Healthcare Challenges and Future Solutions in Dental Practice: Assessing Oral Antibiotic Resistances by Contemporary Point-Of-Care Approaches

Antibiotic resistance poses a global threat, which is being acknowledged at several levels, including research, clinical implementation, regulation, as well as by the World Health Organization. In the field of oral health, however, the issue of antibiotic resistances, as well as of accurate diagnosis, is underrepresented. Oral diseases in general were ranked third in terms of expenditures among the EU-28 member states in 2015. Yet, the diagnosis and patient management of oral infections, in particular, still depend primarily on empiric means. On the contrary, on the global scale, the field of medical infections has more readily adopted the integration of molecular-based systems in the diagnostic, patient management, and antibiotic stewardship workflows. In this perspective review, we emphasize the clinical significance of supporting in the future antibiotic resistance screening in dental practice with novel integrated and point-of-care operating tools that can greatly support the rapid, accurate, and efficient administration of oral antibioticspublishedVersio

Expert Guidance on Target Product Profile Development for AMR Diagnostic Tests

Diagnostics are widely considered crucial in the fight against antimicrobial resistance (AMR), which is expected to kill 10 million people annually by 2030. Nevertheless, there remains a substantial gap between the need for AMR diagnostics versus their development and implementation. To help address this problem, target product profiles (TPP) have been developed to focus developers’ attention on the key aspects of AMR diagnostic tests. However, during discussion between a multisectoral working group of 51 international experts from industry, academia and healthcare, it was noted that specific AMR-related TPPs could be extended by incorporating the interdependencies between the key characteristics associated with the development of such TPPs. Subsequently, the working group identified 46 characteristics associated with six main categories (i.e., Intended Use, Diagnostic Question, Test Description, Assay Protocol, Performance and Commercial). The interdependencies of these characteristics were then identified and mapped against each other to generate new insights for use by stakeholders. Specifically, it may not be possible for diagnostics developers to achieve all of the recommendations in every category of a TPP and this publication indicates how prioritising specific TPP characteristics during diagnostics development may influence (or not) a range of other TPP characteristics associated with the diagnostic. The use of such guidance, in conjunction with specific TPPs, could lead to more efficient AMR diagnostics development

Fully automated point-of-care differential diagnosis of acute febrile illness

Background In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk (“FeverDisk” for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device. Methodology/Principal findings A sample volume of 200 μL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae. Conclusions/Significance The FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.Additional co-authors: Sieghard Frischmann, Konstantinos Mitsakaki

Developmental roadmap for antimicrobial susceptibility testing systems

Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions

The successful uptake and sustainability of rapid infectious disease and antimicrobial resistance point-of-care testing requires a complex `mix-and-match' implementation package

The emergence and spread of antimicrobial resistance is one of the major global issues currently threatening the health and wealth of nations, with effective guidelines and intervention strategies urgently required. Such guidelines and interventions should ideally be targeted at individuals, communities, and nations, requiring international coordination for maximum effect. In this respect, the European Joint Programming Initiative on Antimicrobial Resistance Transnational Working Group ‘Antimicrobial Resistance - Rapid Diagnostic Tests’ (JPIAMR AMR-RDT) is proposing to consider a ‘mix-and-match’ package for the implementation of point-of-care testing (PoCT), which is described in this publication. The working group was established with the remit of identifying barriers and solutions to the development and implementation of rapid infectious disease PoCT for combatting the global spread of antimicrobial resistance. It constitutes a multi-sectoral collaboration between medical, technological, and industrial opinion leaders involved in in vitro diagnostics development, medical microbiology, and clinical infectious diseases. The mix-and-match implementation package is designed to encourage the implementation of rapid infectious disease and antimicrobial resistance PoCT in transnational medical environments for use in the fight against increasing antimicrobial resistance.Peer Reviewe

