PHAR4813 Methods for the Detection of Pathogenic Bacteria PDF

 Acknowledgement of Country I would like to acknowledge the Traditional Owners of Australia and recognise their continuing connection to land, water and culture. I am currently on the land of the Gadigal people and pay my respects to their Elders, past, present and emer...

 Acknowledgement of Country I would like to acknowledge the Traditional Owners of Australia and recognise their continuing connection to land, water and culture. I am currently on the land of the Gadigal people and pay my respects to their Elders, past, present and emerging. I further acknowledge the Traditional Owners of the country on which you are on and pay respects to their Elders, past, present and future.  Prof. Paul Groundwater [email protected] PHAR4813 Methods for the Detection of Pathogenic Bacteria The Post Antibiotic Age?  Antimicrobial Treatment Traditional advice is to complete the course of antibiotics But does this always represent the optimum use?  Australian Pharmacy Council Pharmacy Learning Domains Learning domain 1: The health care consumer The pharmacist’s contribution to the promotion of good health and disease prevention. Symptoms recognition and management, the principles of differential diagnosis, important diagnostic methods and tests, and medical terminology. Learning domain 4: Medicines: the medicinal product Medical devices: their types, regulation and, particularly, their use for the measurement and maintenance of physiological function or medicine delivery. Learning domain 6: The wider context Scientific, clinical, health services and social services research; methods, results and their application as they are relevant to pharmacy.  Overview Ø The need for bacterial detection Ø Surveillance techniques Ø Microscopy and staining Ø Chromogenic / fluorogenic methods Ø Molecular diagnostic methods  “The World is facing an Antibiotic Apocalypse”, Prof Sally Davies, UK CMO Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. The Review on Antimicrobial Resistance, J O’Neill (chair) [https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf] “The loss of efficacy of antibiotics and other antimicrobials worldwide can be understood as a tragedy of the commons.” World Bank Drug-resistant infections : a threat to our economic future, 2017 Predicted Annual Deaths (and Mortality Rates) Due to AMR by 2050  Multidrug Resistant Organisms (MROs) and Drug Resistance On any given day in the US approx. 4% of acute care hospital patients have at least one Healthcare Associated Infection [HAI] (1.7M patients affected annually) [Magill et al., New Engl J Med 2014, 370, 1198-1208] In Europe the average prevalence of HAI is 7.1 per 100 patients; the European Centre for Disease Prevention and Control (ECDPC) estimates that more than 4M patients are affected by HAI every year in Europe [Annual epidemiological report on communicable diseases in Europe 2008, European Centre for Disease Prevention and Control, Stockholm] Of particular concern is the transmission of resistance between multi-resistant organisms (MRO), which could ultimately lead to strains which have limited or no susceptibility to antibacterial agents (MDR / XDR / PDR) AMR is a Global Problem: Global Initiatives UK Government re-instituted the Longitude Prize in 2014; UK public voted on which global problem should be the subject of the £10M prize [https://longitudeprize.org/] The successful challenge was ‘to create a cost-effective, accurate, rapid and easy-to-use test for bacterial infections that will allow health professionals worldwide to administer the right antibiotics at the right time’ The 5-year period for teams to submit their applications to the Longitude Prize began in November 2014; assessments of submissions made every 4 months; the first team to meet all of the criteria will win the prize. The $20M Antimicrobial Resistance Diagnostic Challenge seeks ‘new, innovative, and novel laboratory diagnostic tests that identify and characterize antibiotic resistant bacteria and/or distinguish between viral and bacterial infections’ [https://dpcpsi.nih.gov/AMRChallenge] Evidence-Based Prescribing Should be Informed by Diagnostic Testing World Health Organisation (WHO) global action plan on antimicrobial resistance suggests that antibiotic prescriptions are rarely based upon effective diagnoses and that the standard of care should be