fcimb.2020.00308

REVIEW
published: 15 July 2020

doi: 10.3389/fcimb.2020.00308
Modern Tools for Rapid Diagnostics

of Antimicrobial Resistance
Antti Vasala 1*, Vesa P. Hytönen 1,2 and Olli H. Laitinen 1
1
Protein Dynamics, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland, 2 Fimlab Laboratories,
Tampere, Finland
Fast, robust, and affordable antimicrobial susceptibility testing (AST) is required, as

roughly 50% of antibiotic treatments are started with wrong antibiotics and without a
proper diagnosis of the pathogen. Validated growth-based AST according to EUCAST
or CLSI (European Committee on Antimicrobial Susceptibility Testing, Clinical Laboratory
Standards Institute) recommendations is currently suggested to guide the antimicrobial
therapy. Any new AST should be validated against these standard methods. Many rapid
diagnostic techniques can already provide pathogen identification. Some of them can
additionally detect the presence of resistance genes or resistance proteins, but usually
isolated pure cultures are needed for AST. We discuss the value of the technologies
applying nucleic acid amplification, whole genome sequencing, and hybridization as
well as immunodiagnostic and mass spectrometry-based methods and biosensor-based
Edited by: AST. Additionally, we evaluate the potential of integrated systems applying microfluidics to
Francois Vandenesch, integrate cultivation, lysis, purification, and signal reading steps. We discuss technologies
Université de Lyon, France
and commercial products with potential for Point-of-Care Testing (POCT) and their
Reviewed by:
capability to analyze polymicrobial samples without pre-purification steps. The purpose
Jerome Lemoine,
Université de Lyon, France of this critical review is to present the needs and drivers for AST development, to show the
Karsten Becker, benefits and limitations of AST methods, to introduce promising new POCT-compatible
University Medicine
Greifswald, Germany technologies, and to discuss AST technologies that are likely to thrive in the future.
*Correspondence: Keywords: antibiotic resistance, antimicrobial susceptibility test, antimicrobial resistance, point of care test, rapid
Antti Vasala AST
antti.i.vasala@gmail.com
Specialty section: BACKGROUND AND FOREWORD

This article was submitted to
Clinical Microbiology, There is an unmet need for rapid and decentralized diagnostics in outpatient clinics to reduce
a section of the journal the misuse of antibiotics. It is important to identify the etiological pathogen and to differentiate
Frontiers in Cellular and Infection
between viral and bacterial infections, to identify the antimicrobial resistances in microbes, and
Microbiology
to find out which antimicrobial agent should be used for the cure. Thereby the unnecessary
Received: 17 January 2020 use of antibiotics could be minimized and the spread of antibiotic resistance better controlled.
Accepted: 22 May 2020
According to the WHO, antimicrobial resistance (AMR) is the largest global health threat in the
Published: 15 July 2020
21st century and requires urgent measures. Common infections are becoming untreatable due
Citation: to the emergence of AMR. More than 700,000 people die of drug-resistant infections every year,
Vasala A, Hytönen VP and Laitinen OH
and this figure is expected to reach ten million by 2050 (United nations meeting on antimicrobial
(2020) Modern Tools for Rapid
Diagnostics of Antimicrobial
resistance, 2016). According to current understanding, in EU and EEA countries more than
Resistance. 33 000 people are killed every year due to antibiotic-resistant bacteria. They also cause close
Front. Cell. Infect. Microbiol. 10:308. to 900 000 disability-adjusted years (Cassini et al., 2019). Bringing diagnostics closer to the
doi: 10.3389/fcimb.2020.00308 general practitioners and hence to the patient would cause a paradigm shift from empirical to
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 July 2020 | Volume 10 | Article 308
Vasala et al. Rapid Diagnostics of Antimicrobial Resistance
evidence-based treatments of infectious diseases in outpatient Resource for Research and Education: Educational Resources—
clinics. Rapid diagnostics are needed for both pathogen Rapid Antimicrobial Susceptibility Testing | University of Utah)
identification and resistance testing. The prevalence of AMR is particularly useful.
may be very high for some species in certain geographic The innovations in electronics, biosensor techniques,
locations. According to the current recommendations on AST optics, microfluidics, hybridization technologies and DNA
(antimicrobial susceptibility testing), pure culture isolates are amplification technologies have yielded new approaches in
used to test the effect of antimicrobial drugs. This is required AST. Unfortunately, the scientific papers on these technologies
as the sample matrix (blood, urine, mucosal) as well as the do not sufficiently relate these findings to the practical needs
number and proportions of different microbial species may in POCT. The requirement of a microbiology laboratory and
vary a lot in polymicrobial samples. It may be unclear whether the time and resources needed for the enrichment of cultures,
the detected microbe is pathogenic or merely a commensal preparation of pure cultures. and sample treatments is often
species. Despite significant progress in diagnostic technologies not sufficiently considered. These requirements also easily blur
in recent years, most patients with infectious diseases are still the total costs of AST. For these reasons, the deployment of
treated empirically and thus antibiotics are heavily overused new molecular methods for AST has been very slow (Doern,
(Li et al., 2016; Mashalla et al., 2017). Even in Western 2018). The standards of care for antibiotic prescription are quite
countries, 30% of antibiotic prescriptions are considered to be consistent in most European countries and the USA and apply
either unnecessary or suboptimal (Centers for Disease Control evidence-based ID and AST when available. In total, urinary
Prevention, 2018). Current diagnostic tests serve hospitalized and respiratory tract infections form a significant part of acute
patients rather well, but they are often not available in outpatient infections. Quick and accurate diagnosis for these diseases
clinics. For typical growth-based AST, several cultivation rounds already at outpatient settings could efficiently restrict the spread
are required: enrichment cultivations (e.g., blood cultures) to of AMR bacteria and allow an early isolation of the carrier and
increase the number of bacteria, plate cultivations to obtain correct treatment. Rapid diagnosis would also allow the prompt
pure cultures, and finally AST for liquid or plate cultures dismantling of unnecessary patient isolation, saving money
using various antimicrobial loads. Microbiology laboratories and resources. However, technical improvements translate into
apply the EUCAST-accepted breakpoint values to define whether benefits only if the structured communication and interpretation
the microbe is susceptible or resistant to the tested antibiotic. of the results are applied by the clinicians (Maurer et al., 2017)
They use the disk diffusion method or other systems calibrated and the cost of these technologies are reasonable.
to EUCAST standards. Altogether, AST may require several Vital emergency diagnostics for septicaemia has received a lot
days. Rapid molecular diagnostics has been discussed in many of resources (Marco, 2017; Hughes, 2018). New sensitive methods
excellent reviews (Pulido et al., 2013; Plüddemann et al., 2015; Li such as T2MR (T2Biosystems, USA) can quickly detect molecular
et al., 2017b; Maurer et al., 2017; Syal et al., 2017; Maugeri et al., targets directly from clinical samples, enabling rapid pathogen
2019). They present the progress in Nucleic Acid Amplification identification and detection of resistance factors. However,
Technology (NAAT), electrochemical methods, microarrays, growth-based AST for blood requires a fairly high bacterial
micro- and nanoparticles, as well as mass spectrometry count for enrichment cultures and a well-equipped microbiology
applications, but also emphasize that very few of the molecular laboratory. The achievements in blood testing do not necessarily
methods have acquired FDA approval. relate well to the antimicrobial stewardship in the front-line:
The review of David Boyle, “Tuberculosis Diagnostics healthcare settings.
Technology Landscape” is worth reading, since, although not Optimal antimicrobial therapy policy would require (1) Fast
focusing on AST, it presents comprehensively new affordable point of care analysis, (2) Identification of the etiological agent,
molecular diagnostic technologies available in standard (3) Finding of an efficient antibiotic, and (4) Determination of
microscopy stations, particularly in developing countries (Boyle, the functional dosage. According to Prof. Gunnar Kahlmeter
2017). The most up-to-date and concise progress compendia (Chairman, EUCAST general committee) (Kahlmeter, 2016), the
in the field of AST can be found in congress presentations, key questions for any new AST technology are:
lectures and webinars. Prof. Mark Fisher’s webinar “Rapid • Is it generally applicable or suitable only for one infection (for
Antimicrobial Susceptibility Testing” (ARUP, 2020; Scientific example sepsis or one resistance type)?
• What is the capacity? How many organisms/agents per hour
can be processed?
Abbreviations: AMR, Antimicrobial Resistance; AR gene, Antibiotic Resistance
gene; AST, Antimicrobial susceptibility testing; CLSI, Clinical & Laboratory
• Has the technology been validated against reference methods?
Standards Institute; EUCAST, European Committee on Antimicrobial • Are there any reference installations?
Susceptibility Testing; CFU, Colony forming units; FISH, Fluorescence in • Is scientific literature available?
situ Hybridization; ID, Identification; LF, Lateral flow; LCR, Ligase Chain • When will it be on the market?
Reaction; MALDI-TOF, Matrix-Assisted Laser Desorption/Ionization Time-
Of-Flight mass spectrometry; MIC, Minimal Inhibitory Concentration; POCT, Complete answers are hard to dig up, but this review tries to
Point of Care Testing; NA= Nucleic Acid; NAAT, Nucleic Acid Amplification address these questions. We present the needs and drivers for
Technology; PIT, Plasmonic Imaging and Tracking; SERS, Surface Enhanced
AST development, increase the understanding about the role
Raman Scattering; FEED, field effect enzymatic detection; MADM, Multiplexed
Automated Digital Microscopy; PNA, Peptide-Nucleic Acid probe; WGS, Whole of rapid AST in diagnostics of infectious diseases, show the
Genome Sequencing. benefits and limitations of AST methods, introduce the key
POCT-compatible technologies, and contemplate on which AST to obtain pure isolates. Then identification (with MALDI-TOF
technologies are likely to thrive in the future. mass spectrometer, if available) is performed. Thereafter, AST
and MIC determination is performed according to EUCAST or
CLSI standards. This sequence, in total, requires several days.
CURRENT TECHNOLOGIES IN Standard healthcare settings do not have advanced microbiology
ANTIMICROBIAL SUSCEPTIBILITY laboratories with mass spectrometry instruments. Their arsenal
TESTING AND MICROBIAL for the diagnosis of infectious diseases may be limited to
IDENTIFICATION immuno-chromatographic strip tests (aka lateral flow tests = LF
or “dip-sticks”) applied to the detection of viruses (e.g., influenza)
Bacteria can acquire resistance to antibiotics by several and bacterial pathogens causing sexually transmitted diseases.
mechanisms. The antibiotic can be degraded or chemically Quick identification can efficiently restrict the search palette
modified (by acetylation, phosphorylation, nucleotidylation, for certain antibiotics. Hence mass spectrometry has become
ADP-ribosylation, mono-oxygenation, glycosylation). The drug a versatile workhorse in clinical laboratories. It is routinely
intake can be prevented, or efflux can be enhanced. Some applied for bacterial ID as soon as isolated colonies are available.
resistance mechanisms are based on the reprogramming of cell Through the simultaneous measurement of several metabolites
wall synthesis. Even slight changes in the target molecule, e.g., a biochemical signature of microbes can be obtained. Matrix-
a point mutation in the ribosomal protein, can render the assisted laser desorption/ionization time of flight (MALDI-TOF)
antibiotic inefficient. The overwhelming variety of antimicrobials applies laser energy to evaporate the matrix-bound sample, that is
and resistance mechanisms complicates AST. Genotypic (nucleic then immediately analyzed. When frequent sampling is applied,
acid-based) methods can only find resistances that are searched MALDI-TOF can even provide semi-quantitative growth rate
for, and the potentially found resistance genes are not necessarily data (Maxson et al., 2017). Bruker Corp. (Germany) has launched
from the actual pathogenic organism. According to EUCAST test kits such as BT STAR-Carba Assay for AST based on
and CLSI guidelines, reliable antibiotic resistance diagnostics antibiotic degradation monitoring.
requires phenotypic testing, i.e., an experimental test whether AST for blood cultures has been applied after a short-term
the microorganism grows in the presence of the antibiotic. cultivation on agar plates followed by susceptibility testing
These methods work regardless of the resistance mechanism using VITEK AST cards selected on the basis of MALDI-
and give answers to the practical questions: which antibiotic TOF analysis (Idelevich et al., 2014; Mauri et al., 2017). By
is efficient and which dose should be applied in the therapy. applying the MBT-ASTRATM test with MALDI Biotyper for
Classical AST techniques such as broth microdilution, disk ID and AST, identification of mycobacterial strains resistant to
diffusion, gradient tests, agar dilution and breakpoint tests are rifampicin, isoniazid, linezolid, ethambutol, clarithromycin and
based on continuous exposure of a bacterial isolate to a set of rifabutin can be obtained 1 week faster than through routine
antimicrobials, followed by a visual detection of growth. The use cultivation-based AST (Ceyssens et al., 2017). The MS approaches
of advanced optoelectronic systems, fiber optics, microfluidics combined with NAAT or microfluidics will be presented in
and indicator dyes sensitive to redox-state or pH can further “Future technologies” section.
enhance the sensitivity and performance of optical systems.
Several commercial systems have streamlined and partly
automatized the follow-up of AST cultures. Systems like Vitek CURRENT TECHNOLOGIES FOR RAPID
and Microscan perform automated turbidity measurement for AST
multiwell liquid cultures. BD Phoenix systemTM applies a redox
indicator to enhance the detection of organism growth. These Many novel methods claim to perform AST in minutes or in
systems have turnaround times as short as 4 h for ID and 6–8 h few hours. Such statements usually ignore the need of time-
for susceptibility testing (She and Bender, 2019). The CE-marked consuming steps such as enrichment cultures and isolation
Alfed 60 ASTTM system (Alifax, Italy) uses sensitive laser-light of pure cultures (Figure 1). Methods based on NAAT, nucleic
scattering technology to detect bacterial growth in a liquid culture acid hybridization or immunodiagnostics in principle allow the
broth and provides antimicrobial susceptibility results directly use of non-purified polymicrobial clinical samples. A short
from positive blood culture bottles within 4–6 h. cultivation with a pre-determined antibiotic load followed by
Such broth dilution-based systems use ready-made AST NAAT (e.g., isothermal amplification) can reveal AR, and even
cassettes or cards containing positive controls and wells provide a rough estimate of the minimal inhibitory concentration
with increasing concentrations of antibiotics. They provide (MIC) for the tested antibiotics. Most rapid growth-based AST
continuous growth monitoring and can analyze MIC patterns for methods perform end-point analysis only, whilst others rely on
a large group of organisms through their extensive databases. frequent sampling from the cultivation chamber. Some sensitive
Pathogen identification (ID) is usually a preliminary step of immunodiagnostic systems however provide real on-line growth
AST. For blood samples, microscopy and Gram-staining are monitoring (Figure 2). Biosensor technologies detecting changes
nearly always performed, as Gram-positive bacteria in general in microbial metabolism, movement or heat production have not
have a more limited variety of antibiotic resistances and less yet provided convincing clinical demonstrations. Fast, reliable,
problems with multidrug resistance. In AST, the following easy-to-use and inexpensive systems applicable to AST in
sequence is typically applied: First clinical samples are cultured outpatient clinics are still elusive (van Belkum et al., 2019a).
FIGURE 1 | Rapid AST or rapid result? (A) Current technologies. (B) Rapid AST applicable to pure cultures. (C) Rapid AST for clinical polymicrobial samples. The
presented times are rough estimates and generalizations.
FIGURE 2 | Usability landscape of rapid AST technologies. NAAT, nucleic acid amplification technology; TPX, immunodetection based on two-photon excitation
fluorometry; Multipath, immunodiagnostic method applying magnetic spheres for cell separation, fluorescent nanoparticles for labeling and non-microscopic imaging.
Microscopy Clinical laboratories applying mass spectrometry are unlikely to