Developmental roadmap for antimicrobial susceptibility testing systems

Antimicrobial susceptibility testing (AST) technologies help to accelerate the initiation of targeted antimicrobial therapy for patients with infections and could potentially extend the lifespan of current narrow-spectrum antimicrobials. Although conceptually new and rapid AST technologies have been described, including new phenotyping methods, digital imaging and genomic approaches, there is no single major, or broadly accepted, technological breakthrough that leads the field of rapid AST platform development. This might be owing to several barriers that prevent the timely development and implementation of novel and rapid AST platforms in health-care settings. In this Consensus Statement, we explore such barriers, which include the utility of new methods, the complex process of validating new technology against reference methods beyond the proof-of-concept phase, the legal and regulatory landscapes, costs, the uptake of new tools, reagent stability, optimization of target product profiles, difficulties conducting clinical trials and issues relating to quality and quality control, and present possible solutions

Ανάπτυξη ακουστικού βιοαισθητήρα πολλαπλής ανάλυσης για κλινική διαγνωστική

This work focuses on the development of a multi-analyte biosensor, based on a Surface Acoustic Wave (SAW) device. The novelty of the concept lies in the way of achieving multiplicity: instead of the “traditional” way of a sensor element array, multiplicity is induced by compartmentalization of a single sensor, achieved via microfluidics (“microfluidics-on-SAW”, or “μF-on-SAW” setup). Initially, the appropriate SAW device for the microsystem was selected among twelve device configurations (varying in substrate, operating frequency and waveguide thickness) upon loading with different classes of materials (mass, viscous, viscoelastic). In particular, a dual quartz-based SAW biochip was used, operating at 155 MHz with 0.70 μm thick PMMA waveguide. Subsequently, the microfluidic module was designed targeting flexibility and simplicity. Considering functional and geometrical limitations imposed by the SAW biochip, the two components were successfully assembled. The fabrication process for the microfluidic module was soft lithography of PDMS (rapid prototyping and replica molding); 3-, 4-, and 5-channel modules were made, all successfully tested, and the 4-channel one used in the project. Reproducibility and sensitivity tests were carried out using aqueous glycerol solutions, and standard protein biomolecules (neutravidin and biotinylated BSA, as well as protein G and IgG). The standard deviation in the signal values among the sub-areas was less than 10%, in all cases. The proof-of-principle of multi-sample detection was achieved via four biotinylated molecules. Each one was injected in one μF-on-SAW compartment and interacted with pre-adsorbed neutravidin; separate detection of the analytes, kinetics and equilibrium analysis were successfully demonstrated. Maximum multiplexity was achieved when the two devices of the biochip were pre-functionalized with different receptors, and four different samples were injected in each microchannel (altogether 8 probed interactions). The final step was the application of μF-on-SAW in multi-sample detection of clinical significance. In particular, cardiac markers were used, the detection of which was realized via antibody-antigen interactions. The four cardiac markers (CK-MB, CRP, D-dimer, and PAPP-A) were successfully detected individually and in various concentrations; analytical curves were created for each biomarker and correlation to the