evidence-based prescribing and dispensing. Evidence-based prescribing and dispensing could be informed by effective, low-cost diagnostic tests, capable of integration into clinical, pharmacy, and veterinary practices. [World Heath Organisation (2015). Global action plan on antimicrobial resistance; http://www.who.int/antimicrobial- resistance/global-action-plan/en/] The 2016 O’Neill report on ‘Tackling Drug-Resistant Infections Globally’ suggests that by 2020 all clinicians should perform a rapid diagnostic test before prescribing antimicrobials. [J. O’Neill (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations; https://amr-review.org/]  WHO Global Action Plan on AMR Strengthen Reduce the Optimize Use of Improve Make Economic Knowledge and Incidence of Antimicrobials Awareness and Case for Evidence Base Infection Understanding Investment of AMR Develop AMR Further develop Develop and Improved public Obtain required surveillance hygiene and implement a communication funding for systems infection comprehensive implementation prevention action plan where; programs Optimal use is Share Determine and Establish AMR as Undertake guided by information report an element of international diagnostic tests internationally susceptibility of professional research HAI education collaboration Antibiotics only Collect and share Implement best accessed through Elevate AMR to a New models for data on practises for qualified HCP priority in both investment antimicrobial use prevention in governmental and access animal health / Antibiotics are agenda agriculture safe and AMR research Promote efficacious agenda vaccination of animals in food Antibiotic use in chain agriculture is phased out  WHO Global Action Plan on AMR Strengthen Reduce the Optimize Use of Improve Make Economic Knowledge and Incidence of Antimicrobials Awareness and Case for Evidence Base Infection Understanding Investment of AMR Develop AMR Further develop Develop and Improved public Obtain required surveillance hygiene and implement a communication funding for systems infection comprehensive implementation prevention action plan where; programs Optimal use is Share Determine and Establish AMR as Undertake guided by information report an element of international diagnostic tests internationally susceptibility of professional research HAI education collaboration Antibiotics only Collect and share Implement best accessed through Elevate AMR to a New models for data on practises for qualified HCP priority in both investment antimicrobial use prevention in governmental and access animal health / Antibiotics are agenda agriculture safe and AMR research Promote vaccination efficacious agenda of animals in food chain Antibiotic use in agriculture is phased out We Need Rapid Surveillance Techniques for Pathogenic Bacteria to Inform the Use of Antimicrobial Agents Results used to identify colonized / infected patients and inform their directed (rather than empirical) treatment Surveillance should form the basis for an organism- specific approach to transmission-based precautions Effective infection prevention relies on the introduction of contact precautions (such as patient isolation in a single-patient room or cohorting patients with the same strain of MRO in designated patient-care areas)  Antimicrobial Stewardship Ø Systematic approach to optimising the use of antimicrobials in hospitals Ø Includes; implementing clinical guidelines that are consistent with the latest version of Therapeutic Guidelines establishing formulary restriction and approval systems that include restricting broad-spectrum and later generation antimicrobials educating prescribers, pharmacists and nurses about good antimicrobial prescribing practice and antimicrobial resistance Antimicrobial Stewardship Australian Prescriber  MRSA Ø MRSA is susceptible to very few agents, including the glycopeptides (vancomycin and teicoplanin), quinupristin-dalfopristin and linezolid. Cases of methicillin and quinuprustin-dalfopristin resistant Staphylococcus aureus already reported in Europe G Werner, C Cuny, F-J Schmitz, and W Witte Methicillin-resistant, quinuprustin- dalfopristin-resistant Staphylococcus aureus with reduced sensitivity to glycopeptides. J. Clinical Microbiology, 2001, 39, 3586.  