ESCMID Online Lecture Library © by Author ESCMID Online Lecture Library

Ongoing revolution in clinical microbiology: current status and future perspectives

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 Small/middle scale integrated systems

point-of-care tests and 24/7 concept

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

POC = point-of-care © by author ESCMID Online Lecture Library POC = point-of-care

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 % Where is my instrument ???

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 Sci Rep 2016;6:36000

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

Anal Chem 2016; 88: 4651−4660 No electricity ??

Sci Rep 2014;4:4137. Loop-mediated isothermal amplification (LAMP)

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

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

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

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

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

CRISPR-Cas – based diagnostic assays © by author ESCMID Online Lecture Library Sensitivity in atto range -18 Science 2017; 356: 438–442 10

1,000 x more sensitive than other CRISP- based assay developed just a year ago

total laboratory automation in bacteriology

rayon swab flocked swab Automated specimen processors/inoculators

WASP (Copan, Italy)

InoqulA (BD Kiestra, The Netherlands)

Previ Isola (bioMerieux, France) © by author PreLUD (i2a Diagnostics, France) ESCMIDAUTOPLAK (NTE Healthcare,Online Spain) Lecture Library J Clin Microbiol 2009;47:1101–6. J Clin Microbiol 2015;53:2298–307. J Clin Microbiol 2014;52:796–802. J Clin Microbiol 2012;50:2732–6. J Clin Microbiol 2015;53:2298–307.

, laboratory , workload and laboratory costs

© by author author by © ESCMID Online Lecture Library Library Lecture Online ESCMID Demonstrated advantages of automated specimen processing specimen processors compared to manual streaking: specimen - produce more isolated colonies - enhanced reproducibility exhibit - provide decreased hands-on plating time - decreased requirement for subculturing -to result decrease in time significant Incubators/Digital Cameras

- constant and uniform temperature (laminar flow) - internal automated digital imaging system

WASP Kiestra capacity 882/1764 plates 1152 plates camera 48 Mp© by author5 Mp size of image files 20–25 Mb 3 Mb ESCMID Online Lecture Library light sources/background front, back, side lights no or black background Telebacteriology the use of digital imaging and file storage for on-screen reading and decision making

© by author ESCMID Online Lecture Library Disadvantages of laboratory automation complex systems (mechanical, electronical, hardware, software, LIS connectivity) Murphy‘s law frequently on board - anything that can go wrong will go wrong

lab automation requires a huge change in lab organization/workflow © by author ESCMID Online Lecture Library Future developments

- inteligent digital imaging (development of intelligent algorithms and expert systems with different future applications)

- microbial growth detection and quantification (urine cultures) - presumptive identification of species growing on chromogenic agar (MRSA, VRE, ESBL) - automated recognition of sister colonies from chromogenic and nonchromogenic agar

- automated colony-picking modules (for ID by MALDI-TOF and suspension preparation) © by author - fully automated disk diffusion AST ESCMID Online Lecture Library revolution ?

syndrome-specific testing © by author ESCMID Online Lecture Library Syndrome-specific testing

comprehensive panels of probable pathogens causing a particular clinical syndrome highly multiplexed PCR platforms one sample - multiple results designed to directly probe specimens (respiratory, stool, CSF, blood, urogenital) and positive blood culture bottles for an array of microorganisms can have significant impact on patient© careby and author management redefining the diagnosis of infectious disease ? ESCMID Online Lecture Library US Food and Drug Administration – approved syndromic panels for multiple pathogen detection

Hanson, Couturier. Clin Infect Dis 2016;63:1361-7. © by author ESCMID Online Lecture Library

FDA approved: October 12, 2015 © by author ESCMID Online Lecture Library © by author

- overallESCMID specificity ofOnline the assay =Lecture 1,544/1,556 =Library 99.2 %

- 12 positives = 5 true positives and 7 false positives revolution ?

serology © by author ESCMID Online Lecture Library Science 2015;348:aaa0698 et al.