known physiological and pathological values was made. Eventually, by using the μF-on-SAW it was feasible to selectively capture each marker out of a mixture or all four, a proof that the system can potentially be used in body fluids (were many “unwanted” species are present). Finally, from the different groups of biomolecules detected throughout the project, interesting results emerged concerning the interaction of acoustic waves with biomolecules and the correlation of the acoustic signal with inherent properties of biomolecules such as their molecular weight and viscoelastic nature.Η παρούσα εργασία εστιάζεται στην ανάπτυξη ενός βιοαισθητήρα ανίχνευσης πολλαπλών δειγμάτων, βασισμένου σε μικροδιατάξεις επιφανειακών ακουστικών κυμάτων (SAW). Η καινοτομία της ιδέας βρίσκεται στον τρόπο επίτευξης της πολλαπλότητας ανάλυσης: αντί του «παραδοσιακού» τρόπου της χρήσης μιας συστοιχίας από αισθητήρες, εδώ η πολλαπλότητα επιτυγχάνεται μέσω της διαμερισματοποίησης της επιφάνειας ενός μόνο αισθητήρα σε υποπεριοχές, γεγονός που επάγεται από τη χρήση μικρορευστομηχανικών διατάξεων. Αρχικά επιλέχθηκε η κατάλληλη μικροδιάταξη αισθητήρα SAW ανάμεσα σε δώδεκα διαφορετικές διαμορφώσεις (οι οποίες διέφεραν ως προς το υπόστρωμα, τη συχνότητα λειτουργίας και το πάχος του κυματοδηγού) χρησιμοποιώντας διαφορετικής φύσεως υλικά (με ελαστικό, ιξώδη και ιξωδοελαστικό χαρακτήρα) για το χαρακτηρισμό. Τελικά χρησιμοποιήθηκε μια διάταξη διπλού αισθητήρα με υπόστρωμα χαλαζία στα 155 MHz και με πάχος κυματοδηγού 0.70 μm. Ακολούθως, σχεδιάστηκε η μικρορευστομηχανική διάταξη/κυψελίδα, με γνώμονα την ευελιξία και απλότητα στη χρήση του συστήματος και λαμβάνοντας υπόψη λειτουργικούς και γεωμετρικούς περιορισμούς που επιβάλλονταν από τη μικροδιάταξη του διπλού αισθητήρα. Η μέθοδος κατασκευής της κυψελίδας ήταν η λιθογραφία μαλακής ύλης του υλικού PDMS (τα βασικά βήματα της οποίας ήταν η ταχεία δημιουργία πρωτοτύπου και η αντιγραφή του εκμαγείου) με την οποία κατασκευάστηκαν και δοκιμάστηκαν επιτυχώς κυψελίδες με 3, 4, και 5 κανάλια. Η επαναληψιμότητα και η ευαισθησία ελέγχθηκαν χρησιμοποιώντας υδατικά διαλύματα γλυκερόλης και κάποιες συνήθεις πρωτεΐνες (νιουτραβιδίνη και βιοτινυλιωμένη BSA, protein G και IgG). Η απόκλιση του σήματος μεταξύ των σχηματισμένων υποπεριοχών ήταν μικρότερη από 10% σε όλες τις περιπτώσεις. Η απόδειξη της λειτουργίας του μικροσυστήματος ως πολυ-δειγματικού αναλυτή, έγινε με χρήση τεσσάρων διαφορετικών βιοτινυλιωμένων πρωτεϊνών. Κάθε πρωτεΐνη εισήλθε σε έναν υποχώρο του μικροσυστήματος και ανιχνεύθηκε η αλληλεπίδρασή της με την ήδη προσροφημένη νιουτραβιδίνη. Η ανεξάρτητη ανίχνευση των βιομορίων στους υποχώρους καθώς και η ανάλυση της κινητικής τους έγιναν με επιτυχία. Η αξιοποίηση του μικροσυστήματος στο μέγιστο βαθμό του πραγματοποιήθηκε όταν δύο διαφορετικοί υποδοχείς προ-ακινητοποιήθηκαν στους δύο αισθητήρες και τέσσερα διαφορετικά δείγματα εισήχθησαν στα μικροκανάλια. Έτσι ανιχνεύθηκαν συνολικά 8 διαφορετικές αλληλεπιδράσεις. Η εφαρμογή του μικροσυστήματος σε δείγματα κλινικού ενδιαφέροντος πραγματοποιήθηκε με χρήση καρδιακών δεικτών, όπου η ανίχνευση έγινε μέσω της αλληλεπίδρασης αντιγόνου-αντισώματος. Οι τέσσερις δείκτες (CK-MB, CRP, Ddimer, και PAPP-A) αναλύθηκαν σε διάφορες συγκεντρώσεις, διερευνήθηκαν τα όρια λειτουργίας του μικροσυστήματος, και έγινε συσχετισμός με πραγματικές κλινικές τιμές. Επίσης, επιτεύχθηκε επιλεκτική ανίχνευση των πρωτεϊνών μέσα από ένα μίγμα όλων των διαθέσιμων δεικτών. Τέλος, από τα πειράματα που έγιναν με όλα τα βιομόρια, προέκυψαν συμπεράσματα και ενδιαφέροντα δεδομένα σχετικά με το συσχετισμό του ακουστικού κύματος με ιδιότητες των βιομορίων (το μοριακό τους βάρος και την ιξωδοελαστική τους φύση)