Counting of bacteria on agar plates by microscopy techniques need PNA-FISH technology; the Bruker MALDI Septityper R kit
is possible long before they have reached the number providing PBP2A, e.g., can detect the mecA-encoded PBP2A-protein in 1 h
visible colonies. E. coli colonies visible by eye contain roughly with fairly low costs.
5 × 106 bacteria, but by microscopy microcolonies formed by In actively growing cells, RNA is more abundant than
120 cells can already be detected (London et al., 2010). Drug DNA and thus a good target for probing. Especially precursor
susceptibility (MODS) for Mycobacterium tuberculosis can be rRNA (pre-rRNA), the intermediate stage in the formation of
assessed by observing cell aggregates (cords) microscopically mature rRNA, is a good indicator of bacterial metabolism,
in sealed microtiter plates (den Hertog et al., 2014). The viability, and growth rate (Halford et al., 2013). The biosensor-
Growth Direct System by Rapid Micro Biosystems Inc. detects based AST (b-AST) system from Genefluidics Inc. (CA, USA)
microcolonies with digital imaging by illuminating them with measures bacterial growth by quantifying 16s rRNA molecules
blue light and directing the cellular autofluorescence directly with an electrochemical biosensor. This system uses species-
onto a CCD chip without magnification. The average time for E. specific probes and integrates nanotechnology, plastic micro
coli detection by this autofluorescence was 3.1 h compared to an electromechanical system and microfluidics (Mach et al., 2011).
average of 8.5 h for the visual plate counting method. Although This system achieved a detection limit of 104 cfu ml−1 in rapid
the idea of applying this to AST has been declared in the patent AST of clinical urine and blood samples (Liu et al., 2014). Mohan
application, clinical studies for AST have not yet been presented. et al. demonstrated the simultaneous detection of uropathogens
Automated microscopy systems can provide real time and the host biomarker lactoferrin in urinary tract infection,
growth curves, and quantitative bacterial counts have been but reached only 89% sensitivity in the pathogen identification
presented. Multiplexed automated digital microscopy (MADM) (Mohan et al., 2011). In 2018 Genefluidics announced CE-IVD
applying Fluorescent in situ Hybridization (FISH) has been Marking for the UtiMaxTM kit, which provides ID in 30 min and
commercialized by Accelerated Diagnostics (USA) for rapid on- AST in 2 h from urine with an overall sensitivity of 100% and
line AST (Metzger et al., 2014; Chantell, 2015). The Accelerate specificity of 98.2% (GeneFludics Inc., n.d.).
Pheno R system can separate impurities from clinical samples
(e.g., blood or urine) by brief electrophoresis, which runs
impurities into a gel. After this a change of the electric field Nucleic Acid Amplification Technology
polarity repels the microbes back to the liquid. A fluorescence (NAAT) in AST
signal is detected every 10 min from samples taken from the The first generation “molecular tests” such as restriction
bacterial culture multiplying in Mueller-Hinton media (Charnot- fragment length polymorphism, pulsed-field electrophoresis,
Katsikas et al., 2017). This system seems to be currently the multiple locus tandem repeat analysis, multi-locus sequence
only FDA-approved growth-based rapid diagnostic AST system typing and virulence genotyping were suitable rather for
(Doern, 2018). The performance of Accelerate Pheno R has typing and outbreak investigation than for AST. Since
been demonstrated with many clinical studies, e.g., with urinary these technologies require a high amount of purified
tract infections (Charnot-Katsikas et al., 2017) and bloodstream nucleic acid, they do not allow rapid diagnostics. However,
infections (Charnot-Katsikas et al., 2017; Marschal et al., 2017; hybridization-based approaches and molecular beacon
Descours et al., 2018). With 232 positive blood cultures tested, the systems have persisted and are creatively combined with
overall essential agreement with routine methods was 95.1%, and NAAT technologies.
the time needed for AST was decreased by 42 h in comparison NAAT is a very powerful tool for pathogen identification,
to standard growth-based analysis. ID could be obtained in 1.5 h especially when combined with a syndromic approach. Many
and AST in 7 h (Charnot-Katsikas et al., 2017). diagnostic panels provided e.g., by BioMérieux, Elitech, Bosch,
Eplex, Qiagen, or Becton Dickinson, include detection of specific
Hybridization-Based Systems AR-genes. They can provide clinically relevant results, especially
FISH is a highly specific method to visualize the presence of in cases where a detailed antibiogram is not needed. For
the target organism in a quantitative manner. The PNA-FISH example, pathogen such as Bordetella, Legionella, Mycoplasma,
technology applies peptide nucleic acid probes which allow more Chlamydia trachomatis, or Neisseria gonorrhea exhibit quite
rapid and specific binding than DNA or RNA probes (Perry- few antibiotic resistances. The detection of specific AR genes,
O’Keefe et al., 2001; Almeida et al., 2009; Cerqueira et al., 2011). however, cannot give an undisputed proof of antibiotic
It is applied in the commercial QuickFish technology (OpGen, resistance. The identified AR genes do not necessarily relate
USA) which performs ID by targeting 16S rRNA (Enroth to the pathogen causing the disease, or the found resistance
et al., 2019). XpressFish specifically detects the mecA gene in gene may not be functional. NAAT neither defines the MICs
Staphylococcus, allowing, when used subsequent to QuickFish- nor directly indicates which antibiotics should be used. An
based identification, diagnosis of methicillin resistance already advantage of NAAT is that the tests can be relatively quickly
2 h after the blood culture turns positive (Salimnia et al., updated for newly emerging pathogens and resistance factors.
2014). Since a temperature of 55◦ C is needed for target cell Quantitative PCR (qPCR) allows a rough quantification of
permeabilization, fixing and hybridization, PNA-FISH systems microbes. Quantitative reverse transcription PCR (qRT-PCR)
are not applicable to on-line growth monitoring. FDA-approved can additionally assess the expression level of resistance genes
systems are available also from bioMérieux (Durham, NC). after exposure to different antibiotic loads and thus provide
rough MIC values. The cost of devices and reagents for qRT- allowing the use of samples without NA extraction), enabled
PCR are unfortunately currently far beyond the level acceptable miniaturization and cut down the costs of instrumentation
for routine AST. NAAT is a powerful tool for the identification by allowing NAAT at a constant (isothermal) temperature.
of both bacterial and viral pathogens. In principle it allows NUCLISENS R EASYQ R (bioMerieux) was the first automated
the use of patient samples without enrichment cultivations. system to combine NASBA and real-time detection using
However, due to the risk of losing the template during nucleic molecular beacon probes. It enabled the fast detection of
acid extraction and the sensitivity of DNA polymerases to Klebsiella carbapenemase genes (Spanu et al., 2012). LCR has
impurities in the sample matrix, enrichment cultivations and been successfully used for the detection of ciprofloxacin and
nucleic acid purification are often necessary. Due to the vast doxycycline resistance genes in Bacillus anthracis, Francisella
choice of different fluorescent labels, several target genes can tularensis, and Yersinia pestis (Oblath et al., 2013). LCR can be
be conveniently tested in parallel from the same sample. easily integrated into detection systems such as electrochemical
The systems can reach further sensitivity and specificity by and magnetic biosensors, quantum dots, quartz crystal and
applying hybridization to DNA arrays. A good number of FDA- leaky surface acoustic surface biosensors, Surface Enhanced
approved multiplexed diagnostic panels are already available. Raman Scattering (SERS), chemiluminescence and fluorescence
Such commercial systems include Xpert R (Cepheid Inc.), resonance energy transfer (Oblath et al., 2013). High-throughput
ePlex (GenMark Diagnostics), Unyvero (Curetis AG), BD Max multiplex genotyping can be achieved also by DNAzyme (DNA
(Becton-Dickinson), SeptiFast (Roche), Magicplex (SeeGene), oligonucleotides) technology, rolling circle amplification (RCA)
Novodiag (Mobidiag), and GenomEra (Abacus Diagnostics). and strand displacement amplification (SDA) techniques. LAMP
The Xpert R technology combines sample preparation, real-time is especially robust, since it is less sensitive to inhibitors than
PCR and nucleic acid analysis with molecular beacons. The standard PCR. This allows analysis after a minimal processing
ePlex system provides electrochemical detection of the amplified of blood (Curtis et al., 2008), urine or stool (Francois et al.,
sequences, wherein the detection is achieved by hybridizing 2011). LAMP is applicable to low-resource field settings where
ferrocene-labeled probes with the sample DNA (Nijhuis et al., DNA or RNA extraction is not possible. However multiplexing
2017). Hybridization techniques and microfluidics have been approaches are less developed for LAMP than for PCR (Sahoo
implemented also to Novodiag’s GenomEra microarray platform. et al., 2016).
It applies time-resolved fluorescence detection of the amplified
product on a sealable plastic chip packed with dry chemistry. Immunodetection of Pathogens
Also Curetis, Becton-Dickinson, Roche and SeeGene products Immunodetection is a specific and sensitive method for the
apply cartridge-based integrated designs (Hughes, 2018). Fast identification of bacterial pathogens (Verma et al., 2013), toxin
analytics of PCR products enhance the throughput of NAAT proteins (Zhu et al., 2014), and viruses. Since immunodetection
systems. T2Biosystems has recently launched a test panel able does not necessarily require disruption of the target microbes,
to detect 13 resistance genes from both gram-positive and it can potentially provide pathogen identification and growth
gram-negative pathogens directly from blood. The amplification monitoring in a single step. It is applicable as simple lateral
products are detected by magnetic resonance after hybridization flow (LF) tests, but can also be integrated to biosensor
with DNA probes conjugated with superparamagnetic particles technology, microfluidics and even to DNA/RNA-based analysis.
(Hong Nguyen et al., 2019). Companies like Mizuho Medy, Alere, and Beckton Dickinson
PCR/electrospray ionization–mass spectrometry (IRIDICA have launched several easy-to-use stick tests for clinical
PCR/ESI-MS by Abbott Laboratories Inc., USA) allows the diagnostics of (influenza) viruses and bacteria causing sexually
detection of >750 different bacterial species in a single test transmitted diseases. The binders are typically antibodies that are
(Strålin et al., 2016). The High Resolution Melting system immobilized onto strips, micro/nanoparticle beads or biosensor
(HRM, by ThermoFisher) identifies variations in nucleic acid surfaces providing an efficient and specific target binding. The
sequences by detecting small differences in PCR melting curves. detection antibody can be labeled with fluorescent dyes or redox
A melting curve analysis for real-time quantitative PCR or enzymes to provide a quantitative signal.
digital PCR (wherein the sample is partitioned into a large Only few products are available for the direct detection
number of individual wells each containing either 1 or 0 of antibiotic resistance proteins. The LF-test developed by
targets) performed for growing bacterial cultures, revealed Kitao et al. detects chloramphenicol resistance in P. aeruginosa
both ID and antimicrobial susceptibility profiles for E. coli, E. samples (Kitao et al., 2010). Alere Inc. has launched an
faecalis, P. mirabilis and S. aureus in ∼6.5 h, when analyzed immunochromatography test for the detection of MRSA, based
by machine learning algorithms (Athamanolap et al., 2017). on a PBP2a-specific chicken IgY antibody (Yamada et al., 2013).
For routine analytics, such systems are still too expensive, The PBP2a SA Culture Colony Test can identify MRSA in 6 min
and they require separate kits for DNA extraction and PCR. (Trienski et al., 2013; Delport et al., 2016). Coris Bioconcept
However, new enzymes and technologies such as ligase chain (Belgium) has launched tests for the detection of carbapenemases
reaction (LCR) (Barany, 1991), nucleic acid sequence-based (OXA-48-like, KPC, and NDM type) from enterobacterial
amplification (NASBA) (Compton, 1991), strand displacement isolates (Bogaerts et al., 2013; Glupczynski et al., 2017). Boutal
amplification (SDA) (Walker et al., 1992) and loop-mediated et al. have presented LF tests for the carbapenemases TX-M-
isothermal amplification (LAMP) (Notomi et al., 2000) have 15, NDM, OXA-48-like, KPC, IMP, and VIM (Boutal et al.,
simplified nucleic acid amplification, made it more robust (often 2017). LF tests work well with isolated clinical isolates. They are,
however, generally not applicable for direct analysis of clinical Changes in individual cell morphology or size indicate growth
samples. The commercial MultiPathTM platform (First Light long before bacteria have multiplied. Single-cell morphological
Diagnostics Inc., USA) applies non-magnified digital imaging analysis (SCMA) uses bright-field microscopy to determine
for the detection of biomolecules tagged with antibody-coated antibiotic-induced changes in cells immobilized on an agarose
fluorescent nanoparticles. The mariPOC R system by ArcDia channel chip (Choi et al., 2014). The commercial MultiPathTM
Ltd, based on Two-Photon eXcitation fluoroscopy technology platform applies non-magnified digital imaging for the detection
(TPX), is already in diagnostic use for immunogenic detection of biomolecules tagged with antibody-coated fluorescent
of pathogens. These two technologies will be discussed closer in nanoparticles. Antibody-coated magnetic particles are used to
the following sections. bind to target cells and to pull them down to the camera surface,
thus eliminating background signal and enabling a wash-free
assay for clinical samples. The system counts individual targets
FUTURE TECHNOLOGIES UNDER in large areas and performs growth monitoring for multiple
COMMERCIALIZATION targets cells and enables the determination of MIC values. The
MultiPathTM system is currently seeking FDA clearance.
Mass spectrometry is likely to become tightly integrated The Two-Photon eXcitation fluoroscopy technology (TPX),
into other AST technologies, especially in the diagnostics of commercialized by ArcDia Ltd. (Finland), allows separation-free
septicaemia. In the MALDI-TOF Direct-On-Target Microdroplet detection of biological molecules in small reaction volumes by
Growth Assay (DOT-MGA), sample droplets (culture plus immunodetection (Vakkila et al., 2015). The mariPOC R test
antibiotics in 6 µL volume) are spotted directly onto disposable system was developed for rapid pathogen identification. It applies
MS-target plates, incubated for 3–4 h and then analyzed with MS polystyrene microparticles as solid carriers for immunocomplex
(Idelevich et al., 2018). Screening panels for ESBL and AmpC formation, that leads to three-component immunocomplexes
β-lactamases of enterobacteria are already available (Correa- (monoclonal antibody—antigen—labeled monoclonal antibody)
Martínez et al., 2019). The fast progress in microfluidics, on the microspheres in proportion to the analyte concentration.
biosensor technologies, isothermal amplification-based NAAT, The detection of the immunoassay fluorescence signal is
and immunodetection has recently provided several potent achieved by two-photon excitation from the surface of individual
systems which may eventually change the paradigms of AST. microspheres. This mechanism allows the use of unpurified
Gradientech’s QuickMIC system combines microfluidics with clinical samples and on-line monitoring of viable cells in AST
automated time-lapse photomicrography to follow growth cultures. At present this system is under clinical testing for AST.
inhibition along a linear drug gradient. It measures the greyscale Nanostring Technologies Inc. combine genotypic and
intensity changes in the images caused by the formation of phenotypic AST through RNA detection. The GoPhAST-R
microcolonies and provides AST in 2–5 h (Malmberg et al., 2016). platform detects mRNA expression signatures in bacteria after
The system is currently seeking CE-IVD marking and FDA antibiotic exposure. The system can be used directly for positive
approval for AST in blood samples. blood culture bottles. It couples machine learning analysis of
Q-Linea ASTar R applies time-lapse microscopy to fully transcriptional changes with the detection of resistance genes
automated monitoring of blood cultures as well as preparation (Bhattacharyya et al., 2019).
and monitoring of bacterial isolates. It processes 12 samples at a Colorimetric sensor arrays provide an inexpensive method
time and 50 samples a day, delivering true MIC values for up to 48 to detect volatile organic compounds (VOCs) associated with
antibiotics within 6 h. The system does not perform ID, but it can microbial metabolism (Lonsdale et al., 2013; Lim et al., 2014).
be connected to any ID system. Clinical trials will start in 2020. Specific Technologies Inc (USA) has commercialized the small
The BacterioScan 216Dx system (St. Louis, MO, USA) molecule sensor (SMS) array technology which reacts with the
measures both a sample’s optical density (OD) and the scattered metabolic products of bacteria produced during their growth.
intensity by forward laser light scattering, allowing 10–100-fold Their Reveal-AST printed sensor array system responds to the
higher sensitivity compared to normal OD measurements. This volatiles emitted during growth producing a colorimetric pattern.
system can process 16 samples simultaneously and perform real- It reveals also species ID with 94% accuracy (Sharp, 2020).
time continuous growth measurement. It can detect bacterial The SlipChip/dLAMP technology by Caltech integrates
growth in 3 h from clinical urine samples containing >104 seamlessly cultivation and semiquantitative smartphone-based
cfu/ml of E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, visual analysis of NA products (Schoepp et al., 2017). This system,
Bacillus antracis, Yersinia pestis, or Burkholderia pseudomallei currently being commercialized by Talis Inc. (USA), will be
(Bugrysheva et al., 2016; Hayden et al., 2016; Montgomery et al., described closer in the chapter “POCT-compatible technologies.”
2017). The BacterioScan 216R Rapid AST System is currently
under testing. Whole Genome Sequencing
The oCelloscopeTM system (BioSense Solutions, Denmark) Progress in Whole Genome Sequencing (WGS) technologies
applies digital time-lapse angled field microscopy and image has made them a feasible system for pathogen ID and AST
analysis for standard 96-well plates. The time-to-result for (van Belkum and Rochas, 2018). 3rd generation systems such as
positive blood cultures ranged from 1 to 4.2 h (Fredborg et al., Illumina MiniSeq, Pacific Biosciences PacBio Sequel system, or
2015). However, the overall performance for AST has not yet been Oxford Nanopore MiniON and PromethION can provide fairly
sufficiently tested. long reads at high speed. In principle, WGS can simultaneously
provide fast pathogen ID, epidemiological typing, and detection time. smaRT-LAMP was shown to work well with diverse Gram-
of drug susceptibility genes. Since WGS provides a massive negative and Gram-positive pathogens in biological specimens,
amount of data in fragmented form, sophisticated software is giving in ∼1 h results with matched standard cultivation-based
needed to interpret the results (Quainoo et al., 2017). The tests. Reliable pathogen ID was obtained for spiked urea and
European Committee on Antimicrobial Susceptibility Testing blood samples as well as for urea samples of sepsis patients (105
reviewed in 2017 the development status of WGS for AST −108 CFUs). The small sample size (2 µl per reaction), however,
(Ellington et al., 2017). They concluded that, for most bacteria, limits its use for very diluted samples (Barnes et al., 2018).
the available evidence for WGS as an AST tool is still either Priya et al. have successfully coupled loop-mediated
poor or non-existent and thus inadequate for clinical decision isothermal amplification (RT-LAMP) and sensitive quenching
making. They pointed out the urgent need for a single database of unincorporated amplification signal reporters (QUASR)
of all known resistance genes/mutations to facilitate comparison detection technologies to visual detection with a smartphone.
between different systems and bioinformatics tools. The portable “LAMP box” was successfully used for the sensitive
and specific detection of Zika, chikungunya, and dengue viruses
(Priye et al., 2017). This system has not yet been applied to
PROOF-OF-CONCEPT TECHNOLOGIES growth-based AST.
Smartphone-Based Readers Optical or Microscopic Methods