a high-throughput analysis of antiviral antibodies in human sera

VirScan - DNA proteome-wide microarray containing peptides from all human viruses average person had antibodies against 10 viral species; individuals had antibodies against up to 84 species © by author ESCMID Online Lecture Library Nat Med 2017; 23: 548-551

multiplexed assay on a plasmonic-gold platform for measuring IgG and IgA antibodies and IgG avidity against both Zika virus© (ZIKV) by and authordengue virus (DENV) infections pGOLD platform is capable of amplifying near-infrared fluorescence by up to ~100 times, allowingESCMID for sensitive analysis Online of multiple analytes Lecture over a 6–7-log Library dynamic range in contrast to IgM cross-reactivity, IgG and IgA antibodies against ZIKV nonstructural protein 1 antigen were specific to ZIKV infection, and IgG avidity revealed recent ZIKV infection and past DENV-2 infection in patients in dengue-endemic regions antibiotic susceptibility testing ?????????????????

genomic

targeted assays next-generation sequencing alternative© by author assays ESCMID Online Lecture Library phenotypic revolution ? genomic antimicrobial susceptibility testing (targeted assays) © by author ESCMID Online Lecture Library Genomic antibiotic susceptibility testing

nucleic acid amplification-mediated detection of resistance genes or mutations that are correlated with resistance to antibiotics plays an important role in clinical microbiology laboratories and will continue to do so molecular testing will evolve versus syndrome-oriented multiplexed detection of pathogens including genomic AST commercial competition will increase, prices per test will go down and in the end all tests will be of the “sample in – result ©out” formatby author ESCMID Online Lecture Library significant part of antimicrobial susceptibility testing in the near future will be genomic J Clin Microbiol 2017;55:183-198

filter-based cartridge with an integrated sample processing function; testing directly from sputum

INOVATIONS: - four new large-Stokes-shift fluorophores developed - 10-color probe detection in a single PCR tube - a new three-phase, double-nested PCR approach - newly designed sloppy molecular beacons

32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to isoniazid (INH), fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin© (KAN)by author the rate of detection of heteroresistance equivalent to that by Sanger ESCMIDsequencing Online Lecture Library compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN revolution ? molecular antimicrobial resistance testing (next generation sequencing ) © by author ESCMID Online Lecture Library For most bacterial species there is currently insufficient evidence to support the use of whole© genome by sequencingauthor - inferred antimicrobial susceptibility testing to guide clinical decision making. ESCMID Online Lecture Library revolution ? molecular antimicrobial resistance testing (alternative approaches) © by author ESCMID Online Lecture Library J Clin Microbiol 2015;53:425-32 rapid identification of the bacterial species and simultaneous determination of their antibiotic susceptibility profiles - initial short cultivation step in the absence and presence of different antibiotics combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e. resistance) and no growth (i.e. susceptibility) a proof-of-concept study: - urinary tract infections -E.coliciprofloxacin and trimethoprim - 100% accuracy in 3.5 h © by author ESCMID Online Lecture Library phenotypic antibiotic susceptibility testing will remain core technology in clinical microbiology for multiple decades to come © by author ESCMID Online Lecture Library revolution ?

New emerging technologies for phenotypic antimicrobial susceptibility testing © by author ESCMID Online Lecture Library Phenotypic antimicrobial susceptibility testing (short history)

agar plate methods (large macroscopic colonies visible by naked eye required, time consuming)

conventional turbidity methods

7 (10 CFU/mL required – bacterial populations© by must authorgrow 16–20 h before reaching a detectable level) ESCMID Online Lecture Library near future alternatives (use of ultra sensitive alternative approaches for bacterial growth measurement) Accelerate Pheno System ID and AST direct from positive blood culture

© by author ESCMID Online Lecture Library US FDA approved Accelerate Pheno system and Accelerate PhenoTest BC kit for ID and AST testing of pathogens directly from positive blood culture samples on 23 Feb 2017 indicated for AST of pathogenic bacteria most commonly associated with bacteremia/sepsis Accelerate Pheno System How does it work?

