lethal Factor. Anthrax Lethal Factor is a metalloprotease that proteolytes members of the MAPKKs family, thus disrupting the cellular homeostasis and ultimately leading to cellular death. Lethal Factor is composed of four domains, with domains I and IV being of special interest out of their role in the pathogenicity of the disease. Specifically, domain I allows the entrance of the protein into the cell, which is a crucial step for it to manifest its toxic effect, while domain IV includes the metalloprotease catalytic core. Efforts were made to express different polypeptides that represent these two domains, followed by the application of NMR spectroscopy in order to structurally elucidate them. Due to the comparatively large length of the polypeptides, novel protocols had to be applied in both the preparation of the protein samples, so that they fulfill the necessary requirement for acquiring NMR spectra, and the application of NMR spectroscopy. By applying such techniques, the expression and isolation of a polypeptide with MW>27 kDa corresponding to domain I was achieved, followed by acquiring a vast range of homo-and hetero-, 1D, 2D and 3D NMR spectra for the purpose of its structural elucidation. After the analysis of the spectra was carried out, the assignment of the chemical shifts was successfully completed for the nuclei of the backbone. Their values were used for predicting the secondary structure of the polypeptide, which in turn showed significant similarities to the known crystallographic structural data. The polypeptides corresponding to domain IV exhibited poor solubility and/or folding, even after applying novel methods of protein expression and protocols including denaturation agents. Only one polypeptide, consisted of 106 residues, had been previously shown to be soluble and relatively well folded. Thus, it was the only option for monitoring the possible interaction of the catalytic core with a synthetic peptide-analogue of the natural substrate of the enzyme. The possibility of interaction was monitored by using the chemical shift perturbation assay.Η νόσος του Άνθρακα είναι μια οξεία μολυσματική ασθένεια που έγινε ιδιαιτέρως γνωστή από τη χρήση της ως βιολογικό όπλο σε τρομοκρατικές επιθέσεις μετά τα γεγονότα της 11ης Σεπτεμβρίου 2001. Η νόσος οφείλεται στην παθογόνο δράση του βάκιλλου Bacillus anthracis, ενός (+)-Gram βακτηρίου. Η δυσκολία στην αντιμετώπιση της νόσου και η συνεπαγόμενη αυξημένη νοσηρότητα οφείλονται συχνά στην καθυστερημένη διάγνωση και θεραπεία, καθώς τα αρχικά συμπτώματα είναι μη ειδικά. Η τοξική δράση του βάκιλλου οφείλεται στη συνεργιστική δράση 3 πρωτεΐνων που εκφράζει, το Προστατευτικό Αντιγόνο, το Παράγοντα Οιδήματος και τον Θανατηφόρο Παράγοντα. Ο Θανατηφόρος Παράγοντας αποτελεί μια μεταλλοπρωτεάση που πρωτεολύει μέλη της οικογένειας των MAPKKs, δημιουργώντας διαταραχή της κυτταρικής ομοιόστασης που οδηγεί σε κυτταρικό θάνατο. Ο Θανατηφόρος Παράγοντας, που αποτελεί και το αντικείμενο μελέτης της παρούσας ερευνητικής εργασίας, αποτελείται από 4 τομείς, από τους οποίους ο τομέας Ι και ο τομέας IV παρουσιάζουν ιδιαίτερο ενδιαφέρον λόγω του ρόλου του στην παθογένεια της νόσου. Πιο συγκεκριμένα, ο τομέας Ι επιτρέπει την είσοδο της πρωτεΐνης στο εσωτερικό του κυττάρου η οποία είναι απαραίτητη για την άσκηση της τοξικής δράσης, ενώ ο τομέας IV περιέχει το καταλυτικό κέντρο μεταλλοπρωτεάσης. στην παρούσα εργασία, έγινε προσπάθεια έκφρασης πολυπεπτιδικών τμημάτων των δύο αυτών τομέων και ακόλουθη εφαρμογή NMR φασματοσκοπίας για το δομικό χαρακτηρισμό τους. Λόγω του εξαιρετικά μεγάλου μήκους των πολυπεπτιδίων, εφαρμόσθηκαν καινοτόμα πρωτόκολλα που αφορούν τόσο στη διαδικασία παρασκευής των πρωτεΐνικών δειγμάτων ώστε να πληρούν τα κριτήρια για τη διενέργεια NMR πειραμάτων, όσο και στην καθεαυτή εφαρμογή της NMR φασματοσκοπίας και ανάλυσης των φασμάτων. Με τη χρήση των τεχνικών αυτών, ολοκληρώθηκε με επιτυχία η έκφραση, απομόνωση και λήψη όμο- και έτερο-, 1D, 2D και 3D NMR φασμάτων για το δομικό χαρακτηρισμό του πολυπεπτιδίου με MB > 27KDa, που αντιστοιχεί σε ολόκληρο το δομημένο τμήμα του Τομέα Ι. Μετά από ανάλυση των φασμάτων, ολοκληρώθηκε η αποτίμηση των χημικών μετατοπίσεων των πυρήνων του πολυπεπτιδικού σκελετού , καθώς και η πρόβλεψη της δευτεροταγούς του δομής, η οποία έδειξε καταφανείς ομοιότητες με τα μέχρι τώρα γνωστά κρυσταλλογραφικά δεδομένα. Τα πολυπεπτίδια που αντιστοιχούν στον Τομέα ΙV έδειξαν φτωχή διαλυτότητα και αναδίπλωση, παρά τη χρήση καινοτόμων μεθόδων έκφρασης και χρήση αποδιατακτικών μέσων. Το μόνο πολυπεπτίδιο που είχε ήδη δείξει στο παρελθόν σχετικά καλή αναδίπλωση, αν και μήκους μόνο 106 καταλοίπων, χρησιμοποιήθηκε σε μελέτη αλληλεπίδρασης με πεπτιδικό ανάλογο του φυσικού υποστρώματος της καταλυτικής κοιλότητας και η αλληλεπίδραση τους παρατηρήθηκε μέσω καταγραφής της διαφοροποίησης των χημικών μετατοπίσεων