Kadlec et al. combined smartphone technology to a Choi et al. have developed a rapid antimicrobial susceptibility
microphotometric system applying microwell plates coated testing system, dRAST. It can determine the AR from a positive
with antibiotics and the yellow redox indicator dye tetrazolium blood culture bottle in 6 h (Choi et al., 2017). The sample is mixed
salt WST-8, which turns orange by the metabolic activity of with agarose and inoculated into a well of a plastic microchip.
growing cells. The system could correctly monitor the growth of Addition of cultivation medium forms a liquid bridge between
several pathogens associated with urine tract infection. Samples the growth chamber and the satellite well, which contains the
with concentrations of 101 to 106 cfu/mL could be tested directly antibiotic agent. Using microscopic detection of bacterial colony
without preliminary enrichment cultivations (Kadlec et al., formation in agarose, the total time-to-result was only 6 h with a
2014). Feng et al. presented an automated smartphone-based wide range of bacterial concentrations. The tested clinical isolates
device with a 3D-printed attachment holding a microwell plate. (n=206) included 16 Gram-negative species and seven Gram-
A light-emitting diode array and fiber-based optics enabled positive species, and the dRAST system agreed with a standard
detection of turbidity changes in wells already after 1 min (Feng microdilution test with an accuracy rate of 91.11% (Choi et al.,
et al., 2016). This system, tested for 17 antibiotics targeting 2017).
Gram-negative bacteria on clinical isolates of K. pneumoniae, Matsumoto et al. described a microfluidic channel method
provided drug susceptibility interpretation with accuracy of for rapid (3 h) AST for P. aeruginosa by automated microscopic
99.23%. Cui et al. have presented a smartphone-based system detection of cell number and cell morphology (Matsumoto et al.,
for the monitoring of viable bacteria in droplet-based single-cell 2016). Their Drug Susceptibility Testing Microfluidic device
microdroplet cultures (Cui et al., 2018). In this system, single (DSTM) consisted of five sets of four microfluidic channels and
bacteria were encapsulated in monodisperse microdroplets. This allowed simultaneous microscopic observation. Susceptibilities
dSPC (Digital Standard Plate Count) platform could quantify E. to the antibiotics (pre-dried into each channel) were evaluated
coli and B. subtilis in 6 h, compared to 24 h needed for traditional by the differences in cell number and shape between drug-treated
plate counting. These smartphone demonstrations discussed and control cells. Hundred and one clinically isolated strains of P.
above were performed using pure culture isolates. aeruginosa tested with DSTM correlated strongly with the results
Smartphone-based systems applying immunodetection have obtained using the conventional microbroth dilution method
been demonstrated for ID, but so far not for growth- (Matsumoto et al., 2016). This system waits for applicability
based AST. Wang et al. introduced a microwell plate-based testing with other organisms.
microphotometric system, which applied a field-of-view adapter In nanowell AST, morphotyping with a phase contrast
and a microprism array between the mobile phone camera microscopy and optical signal analysis is performed for 0.5
and the 96-well plates. In a serological analysis (771 patient µl cultures. Antibiotic susceptibility data can be obtained for
samples in 12 serology assays for bacterial/viral infections) the uropathogens in <4 h. The system showed a total categorical
system exhibited 97.59∼99.90% analytical accuracy in pathogen agreement of 97.9% with standard disk diffusion assays, but a
identification with costs of ∼50 USD per 96-well plate and careful standardization of cell densities prior to cultivation was
analytical quality sufficient for POCT (Wang et al., 2018). found necessary (Veses-Garcia et al., 2018).
An inexpensive (under 100 USD) smartphone-based
monitoring system for nucleic acid amplification, smaRT-LAMP, Hybridization Methods
was introduced by Barnes et al. The system contains a hot plate Mezger et al. presented a generic method for rapid species
for isothermal amplification, two flexible cables and 96 LED identification and AST after 0.5-2 h cultivation (Mezger et al.,
lights fitted into a cardboard box. The Bacticount software detects 2015). Cultured bacteria from urine samples were lysed by
the emitted green light as a result of a successful amplification sodium hydroxide and heat, and DNA was captured onto
and automatically determines the genome copy number in real magnetic beads. Padlock probes targeting the 16S rRNA gene
were hybridized, ligated and amplified by the circle-to-circle to real-time monitoring of microbial viability in biofilms in the
amplification method. Optical imaging system then performed presence or absence of antimicrobial compounds (Butini et al.,
digital quantification. Antibiotic susceptibility profiles of E. coli 2018). Microcalorimetric methods, although fast and sensitive,
for ciprofloxacin and trimethoprim could be determined with require pure cultures and a fairly high number of bacterial
100% accuracy in 3.5 h (Mezger et al., 2015). cells. In 2017, the Swedish company SymCel announced an
extensive 28-months clinical testing of their microcalorimeter
Detection of Growth-Related Molecules or calScreenerTM for AST. However, currently no clinically validated
Antibiotic Degradation Products microcalorimetry systems are available.
Devices detecting volatile compounds, so-called electronic noses For some antibiotics (beta-lactams, chloramphenicol) AMR
(eNose), can recognize a smell-print characteristic for bacterial can be assayed by the follow-up of antibiotic degradation. The
species and their metabolic profile. Since 1982, electronic noses BYG Carba test detects conductivity changes caused by the
have been applied in diagnostics (Persaud and Dodd, 1982), enzymatic hydrolysis reaction of imipenem antibiotics on an
but mainly for pathogen ID. The Cyranose system (Smiths electrode coated with polyaniline, which is highly sensitive to
Detection) was able to distinguish between controls and samples changes in pH or redox potential. With a loop-full of bacteria (10
positive for S. aureus, S. pneumoniae, Haemophilus influenzae, µl) from a fresh plate as a sample, this home-made inexpensive
and P. aeruginosa in upper respiratory tract infections (Lai instrument could detect carbapenem resistance in <35 min
et al., 2002). Gas chromatography connected to ion mobility displaying 95% sensitivity and 100% specificity in comparison
spectrometry (GC-IMS E-nose) could reliably distinguish to PCR-based analysis (Bogaerts et al., 2016). Mecklenburg
bacterial infections from viral respiratory tract infections (Lewis et al. developed an assay that directly detects the thermal signal
et al., 2017). Saviauk et al. performed an applicability test for generated from the enzymatic breakdown of antibiotics. The
the ChemPro 100i Ion Mobility Spectrometry sensor (Environics system was able to distinguish between penicillinase and metallo-
Inc.). They could discriminate MRSA from MSSA with 83% β-lactamase (Mecklenburg et al., 2017). Its value for clinical work
sensitivity and 100% specificity (Saviauk et al., 2018) and were needs to be evaluated, as it requires pure cultures and does not
able to identify also other pathogens (P. aeruginosa, Enterococcus, provide pathogen ID.
E. coli, and Clostridium perfringens) from culture plates with 78% A variety of electrochemical reporters for cell viability
accuracy. In order to access the AST market, the eNose systems have been applied to viability analysis and drug susceptibility
should outperform the simple and inexpensive colorimetric measurements. The system by Besant et al. uses resazurin dye
sensor array system “Reveal-AST” (Specific Technologies, USA) (an oxidation-reduction indicator) to monitor cells trapped
which is applicable to growth-based AST. in nanoliter wells (Besant et al., 2015). Within 1 h the
microfabricated device could detect the response of E. coli and
NAAT in Growth Monitoring K. pneumoniae exposed to ampicillin and ciprofloxacin in urine
Real-time qPCR can detect quantitative differences between samples spiked with bacteria in concentrations as low as 1 cfu/µL.
cultures exposed to various antibiotics and different The level of commercialization of this technology is not known.
concentrations. Already a 15 min cultivation can provide In microelectromechanical systems (MEMS) the deflections
a detectable increase of nucleic acids (Schoepp et al., associated with the micromotions of bacteria attached to a
2017). Although qPCR devices are expensive and require microcantilever provide a signature of bacterial metabolism. Such
experienced personnel for their operation, low-cost devices changes can indicate growth long before the bacteria replicate.
based on isothermal amplification might change the game With bacteria captured in bi-material microchannel cantilevers,
thoroughly. Applying chip electronics and microfluidics, quantitative antibiograms have been obtained for E. coli and
CalTech (the Technical University of California) has developed S. aureus within 2 h (Etayash et al., 2016). The bacteria absorb
a device applicable to AST. This system is currently under infrared photons and release heat to the support matrix by a
commercialization by SlipChip Corp and will be described closer process of vibrational energy relaxation, inducing bending of the
later in this review in the section “POC-compatible technology.” bimetallic cantilever proportional to the quantity of the released
energy. High sensitivity, corresponding to a single cell per µl,
Biosensor Systems was obtained with Listeria-containing samples. The researchers
Biosensors are devices that measure biological or chemical plan to further integrate sample separation techniques into this
reactions by generating signals proportional to the concentration BioMaterial Cantilever platform. LifeScale Analytics (NC, USA)
of an analyte in the reaction. Exposure to antibiotics causes has already launched a commercial product which correlates
detectable changes in bacterial membranes, morphology, cantilever vibration to biomass for MIC determination (Burg
metabolism, movements, mass, heat production and nucleic et al., 2007). This system performs automated cell counting,
acid content. In microcalorimetry approaches, heat production mass measurement, and visual observation of liquid samples for
correlates with the number of cells arising over time (von AST in <3 h, but requires cell concentrations above 104 cells/ml.
Ah et al., 2009). This approach is applicable to both solid The company has not yet presented peer-reviewed clinical AST
and liquid cultures (Howell et al., 2012). Dynamic heat flow studies for this instrument. Micromotions are affected by flowing
patterns have served species identification from urine samples liquids, and inefficient transfer of antibiotics to immobilized
(Bonkat et al., 2012). Isothermal microcalorimetry revealed bacteria can distort the results. Therefore pre-enrichment and
vancomycin-resistant Staphylococcus aureus in <8 h (Entenza pre-purification of bacteria may be necessary (Li et al., 2017b; Syal
et al., 2014). Butini et al. applied isothermal microcalorimetry et al., 2017).
A low-cost and rapid biosensor has been developed employing dried antibiotics. Following on-chip cultivation, the channels
photoluminescence emission of photo-corroded GaAs/AlGaAs can be monitored with a detection device such as a phase
biochips. Growing bacteria protect the biosensor surface contrast microscope. Some systems use agar to immobilize the
against photo-corrosion, while non-growing or dead cells give bacteria and to form growth chambers. Also gradient-forming
a higher signal. These biochips exposed to a E. coli and microfluidic platforms have been applied (Luka et al., 2015;
Legionella pneumophila cultures (w/o antibiotics) were capable Campbell et al., 2016). He et al. introduced a chip applying
of quantifying electrically charged bacteria in 4.5 h (Nazemi et al., antibody-coated glass beads to capture E. coli O157 and to
2017). provide fluorescence detection for ID and AST. Their device had
The Field Effect Enzymatic Detection (FEED) biosensor an integrated antibiotic release system, which could identify this
platform can detect extremely low bacterial concentrations strain at a range of 104 -108 cfu/ml within 30 min (He et al.,
(below 10 c.f.u./ml). An electrical field between the working 2014). Li et al. integrated antibody-coated carbon nanotubes with
electrode and the immune complex multiplies the biocatalytic isothermal amplification. The carbon nanotube multilayer could
output current, enabling a direct detection of bacteria without perform selective capture, cultivation and release of the bacteria.
sample processing (Shi et al., 2018). These biosensors apply After cultivation the antibody-entrapped bacteria were lysed, and
horse radish peroxidase (HRP) as a redox source in the their DNA was accurately quantified by LAMP. This system could
sandwich hybridization complex. Commercial devices detect E. coli O157:H7 and its toxin at concentrations as low
applying this technology for AST may potentially emerge as 1 cfu/ml without any complicated instrumentation (Li et al.,
in coming years. 2017a). Its use for growth-based AST has not yet been presented.
Researchers at Hong Kong Baptist University have developed a
Other Proof-of-Concept Technologies multidimensional AST system for growth-based AST, providing
Flow cytometry (FC) can provide excitation/emission spectra of detection through automated microscopy in 4 h. They established
cells and give information about cell-counts, morphology and a hydrogel microfluidic chip which simulates drug diffusion
viability, enabling AST in 2–3 h. Due to the large amount of raw and pathogen killing processes inside the human body. Due to
data, mathematical methods such as adaptive multidimensional the chip’s multidimensionality, several antibiotics, nutrients or
statistics must be applied for analysis (Huang et al., 2015). immunologic substances could be tested simultaneously. This
Successful demonstrations with clinical polymicrobial samples system is currently under commercialization (Sun et al., 2016;
are currently missing. Flow cytometric assays struggle with Liu et al., 2017a,b). Weibull et al. have presented a nanowell
complex patient samples, inefficient staining, the presence of AST device capable of real-time optical reading and growth
autofluorescence, the inability to differentiate cellular damage data analysis. This “stationary nanoliter droplet array” (SNDA)
for the influence of antibiotics, and lack of clinical databases for system can provide ID and AST for urine samples within one
validation. Flow cytometry instruments are very costly, making working shift. A filtering process first isolates bacteria from the
this technology an unlikely candidate for POCT. Atomic force clinical urine samples into a growth medium, supplemented
microscope (AFM) allows real time monitoring of bacterial with 10% resazurin (oxidation-reduction indicator). Applying
activity. This principle is applicable to both cultivable and 12 bacteria–antibiotic combinations precise MIC determinations
non-cultivable cells (Longo et al., 2013), but is an expensive could be obtained in 3 h, which is 6-fold faster than traditional
and tedious system for clinical work. Plasmonic imaging and broth microdilution assays in 96-well plates. However, follow-up
tracking (PIT) can be used for the monitoring of nanometer- studies with clinical isolates of both Gram-negative and Gram-
scale motions of single bacterial cells before and after antibiotic positive bacteria are needed (Weibull et al., 2014). Veses-Garcia
addition. The observed image contrast fluctuations were found et al. applied 0.5 µl cultures for uropathogens on a 672-nanowell
to indicate changes in bacterial metabolism long before cell slide equipped with optical signal analysis. They were able to
replication (Syal et al., 2016). Asynchronous magnetic bead define a precise minimum inhibitory concentration for 70 clinical
rotation (AMBR) is a non-microscopy-based approach capable E. coli isolates. Algorithm-assisted optical analysis determined
of monitoring individual cells for elongation, generation time, antibiotic susceptibility in 3 h 40 min, showing a total categorical
lag time, division, as well as sensitivity to antibiotics. It has agreement of 97.9% (Veses-Garcia et al., 2018).
been successfully tested with E. coli attached to anti-E. coli The “Integrated Comprehensive Droplet Digital Detection”
functionalized beads (Kinnunen et al., 2011). This system is (IC 3D) system is capable of detecting bacteria directly from
still at the research stage and a complex technology regarding diluted blood within 1.5–4 h. It consists of bacteria-specific
handling, setup and the need for expertise (Schumacher et al., DNAzyme-based sensors, a droplet microencapsulation system,
2018). lysozyme and a 3D particle counter system. The ongoing
work aims to develop an automated, portable device for
multiplexed and rapid detection of antibiotic-resistant strains
MINIATURIZED AND CHIP-BASED (Kang et al., 2014). The Ultrafast Parallelized Microfluidic
GROWTH MONITORING SYSTEMS Platform consists of four arrays (each holding 110 pL droplets
with 1–4 bacteria) which are screened by dynamic imaging over
Lab-on-a-chip systems combine one or several laboratory 2 h. This imaging-based AST was successfully tested with four
functions into a single integrated circuit. The microfluidic types of pathogens causing urinary tract infection (UTI) (Kang
AST platforms typically apply channels containing pre-loaded et al., 2019).
POCT-COMPATIBLE DEVICES FOR ID AND loop-mediated isothermal amplification) or dPCR (droplet-PCR)