1. lysis of red blood ©cells by & solubilization author of blood components 2. filtration ESCMID3. immobilization Online Lecture Library 4. identification: fluorescent in-situ hibridisaton (FISH) 5. antimicrobial susceptibility: time-lapse imaging New emerging technologies with great potential for phenotypic antibiotic susceptibility testing

flow cytometry resonate mass measurement microbial cell weighing by vibrating cantilevers + atomic force microscopy isothermal microcalorimetry asynchronous magnetic bead rotation testing in microdroplets + epifluorescence digital time-lapse microscopy time-lapse single-cell imaging (SCMA) high-throughput nanowell antibiotic susceptibility testing forward© laser by light scatterauthor technology phase-shift reflectometric interference spectroscopy + micropillar architectures gradient-generating microfluidic AST devices – chip based ESCMIDgradient-generating Online microfluidic Lecture AST devices – hydrogel Library based

AST methods based on bacterial death Forward laser light scatter technology (BacterioScan)

J Clin Microbiol 2016;54:2701-6 combines laser forward light scattering and optical density measurements of bacterial growth prior to visual assessment limit of detection: 1x104 CFU/ml undergoing FDA clinical trials

216Dx UTI System – UTI screening

BacterioScan 3100R/AST/Dx © by author ESCMID Online Lecture Library Resonate mass measurement (LifeScale/Affinity Biosensors)

- mechanically resonant structure with with a microfluidic channel - microbes suspended in broth and passed one by one through a microfluidic channel - mass (and growth curve) measured by the change in resonate energy by ultra-high resolution weighing

- FDA clinical trials started Q4 2016 - available late-2017 Microbial cell weighing by vibrating cantilevers + atomic force microscopy

© by author cantileversESCMID containing small canals Online which facilitate miLecturecrobial passage can Librarybe made to vibrate continuously when metabolically active bacteria pass through, their weight (in the femtogram range) will cause a change in the frequency of cantilever movement atomic force microscopy capable of detecting movements of biologic samples at the nanoscale Atomic force microscopy

BMC Microbiology 2009, 9:106

dynamic technique that allows the measurement of heat production either as a flow rate (W/unit time) or as total accumulation over time (Joules/unit© time)by st emmingauthor from the metabolism of actively growing cells cumulative heat production generally parallels conventional growth curves in that the slope and shape of the accumulating heat production correspond with classical lag, log, and stationary phases - maximum heat values representESCMID the total number Online of cells produced Lecture over time Library process requires specific instrumentation (e.g., TAM air, TAM III, Tam IV, TAM IV-48) for real-time measurement of heat generation with a detection limit in the 0.2 µW range Asynchronous magnetic bead rotation

Anal Chem 2012;84:5250–6

BMC Microbiology 2009, 9:106

when magnetic beads are brought into a revolving magnetic field, they self-assemble and assume a specific rotational spin - the frequency of rotation can be influenced by the binding of bacteria if the beads are equipped with a ligand© that byspecif icallyauthor captures bacterial cells, the rotation of the beads changes at the moment of capture - this change can be measured an increase in the rotational period suggests bacterial growth/proliferation whereas a constant rotational periodESCMID suggests growth inhibition Online Lecture Library this process can be observed directly by illuminating the culture broth (in the form of a hanging drop) with a light-emitting diode or laser - the hanging-drop format also acts as a lens to create magnification of up to 100 x such that the structure and rotational rate of the bead aggregates can be observed microscopically or when projected onto a detector Phase-shift reflectometric interference spectroscopy + micropillar architectures

BMC Microbiology 2009, 9:106 ACS Nano: in press designed biofunctionalized silicon micropillar arrays to provide both a preferable solid−liquid interface for bacteria networking and a simultaneous transducing element that monitors the response of bacteria when exposed to chosen antibiotics in real time - culture-free, label-free phenotypic AST

non-microorganism detection based molecular diagnostic approaches (host response diagnostics) © by author ESCMID Online Lecture Library Non-microorganism detection based molecular diagnostic approach ? (i) © by author ESCMID Online Lecture Library direct detection of the presence of microorganism(s) in clinical specimens

determination of gene© expression by author patterns in patient‘s blood mononuclear cells specific for particular microorganism(s) ESCMID Online Lecture Library signature of host response that distinguishes between bacteria and viruses and between infection and non-infection identification of several expression-based “classifiers“

low-density arrays reduction of expression-based© by “classifiers“ author FilmArray style tests validation on well-characterized cohorts ESCMID Online Lecture Libraryreal-time assays

real-life validation point of care tests 102 adults vs. 41 Sci Transl Med 2013;5:203ra126 healthy volunteers

sensitivity 89% specificity 94%

Sci Transl Med 2016;8:322ra11 overall accuracy 87% - 238/273 concordant with clinical adjudication