Diagnostic tools for tackling febrile illness and enhancing patient management

Most patients with acute infectious diseases develop fever, which is frequently a reason to visit health facilities in resource-limited settings. The symptomatic overlap between febrile diseases impedes their diagnosis on clinical grounds. Therefore, the World Health Organization promotes an integrated management of febrile illness. Along this line, we present an overview of endemic and epidemic etiologies of fever and state-of-the-art diagnostic tools used in the field. It becomes evident that there is an urgent need for the development of novel technologies to fulfill end-users' requirements. This need can be met with point-of-care and near-patient diagnostic platforms, as well as e-Health clinical algorithms, which co-assess test results with key clinical elements and biosensors, assisting clinicians in patient triage and management, thus enhancing disease surveillance and outbreak alerts. This review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem

Validation and verification of predictive salivary biomarkers for oral health

WOS:000632046500002PubMed: 33742017Oral health is important not only due to the diseases emerging in the oral cavity but also due to the direct relation to systemic health. Thus, early and accurate characterization of the oral health status is of utmost importance. There are several salivary biomarkers as candidates for gingivitis and periodontitis, which are major oral health threats, affecting the gums. These need to be verified and validated for their potential use as differentiators of health, gingivitis and periodontitis status, before they are translated to chair-side for diagnostics and personalized monitoring. We aimed to measure 10 candidates using high sensitivity ELISAs in a well-controlled cohort of 127 individuals from three groups: periodontitis (60), gingivitis (31) and healthy (36). The statistical approaches included univariate statistical tests, receiver operating characteristic curves (ROC) with the corresponding Area Under the Curve (AUC) and Classification and Regression Tree (CART) analysis. The main outcomes were that the combination of multiple biomarker assays, rather than the use of single ones, can offer a predictive accuracy of > 90% for gingivitis versus health groups; and 100% for periodontitis versus health and periodontitis versus gingivitis groups. Furthermore, ratios of biomarkers MMP-8, MMP-9 and TIMP-1 were also proven to be powerful differentiating values compared to the single biomarkers.European UnionEuropean Commission [633780]; Karolinska InstitutetKarolinska Institutet; Baden-Wuerttemberg Ministry of Science, Research and Art; University of FreiburgThis study was funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No 633780 ("DIAGORAS" project) and was partially supported by strategic funds from Karolinska Institutet. The article processing charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the University of Freiburg in the funding programme Open Access Publishing. We would like to thank all the study participants who donated samples

Magnetophoresis in Centrifugal Microfluidics at Continuous Rotation for Nucleic Acid Extraction

Centrifugal microfluidics enables fully automated molecular diagnostics at the point-of-need. However, the integration of solid-phase nucleic acid extraction remains a challenge. Under this scope, we developed the magnetophoresis under continuous rotation for magnetic bead-based nucleic acid extraction. Four stationary permanent magnets are arranged above a cartridge, creating a magnetic field that enables the beads to be transported between the chambers of the extraction module under continuous rotation. The centrifugal force is maintained to avoid uncontrolled spreading of liquids. We concluded that below a frequency of 5 Hz, magnetic beads move radially inwards. In support of magnetophoresis, bead inertia and passive geometrical design features allow to control the azimuthal bead movement between chambers. We then demonstrated ferrimagnetic bead transfer in liquids with broad range of surface tension and density values. Furthermore, we extracted nucleic acids from lysed Anopheles gambiae mosquitoes reaching comparable results of eluate purity (LabDisk: A260/A280 = 1.6 ± 0.04; Reference: 1.8 ± 0.17), and RT-PCR of extracted RNA (LabDisk: Ct = 17.9 ± 1.6; Reference: Ct = 19.3 ± 1.7). Conclusively, magnetophoresis at continuous rotation enables easy cartridge integration and nucleic acid extraction at the point-of-need with high yield and purity