Does Universal Surveillance Work? Ø US study looked at rates of MRSA clinical disease in 3 consecutive periods ― baseline (no surveillance), MRSA surveillance for all ICU admissions, universal MRSA surveillance (all admissions) ― in 3-hospital organization with 40,000 annual admissions Ø PCR-based nasal surveillance for MRSA followed by topical decolonization and contact isolation for patients who tested positive for MRSA Ø Baseline prevalence density of MRSA disease was 8.9 cases per 10,000 patient days A Robicsek, JL Beaumont, SM Paule et al., Ann. Internal Med., 2008, 148, 409  Does Universal Surveillance Work? YES Ø During ICU surveillance prevalence density fell to 7.4 and during universal surveillance to 4.7 per 10,000 days Ø Universal surveillance also led to a reduction in disease occurring up to 30 days after discharge Ø If no surveillance estimated that further 1319 transmissions of MRSA would have occurred (0.14 patient-per-day rate of MRSA transmission if patient not subject to contact isolation) JA Jernigan, MG Titus, DH Gröschel et al., Am. J. Epidemiol., 1996, 193, 496  Universal MRSA Surveillance is No Longer Recommended by the Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infection (ARHAI) From 2010 The Department of Health (DoH) introduced mandatory screening for MRSA for all admissions to English hospitals A national audit in 2011 [C Fuller et al., PLoS ONE, 8, e74219] identified poor compliance (61%), that only approx. 50% of new positive patients were isolated, with approx. 25% not receiving decolonization therapy, and that 37% of newly identified MRSA patients were discharged prior to their test result being available (as a result of the mean time to a positive result of 2.87 days). ARHAI now recommends that in England, only ‘checklist-activated screening’ (i.e. of patients with high MRSA risk factors or admission to high risk units) is employed.  Australia Healthcare Associated Infections Clostridium difficile MRSA VRE Clinical educators guide for the prevention and control of infection in healthcare  Elsewhere (UK) Ø ‘The Health and Social Care Act 2008, Code of Practice for the NHS on the prevention and control of healthcare associated infections and related guidance’ (the ‘hygiene code’) was published in January 2009 Ø Relevant NHS bodies must have, and adhere to, policies for the control of outbreaks and infections associated with both MRSA and C. diff, while acute NHS trusts must have similar policies for the other specific alert organisms. Screening of all patients on admission should be used to inform the need for decontamination and / or isolation of colonized patients Requirements of a Bacterial Surveillance Method Sensitive (correctly identifies microorganism) Specific (identifies ONLY microorganism of interest) Rapid Reliable Cost-effective Simple to perform Does not require specialist interpretation  Current Detection Methods Phenotypic Methods Biochemical testing Chromogenic media MALDI-TOF MS Genotypic (Molecular Diagnostic) Methods Real time polymerase chain reaction (PCR) Whole genome sequencing (WGS) Multi-omics approaches [Methods for the Detection and Identification of Pathogenic Bacteria: Past, Present, and Future, Váradi et al., Chem. Soc. Rev., 2017, 46, 4818-4832]  Microscopy / Staining Cocci Bacilli Spirillum http://faculty.capebretonu.ca/cglogowski/2008%20BIOL101LAB2.htm  Microscopy / Staining Gram positive Cocci and Gram negative Coccobacilli http://archive.microbelibrary.org/asmonly/details.asp?id=2419 Microscopy / Staining  Chromophore / fluorophore Detection Cleavage by a specific bacterial enzyme Chromophore Enzyme-targeting Chromogen / Linker / portion Fluorogen Fluorophore Enzyme substrate; colour / fluorescence Chromophore / fluorophore released muted or absent due to linkage to targeting only if specific bacterium present; should molecule; must be stable in medium be retained by bacterial colonies  The Visible Region WAVELENGTH (nm) 750 700 650 600 550 500 450 400 350 RED ORANGE GREEN BLUE VIOLET YELLOW Colour absorbed Perceived colour Wavelength of absorbed light (nm) Violet Green–yellow 400–424 Blue Yellow 424–491 Green Red 491–570 Yellow Blue 570–585 Orange Green–blue 585–647 Red Green 647–700  Chromophores Ø Part of the molecule which contains the electrons involved in an electronic transition is called the chromophore Ø Electronic transitions correspond to electrons in molecule being excited from one energy level to another Ø As conjugation increases, the difference in energy between the highest occupied (HOMO) and lowest unoccupied (LUMO) decreases so lmax increases Ø The wavelength of the maximum in the absorption ― wavelength curve is called lmax  Chromophores Ø Part of the molecule which contains the electrons involved in an electronic transition is called the chromophore Ø Electronic transitions correspond to electrons in molecule being excited from one energy level to another Ø As conjugation increases, the difference in energy between the highest occupied (HOMO) and lowest unoccupied (LUMO) decreases so lmax increases Ø The wavelength of the maximum in the absorption ― wavelength curve is called lmax UV-VIS Spectrum of b-Carotene in Ethanol 1.2 1 Absorbance 0.8 0.6 0.4 0.2 0 410 430 450 470 490 Wavelength (nm) Phenolphthalein 3 2.5 Absorbance 2 1.5 1 0.5 0 220 320 420 520 Wavelength (nm) pH4 dashed black, pH8 dashed red pH9 upper black, pH13 upper red  Requirements for Chromogen Ø Free chromogenic molecule must have strong colour Ø Colour must be muted or absent when attached to targeting molecule Ø Targeting molecule recognised by specific bacterial enzyme Ø Link between targeting molecule and chromogen broken by a specific bacterial enzyme Ø Coloured chromogen released thus confirming presence of specific bacteria  Specific Bacterial Enzyme Targets Ø Peptidases (aminopeptidases cleave N-terminal amino acid from a peptide) Ø L-alanyl aminopeptidase Ø L-pyroglutamyl aminopeptidase Ø L-glutamyl aminopeptidase Ø Glycosidases Ø b-Glucuronidase Ø a- or b-Glucosidase Ø Other enzymes, e.g. deaminase, nitroreductase  Chromophore / fluorophore Detection Cleavage by a specific bacterial enzyme Chromophore Enzyme-targeting Chromogen / Linker / portion Fluorogen Fluorophore Enzyme substrate; colour / fluorescence Chromophore / fluorophore released muted or absent due to linkage to targeting only if specific bacterium present; should molecule; must be stable in medium be retained by bacterial colonies 9-(4-N ‘-L-Ala-L-Ala-L-Ala-aminophenyl)- 10-methylacridinium bis(trifluoroacetate) Differentiates between Gram negative (hydrolysed to red) and Gram positive (grow but no hydrolysis, so colourless)  9-(4-N ‘-L-Ala-L-Ala-L-Ala-aminophenyl- 10-methylacridinium bis(trifluoroacetate) RJ Anderson, PW Groundwater, Y Huang, AL James, S Orenga, A Rigby, C Roger-Dalbert, JD Perry, Bioorg. Med. Chem. Lett., 2008, 18, 832-835 9-Aminophenyl-10-methylacridine  Natural Phenoxazinones Red colour found in fungi Red colour in eyes of insects Coloured antibiotic metabolite from Streptomyces species  Target Molecules: 1,2-Substituted-7-aminophenoxazinones R1 or R2 = H, alkyl C1-C12, aralkyl C6-C14, aryl, CO2H, CO2R2, or NR3R4 Colour muted when N 7-acylated Colour released when amide hydrolysed All incorporate unnatural amino acid, b-alanine Dans la Détection de Micro-organismes à Activité Peptidase, RJ Anderson, PW Groundwater, AL James, D Monget and AV Zaytsev, PCT/FR05/02249 (WO/2006/030119), 2006/3/23.  Pseudomonas aeruginosa Ø Pseudomonas aeruginosa is a pathogenic bacterium that secretes thick mucous (biofilm) and is a complicating infection in lungs of cystic fibrosis patients, burns victims and AIDS patients Ø Difficult to treat once established and leads to high mortality Ø Greater clinical success in treating P. aeruginosa infection if detect and treat early  Why b-Ala-Containing Substrates? Ø L-Alanyl aminopeptidase: bacterial enzyme Ø Gram negative bacteria have L-alanyl aminopeptidase and hydrolyse substrate to release colour Ø Differentiates Gram positive (no colour) from Gram negative bacteria Ø b-Alanyl aminopeptidase Ø Much less common in bacteria but expressed in Pseudomonas aeruginosa Ø Greater selectivity for detection  7-N-(b-Alanyl)amino-1- pentylresorufamine (β-Ala-1-PRF) 50 mg / l for 24 hours Left side (top and bottom) Pseudomonas aeruginosa Top right Burkholderia cenocepacia Bottom right Escherichia coli  7-N-(b-Alanyl)amino-1- pentylresorufamine (β-Ala-1-PRF)  Origin of Colour Ø Effective detection of Pseudomonas aeruginosa Ø Slight detection of Burkholderia cenocepacia Ø Good chromogen with bright purple colour AV Zaytsev, RJ Anderson, PW Groundwater, Y Huang, JD Perry, S Orenga, C Roger-Dalbert, and A James, Org. Biomol. Chem., 2008, 6, 682 - 692 Nouveaux Substrats Enzymatiques Dérivés de Phénoxazinone et Leur Utilisation Comme Révélateur Dans la Détection de Micro-organismes à Activité Peptidase, RJ Anderson, PW Groundwater, AL James, D Monget