AST is applied. The changes in DNA concentrations (control vs.
antibiotic-treated samples) were determined by a “digital single-
Systems with a low price and high speed are needed in molecule counting” system after incubation. In dLAMP, the
all healthcare sectors, but especially in outpatient clinics target molecules or lysed cells are partitioned into thousands of
and in developing countries. Point-of-Care Tests (POCT) nanodroplets so that each compartment contains approximately
could bring the diagnostics into outpatient clinics and thus a single molecule (Schoepp et al., 2017). The 1 nl droplets with
facilitate evidence-based medication in places where extensive DNA concentrations relevant to clinical urine tract infection
prescription of antibiotics happens. Very few rapid AST samples showed 1.23-fold difference in amplification products
systems are currently available, but the ongoing development between resistant and susceptible strains, and 98.1% of the
is promising. David Boyle has made an excellent review of tested samples matched the standard AST results. The LAMP
affordable technologies and devices applicable to “standard chemistry was optimized and applied to a SlipChip microfluidic
microscopy stations” (Boyle, 2017). The devices presented below device equipped with an electrophoretic system concentrating
can provide a fast pathogen identification. Some of these are the bacteria in the sample. This concept was tested for AST
already now applicable to AST, and some devices can be even of positive blood cultures. The results were consistent with
applied to diagnostics of non-infectious diseases as well. standard microdilution tests or the BD Phoenix System when
Scanogen Inc. (USA) develops DNA-based (non- several broad-spectrum antibiotics and clinical E. coli samples
amplification-based) “single-molecule biosensors” bound to as well as the S. aureus ATCC 6538 strain were tested (Yi
microparticles, which can convert the hybridization signal to an et al., 2019). The Talis Biomedical Corporation (CA, USA)
optical signal. The device contains an inexpensive and low-power is currently commercializing dAST/SlipChip technology to a
light-emitting diode ring and uses disposable sample cartridges, product which combines a single-use cartridge integrating fluid
abolishing the need for manual sample preparation. The partitioning for parallel treatment with different antimicrobials,
company is currently developing diagnostic assays for infectious reagent additions, target lysis, extraction and amplification. The
diseases and drug resistance. The MicrobeDx technology NAAT-based system presented by Priye et al. (2017) applies
(MicrobeDx Inc., CA, USA) applies a transformational ribosomal a reverse-transcription loop-mediated isothermal amplification
RNA-based assay on a microfluidic disc platform. The disc holds (RT-LAMP) coupled with the QUASR technique (quenching of
150 nucleic acid capture probes spotted onto a glass slide. This unincorporated amplification signal reporters). The device was
system can discriminate four clinically relevant Staphylococcus found to be five times more sensitive than traditional POCT
species that differ by a single nucleotide polymorphism for the detection of the dengue, chikungunya, or Zika viruses.
(SNP) in diagnostic probe sequences. The protocol includes Hassibi et al. have presented a fully integrated, miniaturized
hybridization, washing, rinsing, and drying steps and does semiconductor biochip with closed-tube detection chemistry
not require purification of the target nucleic acids (Peytavi, for multiplex NA amplification and sequence analysis, which
2005). In a clinical testing phase financed by NIH during the they claimed to have a high dynamic quantification range
years 2018-2019 (so far unpublished), MicrobeDx has tested for the microbial load, while at the same time performing
UroLogic, a highly automated instrument and cartridge system comprehensive mutation analysis on up to 1,000 sequences or
that performs rapid pathogen ID and AST in 30 and 150 min, strands simultaneously in <2 h. This chip was able to correctly
respectively. The Alveo platform (Alveo Technologies Inc, detect and quantify multiple DNA and RNA respiratory viruses
CA) applies hybridization-based electrochemical nucleic acid in clinical samples, while at the same time detecting 54 drug-
detection in single-use cartridges. The cloud-connected device is resistance-associated mutations in six genes of Mycobacterium
capable of analyzing 100 infectious diseases. Sample preparation, tuberculosis (Hassibi et al., 2018). QuantuMDx Ltd (UK) aims
nucleic acid amplification, and real-time hybridization-based to launch a miniaturized portable diagnostic platform that
detection are performed in a single isothermal microfluidic runs on battery power. This Q-POCTM device would be a
channel. This concept has not yet been applied to growth-based portable molecular diagnostic instrument with built-in NA
AST. The LoopampTM technology by Eiken Chemical Corp. amplification for multiplexed diagnostics and drug susceptibility
(China) has already received WHO endorsement for tuberculosis testing within 20 min. It applies nanowires coated with specific
diagnostics (Noncommercial culture drug-susceptibility testing probes for the detection of genetic variants and has cloud-
methods for screening patients at risk for multidrug-resistant based connectivity to share and utilize epidemiologic data. This
tuberculosis: policy statement, 2011). The Loop-mediated technology is currently under clinical evaluation for diagnostics
isothermal amplification (strand displacement reaction) employs of malaria (protozoan Plasmodium falciparum) and tuberculosis
four different primers designed for six distinct regions on the (Mycobacterium tuberculosis). The device, although not designed
target gene. It has been used successfully for the detection of for growth-based AST, might have a very wide applicability
Neisseria meningitidis in clinical cerebrospinal fluid samples (Lee range including non-infectious diseases such as cancer or genetic
et al., 2015) and for the detection of the OXA-23 carbapenemase disorders. The outcome of the clinical tests has not yet been
(Yang et al., 2018) gene of Acinetobacter baumannii. published. The Multipath instrument (First Light Diagnostics
Schoepp et al. (2017) demonstrated with 51 clinical samples Inc., USA) applies fluorescence-labeled nanoparticles attaching
that the antibiotic exposure time in phenotypic AST can to the target cells by immunobinding. The nanoparticle/cell
be shortened to 15 min when dLAMP (digital real-time complex can then be pulled toward the detection system
by magnetic beads conjugated with another microbe-specific benefit from methods concentrating the cells and removing the
antibody. Thereby the system is capable of monitoring viable cells impurities. The FISH-based Accelerate Pheno system applies
using non-magnified digital imaging. A POCT-compatible device electrokinetic/electrophoretic system for these purposes. Coarse
is currently under testing. Immunodiagnostic TPX-technology cell sorting for blood samples can be achieved through simple
(two-photon excitation fluorometry), commercialized by ArcDia inertial microfluidic systems, where centrifugal (inertial) force
Ltd (Finland) to mariPOC and mariAST platforms, can analyze drives bacteria to the outer side of a spiral-formed microcapillary
40–100 samples a day. It has a cloud-based connectivity allowing tube, while blood cells stay on the inner side (Bhattacharyya
efficient sharing of epidemiologic data. TPX allows the use et al., 2017). Some systems bypass the problem of impurities by
of untreated polymicrobial clinical samples on-line, providing bringing the target close to the biosensor surface with magnetic
bacterial ID in 20 min and antibiotic resistance in a few hours. and antibody-coupled beads or nanoparticles. Miniaturization
Unlike most other AST systems, it can also provide sensitive sets high demands to the standardization of the conditions, as
immunodetection of viruses Sanbonmatsu-Gámez et al., 2015; the samples should represent similar growth states and culture
Bruning et al., 2018. densities. Robots may be needed for accurate pipetting.
POCT devices using smartphone technology have long been Serious doubts concerning the ability of accelerated cultures to
used for personalized medication applications such as the fully substitute standard growth-based tests have been presented.
colorimetric analysis of urine strips (Barnes et al., 2018). The use Accelerated cultivations may struggle to differentiate between
of smartphones in growth-based AST for miniaturized cultures wild type and resistant strains (Leclercq et al., 2013; Kahlmeter,
has been presented in several studies (Kadlec et al., 2014; Schoepp 2014; Maurer et al., 2017). This is especially a concern in the
et al., 2017; Cui et al., 2018; Hernández-Neuta et al., 2019), case of induced expression of the resistance factors, e.g., in the
although instrument manufacturers seem to be reluctant to detection of AmpC in enterobacteria or macrolide-resistance in
incorporate standard commercial smartphones to their systems. streptococci (Leclercq and Courvalin, 2002; Jacoby, 2009; Harris
Photolithography, plotting, plasma etching, inkjet printing, and Ferguson, 2012).
cutting, and wax printing can be used to pattern paper for
inexpensive diagnostic tests (López-Marzo and Merkoçi, 2016).
Paper-based systems have been applied for the immunodetection ECONOMIC DRIVERS FOR RAPID AST
of Helicobacter pylori, Chlamydia infections and Zika virus
infections, but platforms integrating LF-testing into growth- Substantial overall savings can be obtained with rapid AST
based AST have not been presented. through the reduction of hospital days, disability days and the
saving of lives (Cassini et al., 2019). Despite the potential total
cost savings, the high price of molecular testing forms an efficient
PROBLEMS WITH MINIATURIZATION AND barrier for the installation of new technologies. A price tag
RAPID AST ranging from $100 to $250 per test is common for molecular
tests (Li et al., 2017b). For a typical 500-bed community hospital,
Due to the small sample size (at lowest a single bacterial detailed multiplex testing of positive blood cultures by NAAT
cell), rapid AST may not give results representing the whole could cost more than $500,000/year in reagents alone (She and
bacterial population in the diagnostic sample. This problem can Bender, 2019). Also the instrument prices may be significant.
be mitigated by analyzing a large number of individual cells. The total costs of testing are quite difficult to estimate. The
An example of such a strategy is the stochastic confinement prices of test kits and diagnostic instruments are based on
of bacteria in nanoliter droplets, so that a high number of the distributors’ offers. Testing costs include clinical sampling
individual droplets can be analyzed quickly and efficiently and usually also cultivations in microbiology labs (enrichment,
(Boedicker et al., 2008). The direct analysis of clinical samples preparation of bacterial isolates) before AST can be performed.
also demands the use of valid internal standards. Additionally As an example, Patel et al. estimated the total costs for mass
any growth-based analysis may utterly fail if the growth rate spectrometry-based AST to be roughly 79 e per patient, when
is very low or the microbes cease to grow due to the lack the cost of the MALDI-TOF device, reagents, pharmacist time
of specific growth factors, a non-optimal atmosphere, or the and the antimicrobial stewardship program are pooled together
accumulation of growth-inhibiting substances. For intracellular (Patel et al., 2017). Yet the reagent costs for one MS-sample can
pathogens the use of NAAT may be necessary. Compared to be pressed close to 1 e. A typical MALDI-TOF MS system with
standard microbial cultivations, favorable growth conditions may all the accessories, software databases and maintenance is costly
be difficult to establish for miniaturized set-ups. These problems (up to e200,000 per year) (Wieser et al., 2012). This implies that
can be mitigated by analyzing cell viability, morphology or the use of the instrument must be high for an acceptable cost
movements. Miniaturization can improve the performance and efficiency. This unfortunately rules out their use in outpatient
capacity of cultivation-based AST only if the need for manual clinics. MS-instruments are, however, very versatile and can be
steps is minimized. As reagent dilutions are often done before applied also to other routine diagnostics in central laboratories
loading onto the chip device, set-up complexity may stay a (Vrioni et al., 2018). Direct inoculation from plates to automated
similar level as in broth dilution methods. Due to the relatively identification systems such as Vitek, Microscan and others has
low number of pathogens in most clinical samples and the been validated and used in many clinical laboratories. These
impurities of the sample matrices, miniaturized cultivations systems apply multiwell liquid cultures and have a turnaround
time as short as 4 h for ID and 6–8 h for AST (She and Bender, While central laboratories have resources to validate new tests
2019), but the prices of these instruments are high. The cost against the standardized tests, smaller healthcare units are bound
of any rapid AST should not significantly exceed the cost level to use tests validated by authorities such as the FDA. The few
currently considered acceptable for routine testing: this might systems currently capable of providing simultaneous ID and
be roughly 30 to 50 e, including sampling, culturing and AST growth-based AST directly from clinical samples are based either
(EU, 2013). Five phenotypic tests for a targeted detection of on FISH (Accelerate Pheno), immunodiagnostics (e.g., mariAST,
enterobacterial carbapenemases have been recently evaluated for MultiPath, immunobiosensors), or digital AST (dAST) (Table 1).
their performance and costs. All these tests required the use As their properties and application areas differ, all these systems
of culture isolates. Per sample, the costs of multiplexed PCR- may have a good future. Rapid growth-based diagnostic systems
based analysis was 30 e, immunochromatographic methods rely on accelerated cultivations. For this reason, it is necessary to
∼15 e, a colorimetric assay 5 e and carbapenem hydrolysis validate every new technology carefully against the standard tests
test 1 e per sample (Baeza et al., 2019). Apart from turnover accepted by EUCAST and CLSI. The MIC breakpoints (sensitive,
time, also the capacity of the test system is important. Among resistant, or in-between) must be re-defined for each system
automated microscopy systems, Accelerate Pheno handles one and each tested organism. This requirement definitely retards
sample per unit module (max. four modules per device, cost per the deployment of new technologies. While most novel AST
sample ∼250 e), whilst the Q-Linea ASTar system can handle systems only perform an endpoint analysis, the systems based
50 samples per day. The capacities of NAAT-based systems can on microscopy, heat production, movements, immunodetection
be even higher. Automated MIC-determination systems such as or detection of mass changes in principle facilitate on-line
VITEK-2 can manage tens of samples per time. For POCT usage, monitoring. Biosensor-based AST systems, however, yet miss
however, a throughput capacity of a few tens of samples per time convincing clinical demonstrations.
would be sufficient. In practice clinical diagnosis should be obtained during office
hours, meaning <8 h from sampling to results. This does not
leave time for enrichment cultivations or preparation of culture
SUMMARY: HOW DO THE AST isolates. The strategy “take a sample, store and analyze later”
TECHNOLOGIES MEET THE works fine for diagnosis of slowly advancing infections, but also
REQUIREMENTS? for NAAT-based systems which do not require viable microbes.
Growth-based AST, on the other hand, need fresh samples to
Several identification systems based on NAAT, NA hybridization ensure pathogen survival until the point of analysis.
and immunodiagnostics are already available for rapid Regarding the speed and the need to handle clinical
diagnostics, but only few are applicable to POCT. This is polybacterial samples, the immunodiagnostic TPX-technology
due to high costs, lack of appropriate facilities or expert (ArcDia Ltd), growth-based FISH (Accelerate Pheno), the
labor, or insufficient performance with clinical samples. Multipath digital imaging technology based on nanoparticles
Multiplexing and high-throughput capacities are important for labeling and magnetic beads for capturing (First Light
for central laboratories, but most healthcare settings process Diagnostics Inc.), and the NAAT-based dAST (Talis Inc.)
only few samples per time. POCT use rules out expensive seem promising options for rapid point-of-care testing of
devices, systems requiring demanding sample preparation, and antimicrobial susceptibility. The Talis system is, however, not yet
systems requiring standard microbiology laboratory facilities. on the market. Immunobiosensors are still in their infancy, but
NAAT serves well for the identification of viral and bacterial may in the future become important in testing non-culturable
pathogens, and in some cases provides also the detection of microbes. Lateral Flow (immuno-chromatographic “dip-sticks”)
AR genes. However, the commercial multiplexed diagnostic systems would be an ideal product format for clinical work,
NAAT panels are far too expensive for routine diagnostics. but their applicability has been so far proven only with isolated
Immunochromatographic tests, especially dip-sticks, are a cultures (i.e., colonies on plate).
low-cost and handy option for virus diagnostics and detection Due to the plethora of different resistance mechanisms, NAAT
of inflammatory factors. Unfortunately systems capable of struggles with the detection of antibiotic resistances in Gram-
detection of antibiotic resistance proteins directly from clinical negative bacteria (Maurer et al., 2017) and with the analysis
samples without enrichment cultures, culture isolation or sample of samples containing commensal flora. NAAT may fail to
purification do not yet exist. The routine diagnostic tools for identify several ESBL genes and genes providing fluoroquinolone
infectious diseases should serve a wide field of requirements: or aminoglycoside resistance. NAAT is, however, a powerful
both the identification of the pathogen (viral and bacterial), and necessary technology for the detection of fastidious, slow-
the detection of antibiotic resistance and the determination of growing or intracellular pathogens, toxin-producing bacteria
the correct antibiotic dosing. Systems based on microscopy are such as E. coli O157:H7, and viruses (Miller et al., 2018). Still,
not compatible with the detection of viruses or inflammatory the increased use of NAAT has raised a concern about the fate
factors. Diagnostic systems differ very much in their potential to of bacterial samples required for further studies (Marder et al.,
provide all relevant data for medication (Table 1). Multiplexing 2017; McAdam, 2017). Since NAAT does not require viable
NAAT-based systems can seamlessly incorporate the detection samples, subsequent epidemiologic studies or cultivation-based
of emerging resistance-related genes or mutations. However, confirmatory AST may be impossible with the stored samples.
every update for a diagnostic panel requires new validations. Whole genome sequencing (WGS) is still in its infancy regarding
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
Vasala et al.
TABLE 1 | Properties of technologies applicable to rapid identification and AST.
Technology Company or Time for AST (h) Simultaneous ID Clinical Online AST Provides Detects new AST for non- Enables Level of References
product and AST polymicrob. MICs resistances culturable virus ID commercialization
(examples) samples microbes
Standard cultivation tests