ESCMID Online Lecture Librarysensitivity 95% J Infect Dis 2015;212:213-22 specificity 92% JAMA 2016;316:835-45

2-transcript signature (FAM89A and IFI44L)

23/23 patients with microbiologically confirmed bacterial infection classified as bacterial (sensitivity, 100% [95%CI, 100%-100%]© by) author 27/28 patients with definite viral infection classified as viral ESCMID(specificity, 96.4% Online[95%CI, 89.3%-100%] )Lecture Library culture-confirmed tuberculosis vs. culture-negative tuberculosis, diseases other than tuberculosis, latent tuberculosis

51-transcript signature sensitivity: 82.9% (68.6 to 94.3) specificity: 83.6% (74.6 to 92.7) for© the by diagnosis author of culture-confirmed tuberculosis

XpertESCMID MTB/RIF Online Lecture Library sensitivity: 54.3% (37.1 to 68.6) specificity: 100% (100.0 to 100.0) for the diagnosis of culture-confirmed tuberculosis Non-microorganism detection based molecular diagnostic approach ? (ii) © by author ESCMID Online Lecture Library direct detection of the presence of microorganism(s) in clinical specimens

uses deep sequencing to monitor gene expression at the level of translation rather than transcription and/or complex protein analysis

(sequencing a cDNA library derived from the short fragments of mRNA covered by the ribosome)

providing novel insights into the identities and amounts of proteins being produced in cells© infectedby author with microorganism(s) ESCMID Online Lecture Library

J Virology 2015; 89: 6164-6166 Non-microorganism detection based molecular diagnostic approach ? (iii) © by author ESCMID Online Lecture Library direct detection of the presence of microorganism(s) in clinical specimens

treatment of infectious diseases© by with author non-traditional drugs, compounds which were originally not developed as antimicrobial agents ESCMID Online Lecture Library Nat Med 2017; 23: 703-713

large institution-wide, prospective clinical sequencing effort to identify genomic alterations in genes and pathways that can be targeted therapeutically with existing drugs

11% of patients enrolled on genomically matched clinical trials

mBio 2014; 5:e01534-14 six FDA-approved drugs identified that could potentially be repurposed to treat influenza viruses

“The use of existing drugs is a very tempting approach because you are accelerating everything.”

host-proteins measured using ELISA, Luminex, protein-arrays and flow-cytometry ImmunoXpert Lancet Infect Dis 2017;17: 431–40

20-second noninvasive diagnosis of Plasmodium falciparum infection without drawing blood or using any reagent Clin Infect Dis 2014;59:1733-40

- thermal desorption-gas chromatography mass spectrometry - prospectively collected breath samples - patients with proven or probable invasive aspergillosis vs. patients without aspergillosis © by author

- 94% sensitivity (95% CI, 81%–98%)

Open Forum Infectious Diseases 2016; 9:ofw051. our technical capabilities are exceeding our ability to apply them effectively and economically to human problems © by author ESCMID Online Lecture Library

Bartlett RC, 1974 a survey of 700 infectious disease physicians in North America

67.5% think that new diagnostic testing is becoming too complex for non-infectious disease physicians © by author ESCMID Online Lecture Library Conclusions the clinical microbiology laboratory is in the middle of a diagnostic revolution continuous introduction of newer technologies and approaches over years more rapid; more sensitive; lower cost? adequate training needed; new skills needed; new profile of microbiologist needed? several open quality and regulatory issues more data on hard endpoints needed; integration into clinical care challenging © by author change management; project management; team management continuousESCMID education and implementatoion Online ofLecture diagnostic and antimicrobial Library stewardship are necessary to ensure that new technologies conserve, rather than consume, additional health care resources and optimally affect patient care