and AV Zaytsev, PCT/FR05/02249 (WO/2006/030119), 2006/3/23 4-Methylumbelliferyl b-D-galactopyranose – use in detection of coliform bacteria 4-MU Highly fluorescent KR Gee et al., Anal. Biochem., 1999, 273, 41. Hydrolysis of b-alanylaminonaphthyridine by Pseudomonas aeruginosa C5H11 C5H11 O b-alanyl aminopeptidase H3N N N N H2N N N H CF3CO2 (weakly fluorescent) (fluorescent; lexcitation 365 nm; lemission 420 nm) L. Váradi, M. Gray, P.W. Groundwater, A.J. Hall, A.L. James, S. Orenga, J.D. Perry, R.J. Anderson, Org. Biomol. Chem., 2012, 8, in press  Fluorogenic Detection using Self- Immolative Linkers (Agar media) Fluorescence generated only by BAP producers P. aeruginosa  Fluorogenic Detection using Self- Immolative Linkers (Liquid media) Váradi, Hibbs, Orenga, Babolat, Perry, Groundwater, RSC Advances, 2016, 6, 58884-58889 Requirements of a Bacterial Surveillance Method Sensitive (correctly identifies microorganism) Specific (identifies ONLY microorganism of interest) Rapid Reliable Cost-effective Simple to perform Does not require specialist interpretation  Molecular Diagnostic Methods Ø Often based on polymerase chain reaction (PCR) technology Ø Mass spectrometry-based methods also offer promise Ø These techniques offer specificity and speed but are complex, costly and require specialist interpretation FC Tenover, Clinical Infectious Diseases, 2007, 44, 418-423  PCR Technology Ø Produces millions of copies of DNA sequence in 2 hours Ø Series of cycles involving; Ø Denaturing of double stranded DNA at > 90 oC Ø Annealing of upstream and downstream primers for DNA sequence of interest Ø Thermus aquaticus (Taq) DNA polymerase then catalyses DNA replication Ø PCR video (DNA Learning Centre)  Real-time PCR Detection of MRSA in a Mixture of Staphylococci Ø Employs DNA primers and a series of molecular beacon probes Ø Primers are specific for the mecA and S. aureus specific orfX genes to allow for variations in the staphylococcal cassette chromosome (SCCmec) Ø SCCmec is a mobile genetic element which carries the mecA gene (which encodes the b-lactam-resistant penicillin binding protein PBP2') Drawbacks with quantitative PCR (qPCR) Ø Involves high levels of amplification so contamination or the detection of nucleic acids which remain from a previously cleared infection is a concern. Ø The discrimination between an asymptomatic colonization and a clinically relevant infection relies upon the development of standardized quantitative cut off cycle-threshold (Ct) values but no clear relationship between number of microorganisms present in specimen and the presence of healthy carriage or disease within the individual. [Cycle-threshold (Ct) value - number of PCR cycles required for the fluorescence signal to cross the threshold (background level). Lower values suggest higher pathogenic bacterial loads.]  PCR Technology Ø Requires some means of detection of amplified DNA sequence. (Traditional PCR assays utilized immunoassays or agarose gels but these added 1-3 hours to assay) Ø Real-time (qPCR) assays use molecular beacons ― single- stranded probes which have a DNA recognition sequence with a fluorescent dye at one end and quencher at other. If recognition sequence matches DNA, probe opens up and fluorescence is no longer quenched Figure courtesy of Wikimedia (http://en.wikipedia.org/wiki/File:Mole cular_Beacons.jpg)  Real-time PCR Detection of MRSA in a Mixture of Staphylococci Ø 98.7% of 1,657 MRSA isolates were detected in under 1 hour using this technique Ø Only 26 of 569 methicillin-susceptible S. aureus (MSSA) strains were mistakenly identified as MRSA MG Bergeron et al., J. Clin. Microbiol., 2004, 42, 1875-1884  Real-time PCR Detection of Other Organisms Ø BD GeneOhm VanR assay: Detection of vancomycin resistant enterococci (VRE) based on the VanA gene Ø Oxazolidinone-resistant E. faecalis and E. faecium (based on G to U mutation at residue 2576 of 23S ribosomal DNA) N Woodford et al., J. Clin. Microbiol., 2002, 40, 4298-4300 Ø P. aeruginosa (based on outer membrane lipoprotein genes, oprI and oprL) P Cornelis et al., J. Clin. Microbiol., 1997, 35, 1295-1299  Vancomycin (Vancocin) Ø Vancomycin is hydrophilic and forms hydrogen bonds to the terminal D-Ala-D-Ala sequence — preventing crosslink formation and blocking the release of the disaccharide from the carrier lipid Ø Resistance to vancomycin occurs through alteration in the ligase activity