Broth dilution test Several 18–36 – – X X X – – Gold standard
Disk diffusion test and Several 18–24 – – X X X – – Gold standard
E-test
Automated readers for cards or microtiter plates
Broth bioMerieux, BD, 5–16 h – – X X X – – Commercial
microdilution-based Siemens
instruments
Disk diffusion-based Giles Scientific, 5–16 h – – X X X – – Commercial
instruments Oriana, BioRad,
BD
Mass spectrometry (biochemical profiling, follow-up of antibiotic degradation, detection of anti-microbial protein)
MALDI-TOF (Bruker Bruker, Shimadzu, 2-4 h – – – – X – – Commercial
MBT) Sciex, Waters
MBT-ASTRA (biochem. Bruker Daltonik 2-4 h – – – X X – – Commercial Sparbier et al.,
profiling after antibiotic GmbH 2016
exposure)
14
Direct-On-Target All instrument 4h – – – X X – – Experimental Idelevich et al.,

Microbial Growth Assay providers 2018;
(DOT-MGA) Correa-Martínez
et al., 2019
Fluorescence & hybridization
FISH (fluorescent XpressFISH 2–4 h X X – – X – – Commercial Salimnia et al.,
probes, microscope) 2014
Multiplexed automated Accelerate 6.5 h X X X(fs) X X – – Commercial Hill et al., 2017
microscopy/FISH Diagnostics
Automated NanoString 24 h X X X(fs) X X – X Commercial, under Barczak et al.,
fluorescence detection Technologies testing 2012;
for expression profiling Bhattacharyya
Rapid Diagnostics of Antimicrobial Resistance

et al., 2017, 2019;
Kelley, 2017;
Koehler et al.,
July 2020 | Volume 10 | Article 308
2018
Non-microscopic First Light 4h X X X X X – X Commercial, under https://www.
imaging, fluorescent Diagnostics testing firstlightdx.com/
antibody-bound publications/
nanoparticles,
magnetic beads for
concentrating
(Continued)
Vasala et al.
TABLE 1 | Continued
Other imaging or spectroscopy-based systems

Surface plasmon Biacore 3000 and 0.5–4h – – X X X X – Experimental Chen et al., 2011;
resonance (SPR) exp. devices Tao and Syal,
2016
Raman spectroscopy Several 2h X – X X X – – Experimental Liu et al., 2016;
(SERS) Wang et al., 2018
Smartphone-based Experimental 2–4 h – – X X X – – Experimental Kadlec et al.,
growth monitoring of 2014; Feng et al.,
microplates, capillaries 2016; Cui et al.,
or chips 2018
Cell sorting systems, flow cytometry
Flow cytometry FASTinov 2h – X X X X (X) – Commercial Costa-de-Oliveira
et al., 2017
Sensor-based detection of micromotions or mass changes
Plasmonic imaging and Experimental <1 h – – X X X X – Experimental Syal et al., 2016
tracking for
nanomotions
15
Atomic force Experimental 0.25-4h – – X (X) X (X) – Inverted microscope Longo et al., 2013
microscopy cantilever
SAW and other mass Experimental 0.5-6h – – X (X) X (X) X Experimental Chang et al.,
sensitive biosensors 2007; Hoß and
Bendas, 2017
Heat production
Microcalorimetry SymCel AB, TA Few hours – – X (X) X (X) – Commercial https://www.
Instruments laboratoryequipment.
com/article/2017/
11/how-use-
calorimetry-tackle-

antibiotic-
resistance
Immunochromatography (lateral flow tests, “dip-sticks”)
Resistance factor Coris Bioconcept 0.25–4 – – – – – – – Commercial ECCMID 2015

specific binders Booth #243
Immunodetection, fluorescence-based
Two-photon ArcDia 2–4 h X X X X X – X Commercial, under Koskinen, 2008
fluorescence testing
microscopy TPX
(Continued)
Vasala et al.
TABLE 1 | Continued
Multipath (magnetic First Light 2–4 h X X X X X – X Commercial, under

beads, Diagnostics testing
non-microscopy
imaging)
Electrochemical biosensors or detection of volatile organic compounds
rRNA-hybridization, GeneFluidics 2–5 h X X – X X (X) – Commercial Mach et al., 2011;
peroxidase signaling Liu et al., 2014
Colorimetric sensor Specific 3–4 h X – X X X – – Commercial https://www.
array for VOC detection Diagnostics specific-dx.com/
reveal-ast
Redox-indicator Experimental 1h – – X X X (X) – Experimental Besant et al.,
resazurin 2015; Avesar
et al., 2017
16
Field effect enzymatic Experimental 1–2 h X X X X X (X) – Experimental Shi et al., 2018
immunosensor
Electronic nose: ion Environics, Few minutes (X) – (X) – – – – Commercial Lewis et al., 2017;
mobility spectrometry Olfactomics Saviauk et al.,
sensor 2018
NAAT
PCR, qPCR Several 2–4 h X X – – – X X Commercial
Integrated Several 4h X X – – – X X Commercial
cassette-based NAAT
solutions
Isothermal amplification Several 0.5–4 X X – – – X X Commercial

Whole Genome Several 1–24 h X X – – X X X Commercial
Sequencing
NAAT combined to cultivation
Isothermal Talis Biomedical 0.5 h X X X(fs) X X (X) X Under Schoepp et al.,

amplification, digital commercialization 2017, 2020;
AST https://talis.bio
The marking X is in brackets, if the possible feature lacks experimental demonstrations. – indicates a missing property. fr indicates the need for frequent sampling.
TABLE 2 | Contemplation on Prof. Kahlmeter’s criteria for new technologies (Kahlmeter, 2016).
Criteria Contemplation
Generally applicable or restricted to certain infections? In principle all growth-based rapid AST systems are generic and work with culture isolates.
However, for polymicrobial clinical samples they must be coupled with specific probes or
antibodies which provide ID. Therefore, specific test panels have been developed e.g., for
respiratory, urinary, and blood samples. The pathogen load may not be high enough for
direct analysis, and especially blood samples may require culturing prior to analysis. AST
for fastidious, non-culturable, or intracellular pathogens call for NAAT. The complexity of
the sample matrix affects the choice of the diagnostic system and the methods for sample
preparations.
Capacity: how many organisms/agents per hour can be processed DNA-arrays and PCR systems (including multiplexed cassette designs) have a high
throughput capacity. Mass-spectrometry performed on PCR products can handle
hundreds of samples per hour in central laboratories. In outpatient clinics speed is more
essential than the capacity. High multiplexing (parameters per sample) and
high-throughput capacity (number of samples) may be challenging to combine. Progress
in NAAT, immunodiagnostics, biosensor technologies and microfluidics has yielded several
systems capable of analyzing tens of samples per day or even during a single work shift.
Has the technology been validated against reference methods? So far quite few quick technologies have received FDA-approval. Currently they include
PCR-tests, cartridge-based NAAT-systems and Accelerate Pheno (automated
microscopy). Several clinical trials are in progress to achieve CE-marking or
FDA-clearance.
Are there any reference installations? Commercial analysis systems in general do have, and manufacturers tend to publish
successful clinical trials. However, finding a lab which lines up to the specific needs may
be challenging.
Is scientific literature available? For mature commercial systems scientific references can be fairly easily found. For
near-market products this is much more challenging. Companies often only declare
on-going tests, but provide only limited info about the progress. Scientific articles typically
present proof-of-concept level data obtained with isolated cultures spiked into sample
matrices.
When on market? Many systems are already available, but they may have a limited scope for ID/AST. Due to
lack of clinical data, some systems have a “research use only” status. Some have been
accepted only for veterinary use. Commercially mature products include several NAAT
systems, FISH-systems and immunodiagnostic system.
its use for rapid AST. The required bioinformatics is challenging, Sensitive growth monitoring can be achieved either by frequent
and universal open databases are needed to interpret the results. sampling (applicable to disruptive methods like FISH or NAAT)
In the near future, the progress in chip, microfluidics or by on-line immunodiagnostic methods. dAST with chip-
and biosensor technologies may provide new inexpensive AST based microfluidics devices and isothermal amplification can
systems. Integration of many sophisticated technologies will be potentially revolutionize phenotypic AST. With this approach,
needed to resolve problems with a low initial pathogen number thousands of individual single bacterium droplet samples can be
and the presence of contaminating sample matrices. Several categorized according whether the amount of amplified DNA
scientific publications have already demonstrated the successful reaches the limit defined for growing cells. The FISH-based
use of smartphone optics and telecommunication capacity for Accelerate Pheno system has already reached FDA approval.
monitoring of microwell or microcapillary cultivations, pH and The mariPOC device based on immunodetection with two-
redox changes and for delivering the read-outs of biosensor data photon excitation fluoroscopy allows non-disruptive microbial
(Berg et al., 2015; Feng et al., 2016; Cui et al., 2018; Hernández- identification. Its clinical validation for AST should be followed
Neuta et al., 2019). The obvious lack of IPR protection for with interest, as this technology enables the use of non-
smartphone-based analytic devices and the requirement to purified clinical samples and also allows follow-up studies to
validate analytic devices as an entity unfortunately discourages confirm the results. Additionally it already provides a rapid and
commercialization of these technologies. sensitive identification of both bacterial and viral pathogens.
The Multipath technology (First Light Diagnostics Inc.) may
CONCLUSIONS provide a functional platform for automated on-line detection
by applying fluorescent nanoparticles for signaling, magnetic
Standard growth-based technologies based on disc diffusion beads for binding, and non-microscopic imaging for detection.
and broth dilution still dominate in AST. They are slow and Multiplexing cartridge-based NAAT solutions are likely to reach
require pure cultures, but in other aspects serve the purpose a significant customer base in central laboratories, since they offer
well. Only few rapid growth-based AST methods work directly high speed, work well with non-culturable bacteria and viruses
with polymicrobial clinical samples, which is required in POCT. and possess an excellent high-throughput power for pathogen
ID. However, all new emerging technologies struggle to meet processed by all the authors. VH and OL mediated valuable
all of the criteria Prof. Kahlmeter set for AST technologies contacts to the expert biotech scientists and healthcare experts
(Table 2). As none of the presented technologies is optimal in consulted for this review. All authors contributed to the writing
all aspects, it is probable that many of them will reach a large and accepted the final version.
customer base. Therefore the consensus statement of the PIAMR
AMR- RDT Working Group on Antimicrobial Resistance and FUNDING
Rapid Diagnostic Testing is still valid: “There is no single major,
or broadly accepted, technological breakthrough that leads the This work was supported by the Business Finland project
field of rapid AST platform development” (van Belkum et al., DARE (Dnro 2769/31/2018), which evaluated the current
2019b). and emerging technologies for AST and the possibilities
to develop new innovative products for AST testing. The
AUTHOR’S NOTE authors declare that this study received funding from
Fimlab Laboratories. The funder was not involved in the
The described technologies have been taken into consideration study design, collection, analysis, interpretation of data,
without any pre-selection and have been judged only by their the writing of this article or the decision to submit it
applicability to clinical diagnostics. for publication.
AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS

AV was the main responsible for the acquisition and structuring We thank to Dr. Pierre Bogaerts, who pointed out which practical
of the scientific literature. The text and conclusions were issues are important in clinical diagnostics of microbial diseases.
REFERENCES Besant, J. D., Sargent, E. H., and Kelley, S. O. (2015). Rapid electrochemical
phenotypic profiling of antibiotic-resistant bacteria. Lab. Chip. 15, 2799–2807.
Almeida, C., Azevedo, N. F., Iversen, C., Fanning, S., Keevil, C. W., doi: 10.1039/C5LC00375J
and Vieira, M. J. (2009). Development and application of a novel Bhattacharyya, R., Liu, J., Ma, P., Bandyopadhyay, N., Livny, J., and Hung,
peptide nucleic acid probe for the specific detection of cronobacter D. (2017). Rapid phenotypic antibiotic susceptibility testing through RNA
genomospecies (Enterobacter sakazakii) in powdered infant formula. detection. Open Forum Infect. Dis. 4, S33–S33. doi: 10.1093/ofid/ofx162.082
Appl. Environ. Microbiol. 75, 2925–2930. doi: 10.1128/AEM.02 Bhattacharyya, R. P., Bandyopadhyay, N., Ma, P., Son, S. S., Liu, J., He, L. L.,
470-08 et al. (2019). Simultaneous detection of genotype and phenotype enables rapid
ARUP (2020). Scientific Resource for Research, and Education: Educational and accurate antibiotic susceptibility determination. Nat. Med. 25, 1858–1864.
Resources - Rapid Antimicrobial Susceptibility Testing | University of Utah. doi: 10.1038/s41591-019-0650-9
Available online at: https://arup.utah.edu/education/fisher-rapidAST-2019. Boedicker, J. Q., Li, L., Kline, T. R., and Ismagilov, R. F. (2008). Detecting bacteria
php/ (accessed May 2, 2020). and determining their susceptibility to antibiotics by stochastic confinement
Athamanolap, P., Hsieh, K., Chen, L., Yang, S., and Wang, T.-H. (2017). in nanoliter droplets using plug-based microfluidics. Lab. Chip 8, 1265–1272.
Integrated bacterial identification and antimicrobial susceptibility testing doi: 10.1039/b804911d
using pcr and high-resolution melt. Anal. Chem. 89, 11529–11536. Bogaerts, P., de Castro, R. R., de Mendonça, R., Huang, T. D., Denis, O.,
doi: 10.1021/acs.analchem.7b02809 and Glupczynski, Y. (2013). Validation of carbapenemase and extended-
Avesar, J., Rosenfeld, D., Truman-Rosentsvit, M., Ben-Arye, T., Geffen, spectrum β-lactamase multiplex endpoint PCR assays according to ISO 15189.
Y., Bercovici, M., et al. (2017). Rapid phenotypic antimicrobial J. Antimicrob. Chemother. 68, 1576–1582. doi: 10.1093/jac/dkt065
susceptibility testing using nanoliter arrays. PNAS. 114, E5787–E5795. Bogaerts, P., Yunus, S., Massart, M., Huang, T.-D., and Glupczynski, Y. (2016).
doi: 10.1073/pnas.1703736114 Evaluation of the BYG carba test, a new electrochemical assay for rapid
Baeza, L. L., Pfennigwerth, N., Greissl, C., Göttig, S., Saleh, A., Stelzer, Y., laboratory detection of carbapenemase-producing enterobacteriaceae. J. Clin.
et al. (2019). Comparison of five methods for detection of carbapenemases in Microbiol. 54, 349–358. doi: 10.1128/JCM.02404-15
enterobacterales with proposal of a new algorithm. Clin. Microbiol. Infect. 25, Bonkat, G., Braissant, O., Widmer, A. F., Frei, R., Rieken, M., Wyler,
1286.e9–1286.e15. doi: 10.1016/j.cmi.2019.03.003 S., et al. (2012). Rapid detection of urinary tract pathogens using
Barany, F. (1991). Genetic disease detection and DNA amplification using microcalorimetry: principle, technique and first results. BJU Int. 110, 892–897.
cloned thermostable ligase. Proc. Natl. Acad. Sci. U.S.A. 88, 189–93. doi: 10.1111/j.1464-410X.2011.10902.x
doi: 10.1073/pnas.88.1.189 Boutal, H., Naas, T., Devilliers, K., Oueslati, S., Dortet, L., Bernabeu, S., et al. (2017).
Barczak, A. K., Gomez, J. E., Kaufmann, B. B., Hinson, E. R., Cosimi, L., Development and validation of a lateral flow immunoassay for rapid detection
Borowsky, M. L., et al. (2012). RNA signatures allow rapid identification of NDM-producing enterobacteriaceae. J. Clin. Microbiol. 55, 2018–2029.
of pathogens and antibiotic susceptibilities. PNAS. 109, 6217–6222. doi: 10.1128/JCM.00248-17
doi: 10.1073/pnas.1119540109 Boyle, D. (2017). Unitaid TB Diagnostics - NAAT for Microscopy Stations.
Barnes, L., Heithoff, D. M., Mahan, S. P., Fox, G. N., Zambrano, A., Available online at: http://unitaid.org/assets/2017-Unitaid-TB-Diagnostics-
Choe, J., et al. (2018). Smartphone-based pathogen diagnosis in urinary Technology-Landscape.pdf (accessed March 8, 2019).
sepsis patients. EBioMedicine 36, 73–82. doi: 10.1016/j.ebiom.2018. Bruning, A. H. L., Aatola, H., Toivola, H., Ikonen, N., Savolainen-Kopra,
09.001 C., Blomqvist, S., et al. (2018). Rapid detection and monitoring of
Berg, B., Cortazar, B., Tseng, D., Ozkan, H., Feng, S., Wei, Q., et al. human coronavirus infections. New Microbes and New Infect. 24, 52–55.
(2015). Cellphone-based hand-held microplate reader for point-of-care doi: 10.1016/j.nmni.2018.04.007
testing of enzyme-linked immunosorbent assays. ACS Nano 9, 7857–7866. Bugrysheva, J. V., Lascols, C., Sue, D., and Weigel, L. M. (2016). Rapid
doi: 10.1021/acsnano.5b03203 antimicrobial susceptibility testing of bacillus anthracis, yersinia pestis, and
burkholderia pseudomallei by use of laser light scattering technology. J. Clin. Curtis, K. A., Rudolph, D. L., and Owen, S. M. (2008). Rapid detection
Microbiol. 54, 1462–1471. doi: 10.1128/JCM.03251-15 of HIV-1 by reverse-transcription, loop-mediated isothermal amplification
Burg, T. P., Godin, M., Knudsen, S. M., Shen, W., Carlson, G., Foster, J. S., et al. (RT-LAMP). J. Virol. Methods 151, 264–270. doi: 10.1016/j.jviromet.2008.
(2007). Weighing of biomolecules, single cells and single nanoparticles in fluid. 04.011
Nature 446, 1066–1069. doi: 10.1038/nature05741 Delport, J. A., Mohorovic, I., Burn, S., McCormick, J. K., Schaus, D., Lannigan,
Butini, M. E., Gonzalez Moreno, M., Czuban, M., Koliszak, A., Tkhilaishvili, R., et al. (2016). Rapid detection of meticillin-resistant staphylococcus
T., Trampuz, A., et al. (2018). Real-time antimicrobial susceptibility assay of aureus bacteraemia using combined three-hour short-incubation matrix-
planktonic and biofilm bacteria by isothermal microcalorimetry. Adv. Exp. assisted laser desorption/ionization time-of-flight MS identification and
Med. Biol. 1214, 61–77. doi: 10.1007/5584_2018_291 alere culture colony PBP2a detection test. J. Med. Microbiol. 65, 626–631.
Campbell, J., McBeth, C., Kalashnikov, M., Boardman, A. K., Sharon, doi: 10.1099/jmm.0.000285
A., and Sauer-Budge, A. F. (2016). Microfluidic advances in den Hertog, A. L., Menting, S., Smienk, E. T., Werngren, J., Hoffner,
phenotypic antibiotic susceptibility testing. Biomed. Microdevices 18:103. S., and Anthony, R. M. (2014). Evaluation of a microcolony growth
doi: 10.1007/s10544-016-0121-8 monitoring method for the rapid determination of ethambutol
Cassini, A., Högberg, L. D., Plachouras, D., Quattrocchi, A., Hoxha, A., resistance in Mycobacterium tuberculosis. BMC Infect. Dis. 14:380.
Simonsen, G. S., et al. (2019). Attributable deaths and disability-adjusted doi: 10.1186/1471-2334-14-380
life-years caused by infections with antibiotic-resistant bacteria in the EU Descours, G., Desmurs, L., Hoang, T. L. T., Ibranosyan, M., Baume, M.,
and the European economic area in 2015: a population-level modelling Ranc, A. G., et al. (2018). Evaluation of the accelerate phenoTM system for
analysis. Lancet. Infect. Dis. 19, 56–66. doi: 10.1016/S1473-3099(18)30 rapid identification and antimicrobial susceptibility testing of Gram-negative
605-4 bacteria in bloodstream infections. Eur. J. Clin. Microbiol. Infect. Dis. 37,
Centers for Disease Control and Prevention (2018). Be Antibiotics Aware: Smart 1573–1583. doi: 10.1007/s10096-018-3287-6
Use, Best Care | Features | CDC. Available online at: https://www.cdc.gov/ Doern, C. D. (2018). The slow march toward rapid phenotypic antimicrobial
features/antibioticuse/index.html (accessed May 28, 2019). susceptibility testing: are we there yet? J. Clin. Microbiol. 56, e01999–17.
Cerqueira, L., Fernandes, R. M., Ferreira, R. M., Carneiro, F., Dinis-Ribeiro, doi: 10.1128/JCM.01999-17
M., Figueiredo, C., et al. (2011). PNA-FISH as a new diagnostic method for Ellington, M. J., Ekelund, O., Aarestrup, F. M., Canton, R., Doumith, M., Giske,
the determination of clarithromycin resistance of helicobacter pylori. BMC C., et al. (2017). The role of whole genome sequencing in antimicrobial
Microbiol. 11:101. doi: 10.1186/1471-2180-11-101 susceptibility testing of bacteria: report from the EUCAST subcommittee. Clin.
Ceyssens, P.-J., Soetaert, K., Timke, M., Van den Bossche, A., Sparbier, K., Microbiol. Infect. 23, 2–22. doi: 10.1016/j.cmi.2016.11.012
De Cremer, K., et al. (2017). Matrix-assisted laser desorption ionization- Enroth, H., Retz, K., Andersson, S., Andersson, C., Svensson, K., Ljungström, L.,
time of flight mass spectrometry for combined species identification and et al. (2019). Infectious diseases evaluation of QuickFISH and maldi sepsityper
drug sensitivity testing in mycobacteria. J. Clin. Microbiol. 55, 624–634. for identification of bacteria in bloodstream infection. Infect. Dis. 51, 249–258.
doi: 10.1128/JCM.02089-16 doi: 10.1080/23744235.2018.1554258
Chang, K.-S., Chang, C.-K., and Chen, C.-Y. (2007). A surface acoustic wave Entenza, J. M., Bétrisey, B., Manuel, O., Giddey, M., Sakwinska, O., Laurent, F.,
sensor modified from a wireless transmitter for the monitoring of the growth of et al. (2014). Rapid detection of staphylococcus aureus strains with reduced
bacteria. Sensors Actuat B-Chem. 125, 207–213. doi: 10.1016/j.snb.2007.02.007 susceptibility to vancomycin by isothermal microcalorimetry. J. Clin. Microbiol.
Chantell, C. (2015). Multiplexed automated digital microscopy for rapid 52, 180–186. doi: 10.1128/JCM.01820-13
identification and antimicrobial susceptibility testing of bacteria and Etayash, H., Khan, M. F., Kaur, K., and Thundat, T. (2016). Microfluidic
yeast directly from clinical samples. Clin. Microbiol. Newsl. 37, 161–167. cantilever detects bacteria and measures their susceptibility to antibiotics
doi: 10.1016/j.clinmicnews.2015.10.001 in small confined volumes. Nat. Commun. 7, 1–9. doi: 10.1038/ncomms
Charnot-Katsikas, A., Tesic, V., Love, N., Hill, B., Bethel, C., Boonlayangoor, 12947
S., et al. (2017). Use of the accelerate pheno system for identification and EU (2013). Commission Implementing Decision of, 12 November 2013 as Regards
antimicrobial susceptibility testing of pathogens in positive blood cultures and a Union Financial Aid Towards a Coordinated Control Plan for Antimicrobial
impact on time to results and workflow. J. Clin. Microbiol. 56, e01166–17. Resistance Monitoring in Zoonotic Agents. Available online at: https://eur-lex.
doi: 10.1128/JCM.01166-17 europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32013D0653
Chen, H., Lin, C.-H., Su, C.-Y., Chen, H.-P., and Chiang, Y.-L. (2011). Feng, S., Tseng, D., Di Carlo, D., Garner, O. B., and Ozcan, A. (2016).
Surface Plasmon Resonance Biotechnology for Antimicrobial Susceptibility Test. High-throughput and automated diagnosis of antimicrobial resistance using
Available online at: www.intechopen.com a cost-effective cellphone-based micro-plate reader. Sci. Rep. 6:39203.
Choi, J., Jeong, H. Y., Lee, G. Y., Han, S., Han, S., Jin, B., et al. (2017). doi: 10.1038/srep39203
Direct, rapid antimicrobial susceptibility test from positive blood Francois, P., Tangomo, M., Hibbs, J., Bonetti, E.-J., Boehme, C. C., Notomi,
cultures based on microscopic imaging analysis. Sci. Rep. 7:1148. T., et al. (2011). Robustness of a loop-mediated isothermal amplification
doi: 10.1038/s41598-017-01278-2 reaction for diagnostic applications. FEMS Immunol. Med. Microbiol. 62,
Choi, J., Yoo, J., Lee, M., Kim, E.-G., Lee, J. S., Lee, S., et al. (2014). A rapid 41–48. doi: 10.1111/j.1574-695X.2011.00785.x
antimicrobial susceptibility test based on single-cell morphological analysis. Sci. Fredborg, M., Rosenvinge, F. S., Spillum, E., Kroghsbo, S., Wang, M., and
Transl. Med. 6:267ra174. doi: 10.1126/scitranslmed.3009650 Sondergaard, T. E. (2015). Rapid antimicrobial susceptibility testing of clinical
Compton, J. (1991). Nucleic acid sequence-based amplification. Nature 350, 91–92. isolates by digital time-lapse microscopy. Eur. J. Clin. Microbiol. Infect. Dis. 34,
doi: 10.1038/350091a0 2385–2394. doi: 10.1007/s10096-015-2492-9
Correa-Martínez, C. L., Idelevich, E. A., Sparbier, K., Kostrzewa, M., and Becker, K. GeneFludics Inc. (n.d.). GeneFluidics Announces CE-IVD Marking of UtiMaxTM
(2019). Rapid detection of extended-spectrum β-lactamases (ESBL) and AmpC uropathogen Identification (ID) and Antimicrobial Susceptibility Testing
β-lactamases in enterobacterales: development of a screening panel using the (AST): GenefluidicsLifeScience. Available online at: http://www.genefluidics.
MALDI-TOF MS-based direct-on-target microdroplet growth assay. Front. com/genefluidics-announces-ce-ivd-marking-of-utimax-uropathogen-
Microbiol. 10:13. doi: 10.3389/fmicb.2019.00013 identification-id-and-antimicrobial-susceptibility-testing-ast/ (accessed
Costa-de-Oliveira, S., Teixeira-Santos, R., Silva, A. P., Pinho, E., Mergulhão, August 18, 2019).
P., Silva-Dias, A., et al. (2017). Potential impact of flow cytometry Glupczynski, Y., Jousset, A., Evrard, S., Bonnin, R. A., Huang, T.-D., Dortet,
antimicrobial susceptibility testing on the clinical management of gram- L., et al. (2017). Prospective evaluation of the OKN K-SeT assay, a new
negative bacteremia using the FASTinov R Kit. Front. Microbiol. 8:2455. multiplex immunochromatographic test for the rapid detection of OXA-48-
doi: 10.3389/fmicb.2017.02455 like, KPC and NDM carbapenemases. J. Antimicrob. Chemother. 72, 1955–1960.
Cui, X., Ren, L., Shan, Y., Wang, X., Yang, Z., Li, C., et al. (2018). Smartphone-based doi: 10.1093/jac/dkx089
rapid quantification of viable bacteria by single-cell microdroplet turbidity Halford, C., Gonzalez, R., Campuzano, S., Hu, B., Babbitt, J. T., Liu, J., et al. (2013).
imaging. Analyst 143, 3309–3316. doi: 10.1039/C8AN00456K Rapid antimicrobial susceptibility testing by sensitive detection of precursor
rRNA using a novel electrochemical biosensing platform. Antimicrob. Agents Kang, D.-K., Ali, M. M., Zhang, K., Huang, S. S., Peterson, E., Digman, M. A.,
Chemother. 57, 936–943. doi: 10.1128/AAC.00615-12 et al. (2014). ARTICLE Rapid detection of single bacteria in unprocessed
Harris, P. N. A., and Ferguson, J. K. (2012). Antibiotic therapy for inducible blood using integrated comprehensive droplet digital detection. Nat. Commun.
AmpC β-lactamase-producing Gram-negative bacilli: what are the alternatives 5:5427. doi: 10.1038/ncomms6427
to carbapenems, quinolones and aminoglycosides? Int. J. Antimicrob. Agents Kang, W., Sarkar, S., Lin, Z. S., McKenney, S., and Konry, T. (2019).
40, 297–305. doi: 10.1016/j.ijantimicag.2012.06.004 Ultrafast parallelized microfluidic platform for antimicrobial susceptibility
Hassibi, A., Manickam, A., Singh, R., Bolouki, S., Sinha, R., Jirage, K. B., testing of gram positive and negative bacteria. Anal. Chem. 91, 6242–6249.
et al. (2018). Multiplexed identification, quantification and genotyping of doi: 10.1021/acs.analchem.9b00939
infectious agents using a semiconductor biochip. Nat. Biotechnol. 36, 738–745. Kelley, S. O. (2017). New technologies for rapid bacterial identification and
doi: 10.1038/nbt.4179 antibiotic resistance profiling. SLAS TECHNOLOGY: Translating Life Sciences
Hayden, R. T., Clinton, L. K., Hewitt, C., Koyamatsu, T., Sun, Y., Jamison, Innovation 22, 113–121. doi: 10.1177/2211068216680207
G., et al. (2016). Rapid antimicrobial susceptibility testing using Kinnunen, P., Sinn, I., McNaughton, B. H., Newton, D. W., Burns, M. A.,
forward laser light scatter technology. J. Clin. Microbiol. 54, 2701–2706. and Kopelman, R. (2011). Monitoring the growth and drug susceptibility
doi: 10.1128/JCM.01475-16 of individual bacteria using asynchronous magnetic bead rotation sensors.
He, J., Mu, X., Guo, Z., Hao, H., Zhang, C., Zhao, Z., et al. (2014). A Biosens. Bioelectron. 26, 2751–2755. doi: 10.1016/j.bios.2010.10.010
novel microbead-based microfluidic device for rapid bacterial identification Kitao, T., Miyoshi-Akiyama, T., Shimada, K., Tanaka, M., Narahara, K., Saito,
and antibiotic susceptibility testing. Eur. J. Clin. Microbiol. Infect. Dis. 33, N., et al. (2010). Development of an immunochromatographic assay for the
′
2223–2230. doi: 10.1007/s10096-014-2182-z rapid detection of AAC(6 )-Iae-producing multidrug-resistant Pseudomonas
Hernández-Neuta, I., Neumann, F., Brightmeyer, J., Ba Tis, T., Madaboosi, aeruginosa. J. Antimicrob. Chemother. 65, 1382–1386. doi: 10.1093/jac/dkq148
N., Wei, Q., et al. (2019). Smartphone-based clinical diagnostics: towards Koehler, J. W., Douglas, C. E., and Minogue, T. D. (2018). A highy multiplexed
democratization of evidence-based health care. J. Intern. Med. 285, 19–39. broad pathogen detection assay for infectious disease diagnostics. PLOS Negl.
doi: 10.1111/joim.12820 Trop. Dis. 12:e0006889. doi: 10.1371/journal.pntd.0006889
Hill, B., Beavis, K. G., Boonlayangoor, S., Tesic, V., Charnot-Katsikas, A., Bethel, Koskinen, J. O. (2008). Two-Photon Excitation Fluorometry in Detection (Issue
C., et al. (2017). Use of the accelerate pheno system for identification and January).
antimicrobial susceptibility testing of pathogens in positive blood cultures Lai, S. Y., Deffenderfer, O. F., Hanson, W., Phillips, M. P., and Thaler, E. R. (2002).
and impact on time to results and workflow. J. Clin. Microbiol. 56, 1–13. Identification of upper respiratory bacterial pathogens with the electronic nose.
doi: 10.1128/jcm.01166-17 Laryngoscope 112, 975–979. doi: 10.1097/00005537-200206000-00007
Hong Nguyen, M., Clancy, C. J., William Pasculle, A., Pappas, P. G., Alangaden, Leclercq, R., Cantón, R., Brown, D. F. J., Giske, C. G., Heisig, P., Macgowan, A. P.,
G., Pankey, G. A., et al. (2019). Performance of the T2bacteria panel et al. (2013). EUCAST expert rules in antimicrobial susceptibility testing. Clin.
for diagnosing bloodstream infections. Ann. Intern. Med. 170, 845–852. Microbiol. Infect. 19, 141–160. doi: 10.1111/j.1469-0691.2011.03703.x
doi: 10.7326/M18-2772 Leclercq, R., and Courvalin, P. (2002). Resistance to macrolides and related
Hoß, S. G., and Bendas, G. (2017). “Mass-sensitive biosensor systems to determine antibiotics in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 46,
the membrane interaction of analytes,” in Methods in Molecular Biology. Vol. 2727–34. doi: 10.1128/AAC.46.9.2727-2734.2002
1520. (Clifton, NJ), 145-157. doi: 10.1007/978-1-4939-6634-9_9 Lee, D., Kim, E. J., Kilgore, P. E., Kim, S. A., Takahashi, H., Ohnishi,
Howell, M., Wirz, D., Daniels, A. U., and Braissant, O. (2012). M., et al. (2015). Clinical evaluation of a loop-mediated isothermal
Application of a microcalorimetric method for determining drug amplification (LAMP) assay for rapid detection of neisseria meningitidis
susceptibility in mycobacterium species. J. Clin. Microbiol. 50, 16–20. in cerebrospinal fluid. PLoS ONE 10:e0122922. doi: 10.1371/journal.pone.01
doi: 10.1128/JCM.05556-11 22922
Huang, T.-H., Ning, X., Wang, X., Murthy, N., Tzeng, Y.-L., and Dickson, Lewis, J. M., Savage, R. S., Beeching, N. J., Beadsworth, M. B. J., Feasey,
R. M. (2015). Rapid cytometric antibiotic susceptibility testing utilizing N., and Covington, J. A. (2017). Identifying volatile metabolite
adaptive multidimensional statistical metrics. Anal. Chem. 87, 1941–1949. signatures for the diagnosis of bacterial respiratory tract infection
doi: 10.1021/ac504241x using electronic nose technology: a pilot study. PLoS ONE 12:e0188879.
Hughes, M. D. (2018). Advances and Trends in Sepsis Diagnostics. Available online doi: 10.1371/journal.pone.0188879
at: www.eacorp.com (Accessed March 12, 2019). Li, J., Song, X., Yang, T., Chen, Y., Gong, Y., Yin, X., et al. (2016).
Idelevich, E. A., Schüle, I., Grünastel, B., Wüllenweber, J., Peters, G., and A systematic review of antibiotic prescription associated with upper
Becker, K. (2014). Rapid identification of microorganisms from positive respiratory tract infections in China. Medicine (Baltimore). 95:e3587.
blood cultures by MALDI-TOF mass spectrometry subsequent to very short- doi: 10.1097/MD.0000000000003587
term incubation on solid medium. Clin. Microbiol. Infect. 20, 1001–1006. Li, T., Zhu, F., Guo, W., Gu, H., Zhao, J., Yan, M., et al. (2017a). Selective capture
doi: 10.1111/1469-0691.12640 and rapid identification of E. coli O157:H7 by carbon nanotube multilayer
Idelevich, E. A., Storck, L. M., Sparbier, K., Drews, O., Kostrzewa, M., and Becker, biosensors and microfluidic chip-based LAMP. RSC Adv. 7, 30446–30452.
K. (2018). Rapid direct susceptibility testing from positive blood cultures by the doi: 10.1039/C7RA04583B
matrix-assisted laser desorption ionization-time of flight mass spectrometry- Li, Y., Yang, X., and Zhao, W. (2017b). Emerging microtechnologies
