Ongoing revolution in clinical microbiology: current status and future perspectives © by author ESCMID OnlineMario Lecture Poljak Library Institute of Microbiology and Immunology Faculty of Medicine, University of Ljubljana, Slovenia . Revolution vs. evolution in diagnostic microbiology? molecular diagnostic microbiology MALDI-TOF mass spectrometry total laboratory automation in bacteriology syndrome-specific testing point-of-care tests and 24/7 concept digital PCR next-generation sequencing© by author next-generation antimicrobial susceptibility testing ESCMIDCRISPR-Cas – basedOnline diagnostic Lecture assays Library non-microorganism detection based diagnostic approaches revolution ? molecular diagnostic microbiology © by author ESCMID Online Lecture Library Molecular methods dramatically changed clinical microbiology allowed discovery of several clinically important and previously unrecognized or uncultivable pathogens reduced the dependency ©of laboratory by author on culture-based methods became gold diagnostic standards for several microorganisms ESCMID(C. trachomatis, HSV Online encephalitis, enteroviral Lecture meningitis, CMV Library reactivation, hepatitis C,…) © by author ESCMID Online Lecture Library Molecular diagnostic systems 2.0+ - fully automated sample-to-result fashion - multiple tests performed concordantly - sample number flexibility - STAT test prioritization© by author ESCMID Online Lecture Library - random access © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Small/middle scale integrated systems revolution ? point-of-care tests and 24/7 concept © by author ESCMID Online Lecture Library “3R” rule Rapid (in clinically relevant time frames) Relevant (clinically relevant) Right (specific and sensitive, analytical category) © by author Right > Relevant > Rapid ESCMID Online Lecture Library Relevant = Rapid > Right faster cheaper © by author 24/7 ESCMID Online Lecture Library Two testing places – evolving concept HH H H POC POC POC POC © by author ESCMID Onlineclin micro Lecture lab Library POC = point-of-care © by author ESCMID Online Lecture Library POC = point-of-care © by author ESCMID Online Lecture Library N Engl J Med 2013;368:2319-24. desire is to have self-contained, fully integrated sample-to-report devices that accept raw, untreated specimens, perform all of the molecular steps, and provide© interpreted by author test results in < 1 h ESCMID Online Lecture Library Point-of-care molecular testing entering clinical practice throughout the world paradigm shift towards decentralized testing especially suited for applications: - where fast turnaround is desirable - where centralized laboratory services face limitations - in resource-limited© countries by author - in rural areas and places that are hard to reach - ships, submarines, off-shore platforms….(3D printer technology and remote ESCMIDfault diagnosis will allowOnline reparation of failures Lecture using a small stock of Librarymaterials and versatile components) poses diverse technological, economic and organizational challenges Selected compact “sample in-results out“ molecular diagnostic devices Cepheid GeneXpert GenMark Dx eSensor Roche cobas Liat System Veredus VereChip Alere iSystem Great Basin Portrait Luminex ARIES Focus Dx Simplexa/3M Cycler Atlas Genetics QuidelSavanna & Solana Enigma Diagnostics Meridian Illumigene Micronics BD Max System Cirrus Dx T-COR 8 ©ELITe by InGenius author Systems BioFire FilmArray Biomeme NanosphereESCMID Verigene SP OnlineFluoresentric, Lecture Inc. Library QuantuMDx GeneWEAVE VivoDx Janssen Diagnostics Rheonix Encompass Optimum U.S. $18 billion dollar market by 2016 for POC ! J Clin Microbiol 2016;54:815 cobas Liat strep A assay vs. S. pyogenes LightCycler PCR assay sensitivity = 100% specificity = 98.3 % © by author positive predictive value = 97.7% negativeESCMID predictive value Online = 100.0% Lecture Library Not all POC NAATs are the same! J Clin Microbiol 2016;54:2763 Specificity: 100 % Sensitivity FluA: 71.3 % Sensitivity FluB: 93.3 % Failure rate: 0 % © by author Specificity: 100 % ESCMID OnlineSensitivity FluA: Lecture 100 % Library Sensitivity FluB: 100 % Failure rate: 1.6 % © by author ESCMID Online Lecture Library Where is my instrument ??? Lab-on-a-USB key microfluidic devices integrated© with by USB author key data storage devices a device could be attached to other computational devices such as a cell phone or laptopESCMID computer to control Online molecular assays Lecture being done onLibrary the microfluidic biochip analysis transmitted to central databases for shared use and metaprocessing © by author ESCMID Online Lecture Library Sci Rep 2016;6:36000 © by author ESCMID Online Lecture Library J Clin Virol 2015;69:16-21 successful in-flight replication of two different DNA Lab-on-a-drone targets (16 min in-flight reaction time). Tamb ∼ 23 °C © by author ESCMID Online Lecture Library Anal Chem 2016; 88: 4651−4660 © by author ESCMID Online Lecture Library No electricity ?? Solar thermal PCR system © by author ESCMID Online Lecture Library Sci Rep 2014;4:4137. Loop-mediated isothermal amplification (LAMP) © by author ESCMID Online Lecture Library PATH NINA heater low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material thermal standard deviation <0.5˚C at operating temperature a cost of approximately 0.06 USD per test for heater reaction materials HIV LAMP amplicon detection via Milenia test strips © by author ESCMID Online Lecture Library PLoS One 2014;9:e113693. rapid (<30 min) and sensitive (<10 copies) visual detection of amplified products using pH-sensitive dyes with minimal buffering capacity achieved with loop-mediated isothermal amplification (LAMP) specificity © by author sensitivityESCMID Online Lecture Library J Virol Methods 2016;234:90-5 gold nanoparticles (AuNP) attached to a single-stranded DNA probe for HPV16 and HPV18 LAMP incubation time of 20 min and a temperature of 65°C detection of the LAMP product by AuNP color change after LAMP amplification its products were hybridized with the AuNP probe for 5 min and then detected by the addition of magnesium salt © by author ESCMID Online Lecture Library J Virol Methods 2017; 244: 32-38 turbidity © by author hand-held UV light ESCMID Online Lecture Library blue-spectrum LED J Virol Methods 2016;237:132-7 © by author non-instrumented nucleic acid amplification, single-used disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents NINA-SUDESCMID heating device harnesses Online the heat from Lecture an exothermic chemical Library reaction initiated by the addition of saline to magnesium iron powder lyophilized HIV-1 RT-LAMP reagents stable at 30°C for up to one month No electricity ?? No instrument ??? © by author ESCMID Online Lecture Library Ustar Biotechnologies (Hangzhou, China) Cross Priming Amplification technology developed by Qimin You, while conducting research in Canada & US - instrument free specimen processing - isothermal nucleic acid amplification - visual read-out detection and easy data interpretation - cross contamination prevention - glassified reagents for ambient temperature transport and storage © by author ESCMID Online Lecture Library revolution ? MALDI-TOF mass spectrometry (matrix assisted laser desorption ionization-time of flight mass spectrometry) © by author ESCMID Online Lecture Library Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) Main advantages of MALDI-TOF: - speed (<3 min/isolate; 96 samples/h) - easy to perform - small amount of organism required -low cost © by author ESCMID Online Lecture Library Clin Microbiol Rev 2014; 27: 783-822 MALDI–TOF in clinical microbiology in 2017 identification of bacterial/fungal isolates – revolution (libraries regularly updated/enlarged) direct identification of pathogens from clinical samples (positive blood culture bottles, urine, positive clinical impact proved) antimicrobial susceptibility testing and detection of resistance mechanisms (several protocols for the detection of bacterial resistance published, commercial kits available for certain resistance© by mechanisms author but have not been validated in routine settings yet, recalibration of the instrument required) ESCMID Online Lecture Library typing (experiences vary substantially, standardization needed) revolution ? Direct detection and identification of bacteria using non-molecular, non MALDI-TOF technologies © by author ESCMID Online Lecture Library T2 Biosystems (Lexington, MA) - magnetic resonance technology (supermagnetic nanoparticles coated with target-specific binding agents cluster around the target, altering water molecules and their T2 relaxation signal) - detects DNA, cells, proteins directly from specimens without extraction or amplification - a low limit of detection (1-3 CFU/ml vs. 100-1000 CFU/ml for PCR) - not impacted by the presence of antimicrobials - printer-size detection device © by author ESCMID Online Lecture Library mBio 2013;4:e00865-13 intrinsic fluorescence spectroscopy (IFS) of whole cells a multistage algorithm correctly classified 99.6% of samples to the Gram level, 99.3% to the family level, and 96.5% to the species level © by author ESCMID Online Lecture Library revolution ? CRISPR-Cas – based diagnostic assays © by author ESCMID Online Lecture Library Sensitivity in atto range -18 Science 2017; 356: 438–442 10