Ø The Vancomycin Resistant Enterococci (VRE) Van A phenotype produces a D-Ala-D-Lactate ligase which synthesises an ester (D-Ala-D-Lac) rather than an amide (D-Ala-D-Ala) Ø D-Ala-D-lactate sequence has 1000-fold reduction in affinity for vancomycin but can still be added to L- Lys and act as precursor for crosslink formation  MALDI-TOF Mass Spectrometry Ø Matrix Assisted Laser Desorption Ionization ― Time Of Flight (MALDI-TOF) Mass Spectrometry Ø energy is transferred to matrix from a laser beam Ø matrix employed has a chromophore which absorbs at the wavelength of the laser Ø matrix absorbs a pulse of energy and undergoes rapid heating which leads to the vaporization and ionization of the analyte molecules Ø molecular weights of ions analyzed by time taken to reach detector  TOF Mass Spectrometry Gas phase ions with Ions accelerated to velocities differing m/z values proportional to m/z values Reflectron Ion trajectory 1 2 linear mode KE = mv 2 Detector Detector linear reflectron mode mode Pusher plate Source Field-free Reflectron region or drift region region Ion trajectory reflectron mode All ions given same kinetic energy (KE) so lighter ions travel faster  MALDI-TOF Mass Spectrometry Ø Matrix Assisted Laser Desorption Ionization ― Time Of Flight (MALDI-TOF) Mass Spectrometry Ø Spectral fingerprints vary between microorganisms Ø Among the compounds detected in the spectrum, some peaks (molecular masses) are specific to genus, species, and sometimes to subspecies Ø Spectra can be obtained from intact cells MALDI-TOF Mass Spectrometry A. Minan et al., Analyst, 2009, 134, 1138-1148  MALDI-TOF MS Detection of MRSA Ø Number of studies have attempted to differentiate MRSA and MSSA Ø MS profiles are different, with MRSA containing more peaks Ø No specific profile for MRSA but individual strains have very similar profiles E Carbonnelle et al., Clin. Biochem., 2011, 44, 104-109  MALDI-TOF MS Detection of Carbapenemase activity A, spectrum of meropenem solution; B, spectrum of non-carbapenemase-producing isolate of Klebsiella pneumoniae; C, Escherichia coli isolate producing the New Delhi metallo-β-lactamase (NDM-1) carbapenemase; D, NDM-1-producing Acinetobacter baumannii. J. Hrabák et al., J. Clin. Microbiol., 2012, 50, 2441-2443  Whole Genome Sequencing (WGS) WGS provides comprehensive information allowing for the; i) identification of pathogens, ii) exact profiling of resistance genes, iii) recognition of outbreaks, and iv) the immediate design of PCR probes based on the generated genetic data in the event of outbreaks. Requires culturing of clinical samples Discovery of the mecC gene (a homologue of the mecA gene, that is responsible for methicillin resistance in MRSA) ̶ redesign of PCR assays to improve sensitivity and avoid false negatives N. C. Gordon et al., J. Clin. Microbiol., 2014, 52, 1182-1191 Whole Genome Sequencing (WGS) – Sanger method Methods for the Detection and Identification of Pathogenic Bacteria: Past, Present, and Future, L Váradi et al., Chem. Soc. Rev., 2017, in press (doi: 10.1039/C6CS00693K).  Point-of-Care (POC) Tests / Devices A POC test is one ‘that is performed near the patient or treatment facility, has a fast turnaround time, and may lead to a change in patient management’. Such tests do not require access to centralised laboratory facilities and should be sufficiently rapid to allow clinically meaningful interventions (e.g. the initiation of directed, as opposed to empirical, antibacterial treatment) to be implemented at the place at which the patient is being treated. WHO Criteria for an Ideal Point of Care (POC) Diagnostic Test Affordable Sensitive Specific User-friendly Rapid and Robust Equipment-free Deliverable Biorecognition Elements Used as Biosensors in POC Devices Antibodies Aptamers Nucleic Acids Proteins reproducible small sample requirements high specificity and sensitivity high specificity, sensitivity, and yield portable multiplex low toxicity speed no instrumentation long unrefrigerated stable over temperature requirement shelf-life portability and pH ranges no pre-concentration possible simultaneous isothermal (no steps reproducible production species identification thermocycling required) long shelf-life AND resistance profiling low power consumption no need for fluorescent single gene mutation can microscopy be differentiated large sample sizes required contamination is a at times poor sensitivity