based direct-on-target microdroplet growth assay. J. Clin. Microbiol. 56, and automated systems for rapid bacterial identification and
00913–00918. doi: 10.1128/JCM.00913-18 antibiotic susceptibility testing. SLAS Technol. 22, 585–608.
Jacoby, G. A. (2009). AmpC -Lactamases. Clin. Microbiol. Rev. 22, 161–182. doi: 10.1177/2472630317727519
doi: 10.1128/CMR.00036-08 Lim, S. H., Mix, S., Xu, Z., Taba, B., Budvytiene, I., Berliner, A. N., et al. (2014).
Kadlec, M. W., You, D., Liao, J. C., and Wong, P. K. (2014). A cell phone–based Colorimetric sensor array allows fast detection and simultaneous identification
microphotometric system for rapid antimicrobial susceptibility testing. J. Lab. of sepsis-causing bacteria in spiked blood culture. J. Clin. Microbiol. 52,
Autom. 19, 258–266. doi: 10.1177/2211068213491095 592–598. doi: 10.1128/JCM.02377-13
Kahlmeter, G. (2014). Defining antibiotic resistance-towards Liu, C. Y., Han, Y. Y., Shih, P. H., Lian, W. N., Wang, H. H., Lin, C. H., et al.
international harmonization. Ups. J. Med. Sci. 119, 78–86. (2016). Rapid bacterial antibiotic susceptibility test based on simple surface-
doi: 10.3109/03009734.2014.901446 enhanced Raman spectroscopic biomarkers. Sci. Rep. 1–15. doi: 10.1038/
Kahlmeter, G. (2016). Rapid Phenotypic Susceptibility Testing. in Symposium srep23375
Lecture in Session: New and Rapid Detection of Antimicrobial Resistance. Liu, T., Lu, Y., Gau, V., Liao, J. C., and Wong, P. K. (2014). Rapid
ECCMID Symposium 2016 on Bacterial Susceptibility & Resistance. Amsterdam, antimicrobial susceptibility testing with electrokinetics enhanced biosensors
The Netherlands. Available online at: https://www.escmid.org/escmid_ for diagnosis of acute bacterial infections. Ann. Biomed. Eng. 42.
publications/escmid_elibrary/material/?mid=33585 doi: 10.1007/s10439-014-1040-6
Liu, Z., Banaei, N., and Ren, K. (2017a). Microfluidics for combating antimicrobial McAdam, A. J. (2017). Unforeseen consequences: culture-independent diagnostic
resistance. Trends Biotechnol. 35, 1129–1139. doi: 10.1016/j.tibtech.2017.07.008 tests and epidemiologic tracking of foodborne pathogens. J. Clin. Microbiol. 55,
Liu, Z., Sun, H., and Ren, K. (2017b). A multiplexed, gradient-based, full-hydrogel 1978–1979. doi: 10.1128/JCM.00678-17
microfluidic platform for rapid, high-throughput antimicrobial susceptibility Mecklenburg, M., Chen, Q., Andersson, A., and Xie, B. (2017). A biosensing
testing. ChemPlusChem 82, 792–801. doi: 10.1002/cplu.201600654 strategy for fast profiling of antibiotic resistance. Proc. Technol. 27, 33–34.
London, R., Schwedock, J., Sage, A., Valley, H., Meadows, J., Waddington, M., doi: 10.1016/j.protcy.2017.04.016
et al. (2010). An automated system for rapid non-destructive enumeration of Metzger, S., Frobel, R. A., and Dunne, W. M. (2014). Rapid simultaneous
growing microbes. PLoS ONE 5:e8609. doi: 10.1371/journal.pone.0008609 identification and quantitation of staphylococcus aureus and Pseudomonas
Longo, G., Alonso-Sarduy, L., Rio, L. M., Bizzini, A., Trampuz, A., Notz, J., aeruginosa directly from bronchoalveolar lavage specimens using
et al. (2013). Rapid detection of bacterial resistance to antibiotics using automated microscopy. Diagn. Microbiol. Infect. Dis. 79, 160–165.
AFM cantilevers as nanomechanical sensors. Nat. Nanotechnol. 8, 522–526. doi: 10.1016/j.diagmicrobio.2013.11.029
doi: 10.1038/nnano.2013.120 Mezger, A., Gullberg, E., Göransson, J., Zorzet, A., Herthnek, D., Tano, E.,
Lonsdale, C. L., Taba, B., Queralto, N., Lukaszewski, R. A., Martino, et al. (2015). A general method for rapid determination of antibiotic
R. A., Rhodes, P. A., et al. (2013). The use of colorimetric sensor susceptibility and species in bacterial infections. J. Clin. Microbiol. 53, 425–432.
arrays to discriminate between pathogenic bacteria. PLoS ONE 8:e62726. doi: 10.1128/JCM.02434-14
doi: 10.1371/journal.pone.0062726 Miller, J. M., Binnicker, M. J., Campbell, S., Carroll, K. C., Chapin, K. C., Gilligan,
López-Marzo, A. M., and Merkoçi, A. (2016). Paper-based sensors and assays: a P. H., et al. (2018). A guide to utilization of the microbiology laboratory for
success of the engineering design and the convergence of knowledge areas. Lab. diagnosis of infectious diseases: 2018 update by the infectious diseases society
Chip. 16, 3150–3176. doi: 10.1039/C6LC00737F of America and the American society for microbiologya. Clin. Infect. Dis. 67,
Luka, G., Ahmadi, A., Najjaran, H., Alocilja, E., DeRosa, M., Wolthers, K., e1–e94. doi: 10.1093/cid/ciy381
et al. (2015). Microfluidics integrated biosensors: a leading technology Mohan, R., Mach, K. E., Bercovici, M., Pan, Y., Dhulipala, L., Wong, P. K., et al.
towards lab-on-a-chip and sensing applications. Sensors 15, 30011–30031. (2011). Clinical validation of integrated nucleic acid and protein detection on
doi: 10.3390/s151229783 an electrochemical biosensor array for urinary tract infection diagnosis. PLoS
Mach, K. E., Mohan, R., Baron, E. J., Shih, M.-C., Gau, V., Wong, P. K., et al. (2011). ONE 6:e26846. doi: 10.1371/journal.pone.0026846
A biosensor platform for rapid antimicrobial susceptibility testing directly from Montgomery, S., Roman, K., Ngyuen, L., Cardenas, A. M., Knox, J., Tomaras, A.
clinical samples. J. Urol. 185, 148–153. doi: 10.1016/j.juro.2010.09.022 P., et al. (2017). Prospective evaluation of light scatter technology paired with
Malmberg, C., Yuen, P., Spaak, J., Cars, O., Tängdén, T., and Lagerbäck, P. matrix-assisted laser desorption ionization-time of flight mass spectrometry for
(2016). A novel microfluidic assay for rapid phenotypic antibiotic susceptibility rapid diagnosis of urinary tract infections downloaded from. J. Clin. Microbiol.
testing of bacteria detected in clinical blood cultures. PLoS ONE 11:e0167356. 55, 1802–1811. doi: 10.1128/JCM.00027-17
doi: 10.1371/journal.pone.0167356 Nazemi, E., Hassen, W. M., Frost, E. H., and Dubowski, J. J. (2017). Monitoring
Marco, F. (2017). Molecular methods for septicemia diagnosis. Enferm. Infecc. growth and antibiotic susceptibility of Escherichia coli with photoluminescence
Microbiol. Clin. (English ed.) 35, 586–592. doi: 10.1016/j.eimce.2017.03.023 of GaAs/AlGaAs quantum well microstructures. Biosens. Bioelectron. 93,
Marder, E. P., Cieslak, P. R., Cronquist, A. B., Dunn, J., Lathrop, S., Rabatsky- 234–240. doi: 10.1016/j.bios.2016.08.112
Ehr, T., et al. (2017). Incidence and trends of infections with pathogens Nijhuis, R. H. T., Guerendiain, D., Claas, E. C. J., and Templeton, K. E. (2017).
transmitted commonly through food and the effect of increasing use of culture- Comparison of ePlex respiratory pathogen panel with laboratory-developed
independent diagnostic tests on surveillance — foodborne diseases active real-time PCR assays for detection of respiratory pathogens. J. Clin. Microbiol.
surveillance network, 10 U.S. Sites, 2013–2016. MMWR. Morb. Mortal. Wkly. 55, 1938–1945. doi: 10.1128/JCM.00221-17
Rep. 66, 397–403. doi: 10.15585/mmwr.mm6615a1 Noncommercial culture and drug-susceptibility testing methods for screening
Marschal, M., Bachmaier, J., Autenrieth, I., Oberhettinger, P., Willmann, M., patients at risk for multidrug-resistant tuberculosis: policy statement (2011).
and Petera, S. (2017). Evaluation of the accelerate pheno system for fast World Health Organization. Available online at: http://www.ncbi.nlm.nih.gov/
identification and antimicrobial susceptibility testing from positive blood pubmed/23586116 (accessed March 13, 2019).
cultures in bloodstream infections caused by gram-negative pathogens. J. Clin. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K.,
Microbiol. 55, 2116–2126. doi: 10.1128/JCM.00181-17 Amino, N., et al. (2000). Loop-mediated isothermal amplification of
Mashalla, Y., Setlhare, V., Massele, A., Sepako, E., Tiroyakgosi, C., Kgatlwane, DNA. Nucleic Acids Res. 28:E63. Available online at: http://www.ncbi.nlm.
J., et al. (2017). Assessment of prescribing practices at the primary nih.gov/pubmed/10871386 (accessed March 7, 2019). doi: 10.1093/nar/28.
healthcare facilities in botswana with an emphasis on antibiotics: findings and 12.e63
implications. Int. J. Clin. Pract. 71:e13042. doi: 10.1111/ijcp.13042 Oblath, E. A., Henley, W. H., Alarie, J. P., and Ramsey, J. M. (2013). A microfluidic
Matsumoto, Y., Sakakihara, S., Grushnikov, A., Kikuchi, K., Noji, H., chip integrating DNA extraction and real-time PCR for the detection of bacteria
Yamaguchi, A., et al. (2016). A microfluidic channel method for rapid drug- in saliva. Lab. Chip. 13, 1325–1332. doi: 10.1039/c3lc40961a
susceptibility testing of pseudomonas aeruginosa. PLoS ONE 11:e0148797. Patel, T. S., Kaakeh, R., Nagel, J. L., Newton, D. W., and Stevenson, J. G.
doi: 10.1371/journal.pone.0148797 (2017). Cost analysis of implementing matrix- assisted laser desorption
Maugeri, G., Lychko, I., Sobral, R., and Roque, A. C. A. (2019). Identification ionization-time of flight mass spectrometry plus real-time antimicrobial
and antibiotic-susceptibility profiling of infectious bacterial agents: stewardship intervention for bloodstream infections. J. Clin. Microbiol. 55,
a review of current and future trends. Biotechnol. J. 14:e1700750. 60–67. doi: 10.1128/JCM.01452-16
doi: 10.1002/biot.201700750 Perry-O’Keefe, H., Stender, H., Broomer, A., Oliveira, K., Coull, J., and Hyldig-
Maurer, F. P., Christner, M., Hentschke, M., and Rohde, H. (2017). Advances Nielsen, J. J. (2001). Filter-based PNA in situ hybridization for rapid detection,
in rapid identification and susceptibility testing of bacteria in the clinical identification and enumeration of specific micro-organisms. J. Appl. Microbiol.
microbiology laboratory: implications for patient care and antimicrobial 90, 180–9. doi: 10.1046/j.1365-2672.2001.01230.x
stewardship programs. Infect. Dis. Rep. 9:6839. doi: 10.4081/idr.2017.6839 Persaud, K., and Dodd, G. (1982). Analysis of discrimination mechanisms in
Mauri, C., Principe, L., Bracco, S., Meroni, E., Corbo, N., Pini, B., et al. (2017). the mammalian olfactory system using a model nose. Nature 299, 352–355.
Identification by mass spectrometry and automated susceptibility testing from doi: 10.1038/299352a0
positive bottles: a simple, rapid, and standardized approach to reduce the Peytavi, R. (2005). Microfluidic device for rapid (andlt;15 min)
turnaround time in the management of blood cultures. BMC Infect. Dis. 17, automated microarray hybridization. Clin. Chem. 51, 1836–1844.
1–8. doi: 10.1186/s12879-017-2851-5 doi: 10.1373/clinchem.2005.052845
Maxson, T., Taylor-Howell, C. L., and Minogue, T. D. (2017). Semi-quantitative Plüddemann, A., Onakpoya, I., Harrison, S., Shinkins, B., Tompson, A., Davis, R.,
MALDI-TOF for antimicrobial susceptibility testing in staphylococcus aureus. et al. (2015). “Position paper on anti-microbial resistance diagnostics,” in Centre
PLoS ONE 12:e0183899. doi: 10.1371/journal.pone.0183899 for Evidence-Based Medicine. 1–142. doi: 10.13140/RG.2.1.1135.9846
Priye, A., Bird, S. W., Light, Y. K., Ball, C. S., Negrete, O. A., and Meagher, Sun, H., Liu, Z., Hu, C., and Ren, K. (2016). Cell-on-hydrogel platform
R. J. (2017). A smartphone-based diagnostic platform for rapid detection of made of agar and alginate for rapid, low-cost, multidimensional test of
Zika, chikungunya and dengue viruses. Sci. Rep. 7:44778. doi: 10.1038/srep antimicrobial susceptibility. Lab. Chip. 16, 3130–3138. doi: 10.1039/C6LC0
44778 0417B
Pulido, M. R., García-Quintanilla, M., Martín-Peña, R., Cisneros, J. M., and Syal, K., Iriya, R., Yang, Y., Yu, H., Wang, S., Haydel, S. E., et al. (2016).
McConnell, M. J. (2013). Progress on the development of rapid methods for Antimicrobial susceptibility test with plasmonic imaging and tracking of
antimicrobial susceptibility testing. J. Antimicrob. Chemother. 68, 2710–2717. single bacterial motions on nanometer scale. ACS Nano 10, 845–852.
doi: 10.1093/jac/dkt253 doi: 10.1021/acsnano.5b05944
Quainoo, S., Coolen, J. P. M., van Hijum, S. A. F. T., Huynen, M. A., Melchers, Syal, K., Mo, M., Yu, H., Iriya, R., Jing, W., Guodong, S., et al. (2017). Current
W. J. G., van Schaik, W., et al. (2017). Whole-genome sequencing of bacterial and emerging techniques for antibiotic susceptibility tests. Theranostics 7,
pathogens: the future of nosocomial outbreak analysis. Clin. Microbiol. Rev. 30, 1795–1805. doi: 10.7150/thno.19217
1015–1063. doi: 10.1128/CMR.00016-17 Tao, N., and Syal, K. (2016). US 2017/0045514 A1. Antibiotic Susceptibility Testing
Sahoo, P. R., Sethy, K., Mohapatra, S., and Panda, D. (2016). Loop mediated via Plasmonic Imaging and Tracking.
isothermal amplification: an innovative gene amplification technique for Trienski, T. L., Barrett, H. L., Pasquale, T. R., DiPersio, J. R., and File, T.
animal diseases. Vet. World 9, 465–469. doi: 10.14202/vetworld.2016. M. (2013). Evaluation and use of a rapid Staphylococcus aureus assay by
465-469 an antimicrobial stewardship program. Am. J. Heal. Pharm. 70, 1908–1912.
Salimnia, H., Fairfax, M. R., Lephart, P., Morgan, M., Gilbreath, J. doi: 10.2146/ajhp130118
J., Butler-Wu, S. M., et al. (2014). An international, prospective, United nations meeting on antimicrobial resistance (2016). Bull. World Health
multicenter evaluation of the combination of advanDx staphylococcus Organ. 94, 638–639. doi: 10.2471/BLT.16.020916
QuickFISH BC with mecA XpressFISH for detection of methicillin- Vakkila, J., Koskinen, J. O., Brandt, A., Muotiala, A., Liukko, V., Soittu, S., et al.
resistant staphylococcus aureus isolates from positive blood (2015). Detection of group a streptococcus from pharyngeal swab samples by
cultures. J. Clin. Microbiol. 52, 3928–32. doi: 10.1128/JCM.01 bacterial culture is challenged by a novel mariPOC point-of-care test. J. Clin.
811-14 Microbiol. 53, 2079–2083. doi: 10.1128/JCM.00018-15
Sanbonmatsu-Gámez, S., Pérez-Ruiz, M., Lara-Oya, A., Pedrosa- van Belkum, A., Bachmann, T. T., Lüdke, G., Lisby, J. G., Kahlmeter, G., Mohess,
Corral, I., Riazzo-Damas, C., and Navarro-Marí, J. M. (2015). A., et al. (2019a). Developmental roadmap for antimicrobial susceptibility
Analytical performance of the automated multianalyte point-of- testing systems. Nat. Rev. Microbiol. 17, 51–62. doi: 10.1038/s41579-018-
care mariPOC R for the detection of respiratory viruses. Diagn 0098-9
Micr Infec Dis. 83, 252–256. doi: 10.1016/j.diagmicrobio.2015. van Belkum, A., Bachmann, T. T., Lüdke, G., Lisby, J. G., Kahlmeter, G., Mohess,
07.010 A., et al. (2019b). Developmental roadmap for antimicrobial susceptibility
Saviauk, T., Kiiski, J. P., Nieminen, M. K., Tamminen, N. N., Roine, A. N., testing systems. Nat. Rev. Microbiol. 17, 51–62.
Kumpulainen, P. S., et al. (2018). Electronic nose in the detection of wound van Belkum, A., and Rochas, O. (2018). Laboratory-based and point-of-care testing
infection bacteria from bacterial cultures: a proof-of-principle study. Eur. Surg. for MSSA/MRSA detection in the age of whole genome sequencing. Front.
Res. 59, 1–11. doi: 10.1159/000485461 Microbiol. 9:1437. doi: 10.3389/fmicb.2018.01437
Schoepp, N. G., Liaw, E. J., Winnett, A., WinnettSavela, E. S., Garner, Verma, J., Saxena, S., and Babu, S. G. (2013). ELISA-Based Identification
O. B., and Ismagilov, R. F. (2020). Differential DNA accessibility to and Detection of Microbes. (Springer, Berlin, Heidelberg), 169–186.
polymerase enables 30-minute phenotypic-lactam antibiotic susceptibility doi: 10.1007/978-3-642-34410-7_13
testing of carbapenem-resistant Enterobacteriaceae. PLoS Biology. 18. Veses-Garcia, M., Antypas, H., Löffler, S., Brauner, A., Andersson-Svahn, H., and
doi: 10.1371/journal.pbio.3000652 Richter-Dahlfors, A. (2018). Rapid phenotypic antibiotic susceptibility testing
Schoepp, N. G., Schlappi, T. S., Curtis, M. S., Butkovich, S. S., Miller, S., Humphries, of uropathogens using optical signal analysis on the nanowell slide. Front.
R. M., et al. (2017). Rapid pathogen-specific phenotypic antibiotic susceptibility Microbiol. 9:1530. doi: 10.3389/fmicb.2018.01530
testing using digital LAMP quantification in clinical samples. Sci. Transl. Med. von Ah, U., Wirz, D., and Daniels, A. U. (2009). Isothermal micro
9:eaal3693. doi: 10.1126/scitranslmed.aal3693 calorimetry–a new method for MIC determinations: results for 12 antibiotics
Schumacher, A., Vranken, T., Malhotra, A., Arts, J. J. C., and Habibovic, P. and reference strains of E. coli and S. aureus. BMC Microbiol. 9:106.
(2018). In vitro antimicrobial susceptibility testing methods: agar dilution to doi: 10.1186/1471-2180-9-106
3D tissue-engineered models. Eur. J. Clin. Microbiol. Infect. Dis. 37, 187–208. Vrioni, G., Tsiamis, C., Oikonomidis, G., Theodoridou, K., Kapsimali, V.,
doi: 10.1007/s10096-017-3089-2 and Tsakris, A. (2018). MALDI-TOF mass spectrometry technology for
Sharp, S. (2020). Specific RevealTM — Specific Technologies. Specif. Reveal. AST detecting biomarkers of antimicrobial resistance: current achievements and
Hours not Days. Available online at: https://www.specific-dx.com/reveal-ast future perspectives. Ann. Transl. Med. 6, 240–240. doi: 10.21037/atm.20
(accessed May 17, 2020). 18.06.28
She, R. C., and Bender, J. M. (2019). Advances in rapid molecular blood Walker, G. T., Fraiser, M. S., Schram, J. L., Little, M. C., Nadeau, J. G., and
culture diagnostics: healthcare impact, laboratory implications, and multiplex Malinowski, D. P. (1992). Strand displacement amplification–an isothermal, in
technologies. J. Appl. Lab. Med. 3, 617–630. doi: 10.1373/jalm.2018. vitro DNA amplification technique. Nucleic Acids Res. 20, 1691–6. Available
027409 online at: http://www.ncbi.nlm.nih.gov/pubmed/1579461 (accessed March 7,
Shi, X., Kadiyala, U., Vanepps, J. S., and Yau, S. T. (2018). Culture-free bacterial 2019). doi: 10.1093/nar/20.7.1691
detection and identification from blood with rapid, phenotypic, antibiotic Wang, L.-J., Naudé, N., Demissie, M., Crivaro, A., Kamoun, M., Wang, P.,
susceptibility testing. Sci. Rep. 8:3416. et al. (2018). Analytical validation of an ultra low-cost mobile phone
Spanu, T., Fiori, B., D’Inzeo, T., Canu, G., Campoli, S., Giani, T., et al. (2012). microplate reader for infectious disease testing. Clin. Chim. Acta 482, 21–26.
Evaluation of the new NucliSENS EasyQ KPC Test for rapid detection of doi: 10.1016/j.cca.2018.03.013
klebsiella pneumoniae carbapenemase genes (blaKPC). J. Clin. Microbiol. 50, Weibull, E., Antypas, H., Kjäll, P., Brauner, A., Andersson-Svahn, H., and
2783–2785. doi: 10.1128/JCM.00284-12 Richter-Dahlfors, A. (2014). Bacterial nanoscale cultures for phenotypic
Sparbier, K., Schubert, S., and Kostrzewa, M. (2016). MBT-ASTRA: a multiplexed antibiotic susceptibility testing. J. Clin. Microbiol. 52, 3310–3317.
suitable tool for fast antibiotic susceptibility testing? Methods. 104, 48–54. doi: 10.1128/JCM.01161-14
doi: 10.1016/j.ymeth.2016.01.008 Wieser, A., Schneider, L., Jung, J., and Schubert, S. (2012). MALDI-TOF
Strålin, K., Ehn, F., Giske, C. G., Ullberg, M., Hedlund, J., Petersson, J., MS in microbiological diagnostics—identification of microorganisms
et al. (2016). The IRIDICA PCR/electrospray ionization-mass spectrometry and beyond (mini review). Appl. Microbiol. Biotechnol. 93, 965–974.
assay on bronchoalveolar lavage for bacterial etiology in mechanically doi: 10.1007/s00253-011-3783-4
ventilated patients with suspected pneumonia. PLoS ONE 11:e0159694. Yamada, K., Wanchun, J., Ohkura, T., Murai, A., Hayakawa, R., Kinoshita, K.,
doi: 10.1371/journal.pone.0159694 et al. (2013). Detection of methicillin-resistant staphylococcus aureus using
a specific Anti-PBP2a chicken IgY antibody. Jpn. J. Infect. Dis. 66, 103–108. Conflict of Interest: VH was employed by Fimlab Laboratories.
doi: 10.7883/yoken.66.103
Yang, R., Zhang, H., Li, X., Ye, L., Gong, M., Yang, J., et al. (2018). A multiplex loop- The remaining authors declare that the research was conducted in the absence of
mediated isothermal amplification assay for rapid screening of Acinetobacter any commercial or financial relationships that could be construed as a potential
baumannii and D carbapenemase OXA-23 gene. Biosci. Rep. 38:BSR20180425. conflict of interest.
doi: 10.1042/BSR20180425
Yi, Q., Cai, D., Xiao, M., Nie, M., Cui, Q., Cheng, J., et al. (2019). Copyright © 2020 Vasala, Hytönen and Laitinen. This is an open-access article
Direct antimicrobial susceptibility testing of bloodstream infection on distributed under the terms of the Creative Commons Attribution License (CC BY).
SlipChip. Biosens. Bioelectron. 135, 200–207. doi: 10.1016/j.bios.2019. The use, distribution or reproduction in other forums is permitted, provided the
04.003 original author(s) and the copyright owner(s) are credited and that the original
Zhu, K., Dietrich, R., Didier, A., Doyscher, D., and Märtlbauer, E. (2014). Recent publication in this journal is cited, in accordance with accepted academic practice.
developments in antibody-based assays for the detection of bacterial toxins. No use, distribution or reproduction is permitted which does not comply with these
Toxins (Basel). 6, 1325–1348. doi: 10.3390/toxins6041325 terms.

fcimb.2020.00308

Uploaded by

Copyright:

Available Formats

fcimb.2020.00308

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

fcimb.2020.00308

Uploaded by

Copyright:

Available Formats

REVIEW

published: 15 July 2020

Modern Tools for Rapid Diagnostics

Fast, robust, and affordable antimicrobial susceptibility testing (AST) is required, as

Specialty section: BACKGROUND AND FOREWORD

Microscopy Clinical laboratories applying mass spectrometry are unlikely to

Smartphone-Based Readers Optical or Microscopic Methods

POCT-COMPATIBLE DEVICES FOR ID AND loop-mediated isothermal amplification) or dPCR (droplet-PCR)

Standard cultivation tests

Direct-On-Target All instrument 4h – – – X X – – Experimental Idelevich et al.,

Rapid Diagnostics of Antimicrobial Resistance

Other imaging or spectroscopy-based systems

Rapid Diagnostics of Antimicrobial Resistance

Resistance factor Coris Bioconcept 0.25–4 – – – – – – – Commercial ECCMID 2015

Multipath (magnetic First Light 2–4 h X X X X X – X Commercial, under

Rapid Diagnostics of Antimicrobial Resistance

Isothermal Talis Biomedical 0.5 h X X X(fs) X X (X) X Under Schoepp et al.,

AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.