degradation by nucleases concern due to multiplexing not possible challenging preparation cross reactivity sensitivity lengthy times to signal of Ab pairs generation requires no differentiation release (and result) flow cytometry requires purified target between dead and viable bacteria expertise and instrumentation Novel Diagnostics for Point-of-Care Bacterial Detection and Identification, S Reali, EY Najib, KE Treuerné Balázs, ACH Tan, L Váradi, DE Hibbs, PW Groundwater, RSC Advances, 2019, 9, 21486-21497  Electrochemiluminescence (ECL) detection of antibody sandwich complex M. tuberculosis 5-methythio-D-xylofuranose-lipoarabinomannan (MTX-LAM) epitope target Capture antibody (S4-20) targets M. tuberculosis-specific MTX-LAM and is bound to the electrode. Detection antibody (A194-01) labelled with the commercial MSD SULFO-TAG™ (a). Formation of the sandwich complex with LAM detected through the ECL generated by the Ru(bpy)32+ component of the SULFO-TAG (b). Oxidation of the Ru(bpy)32+and tripropylamine (TPrA) produces a luminescent excited state [Ru(bpy)32+]* which decays to the ground state, involving light emission (c). The optimised antibody pair resulted in femtomolar analytical sensitivity for LAM detection and overall clinical sensitivity and specificity of 93% and 97%, respectively. Aptamer-based Latent TB Infection Test Smartphone app using colorimetric detection of a 3′-biotin-labeled aptamer which recognizes mannose-capped lipoarabinomannan (ManLAM), a glycolipid from the M. tuberculosis cell wall. M. tuberculosis (Mtb) immobilized on a nitrocellulose membrane. Incubated with the biotin-labelled aptamer, followed by streptavidin-labelled horseradish peroxidase (HRP). Quantitation uses the oxidation of the colourless 3,3′,5,5′-tetramethylbenzidene (TMB) reduced (red) to its blue oxidized (ox) form by HRP in the presence of hydrogen peroxide. Aptamer is specific for binding to ManLAM so does not detect other bacteria, including E. coli, S. aureus and E. faecalis; quantitation limit of 104 CFU/mL and can be performed in 5 hours. L. Li, Z. Liu, H. Zhang, W. Yue, C.-W. Li and C. Yi, Sens. Actuators B Chem., 2018, 254, 337-346. Magnetic Nanoparticles for Detection of S. aureus Protease 1 Black coloured MNPs incorporating a terminal carboxyl group are conjugated to the N- terminus of a peptide substrate for S. aureus protease 2 immobilization on a gold sensor platform (Au- S interaction) 3 biosensor exposed to S. aureus, enzymatic cleavage of the amide bond releases the nanoparticle 4 nanoparticles are attracted to external magnets located at the back of the sensor platform and so expose the gold coloured surface The colour change from black to gold occurs in 1 minute and can be detected with the naked eye. G. A. R. Y. Suaifan, S. Alhogail and M. Zourob, Biosens. Bioelectron., 2017, 90, 230-237.  Requirements of a Bacterial Surveillance Method Sensitive (correctly identifies microorganism) Specific (identifies ONLY microorganism of interest) Rapid, reliable, and cost-effective Simple to perform (does not require specialist interpretation) Characteristic Technique Chromogenic media PCR MALDI-TOF MS Sensitivity ++ +++ +++ Specificity + +++ +++ Cost + +++ +++ Complexity ― ++ ++ Direct detection from clinical YES YES NO samples Time to result (h) > 24 2 2-3  WORKSHOP - The Specific Detection of Pathogenic Bacteria using MALDI-TOF Your group will be assigned a topic from the list below and will then research the pathogenic micro-organism and the use of chromogenic media for its identification or detection. You should then prepare a 10 minute presentation which includes the following sections; INTRODUCTION TO THE MICRO-ORGANISM (2 slides*) PRESENTATION OF RESULTS FROM LITERATURE ASSAYS (4 or 5 slides*) SUMMARY (1 slide) REFERENCES (1 slide) *suggested  WORKSHOP - The Specific Detection of Pathogenic Bacteria using MALDI-TOF You should concentrate on the following aspects (if available) of the assays from the literature; analytical sensitivity, specificity, time to detection (time taken for assay), detection of drug resistance, cost effectiveness, and comparisons with other methods. Assessment: Oral presentation (60%), Answers to questions (20%), Questions asked (20%) 8-9 slides recommended, must be presented by all students. The slide outline (two slides per page) must be submitted via Turnitin prior to the start of the workshop session.