Expert Review of Molecular Diagnostics

ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: http://www.tandfonline.com/loi/iero20

A review of novel technologies and techniques

associated with identification of bloodstream
infection etiologies and rapid antimicrobial
genotypic and quantitative phenotypic
determination

Stephen Poole, Stephen P Kidd & Kordo Saeed

To cite this article: Stephen Poole, Stephen P Kidd & Kordo Saeed (2018) A review of novel
technologies and techniques associated with identification of bloodstream infection etiologies
and rapid antimicrobial genotypic and quantitative phenotypic determination, Expert Review of
Molecular Diagnostics, 18:6, 543-555, DOI: 10.1080/14737159.2018.1480369

To link to this article: https://doi.org/10.1080/14737159.2018.1480369

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May 2018.
Published online: 07 Jun 2018.

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EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
2018, VOL. 18, NO. 6, 543–555
https://doi.org/10.1080/14737159.2018.1480369

REVIEW

A review of novel technologies and techniques associated with identification of

bloodstream infection etiologies and rapid antimicrobial genotypic and
quantitative phenotypic determination
Stephen Poolea*, Stephen P Kidda* and Kordo Saeeda,b
a
Hampshire Hospitals NHS Foundation Trust, Department of Microbiology, Basingstoke and Winchester, UK; bUniversity of Southampton, School of
medicine, Southampton, UK

ABSTRACT ARTICLE HISTORY

Introduction: The antimicrobial aspect of management of patients with blood stream infections (BSI) Received 15 March 2018
and sepsis is time critical. In an era of increasing antimicrobial resistance, rapid detection and identifica- Accepted 21 May 2018
tion of bacteria with antimicrobial susceptibility is crucial to direct therapy early in the course of illness. KEYWORDS
Molecular techniques offer a potential solution to this. Blood stream infections;
Areas covered: In the present review the authors have discussed a number of novel solutions utilizing a antimicrobial susceptibility;
variety of molecular techniques for pathogen detection, identification and antimicrobial susceptibility. rapid diagnostics; PCR;
The review is not designed to be an exhaustive literature review covering all diagnostic solutions ever Accelerate
developed, instead the authors have focused on what they have had experience using, evaluating or
currently view as new and exciting with potential to revolutionize BSI diagnosis.
The authors searched PubMed (Medline) and Google Scholar with terms: BSI, Bacteraemia,
Candidaemia, Diagnostics, AST, Rapid, AMR, Novel and Blood Culture. The authors attended recent
clinical microbiology technology congresses.
Expert commentary: There are multiple exciting novel technologies at differing stages of development
with potential to revolutionize diagnosis of BSI. More work is needed as well as a standardized
assessment of different platforms in order to better understand the clinical and financial impacts
these will have in clinical microbiology laboratories.

1. Introduction antimicrobials is an increasingly daunting task due to an

ever-changing landscape of resistance mechanisms respond-
Blood stream infections (BSI) have carried an enormous burden of
ing to an impossibly complex web of selective pressures.
mortality globally since the beginning of recorded history and
Multiple epidemiological studies have shown shifts over
continue to do so [1]. Current gold standards for identification
time in the pattern of organisms causing sepsis and this
and treatment established by the Surviving Sepsis Campaign
will likely to continue in the future [1]. This highlights a
appear to have made some progress toward reducing this burden
need for rapid diagnostic tests that can not only assist
[2–4] but there is still a great deal more to do.
pathogen detection/identification, but also provide data on
Rapid identification and treatment is essential to improve
antimicrobial susceptibility and resistance mechanisms.
outcomes of patients who develop sepsis and septic shock. In
The wish of every infection specialist is to have a Star
this critical period an association has been well described
Trek™ style handheld Tricorder that could be used on septic
between timely administration of antibiotics and survival [5]
patients providing the ability to identify invading pathogens,
leading to the recommendation that these agents should be
determine antimicrobial susceptibility patterns and identify a
given within 1 hour [6].
source of infection in real time. We are a little way off that at
Correct and timely antibiotic administration has implica-
present but technology is rapidly providing new tools in the
tions both for patient survival and on wider antimicrobial
clinical laboratory and at the patient bedside.
resistance. It is frustrating, therefore, that the current standard
Rapid microorganism identification in signal positive
in testing typically takes at least 24 hours and can take up to
blood cultures has been achieved using established mole-
5 days. By this time the window for effective intervention may
cular techniques (Figure 1) like polymerase chain reaction
have closed.
(PCR)[9], fluorescent in situ hybridization (FISH) [10,11] and
A key aspect to improving outcomes is the targeting of
Matrix Assisted Laser Desorption Ionisation Time Of Flight
empirical antimicrobials toward suspected causative organ-
Mass Spectrometry (MALDI-TOF MS). The BioFire Filmarray®
isms and likely susceptibility patterns, especially, in the con-
[12], SeeGene Magicplex™ Sepsis and Bruker Biotyper
text of BSI [7]. Inadequate empirical therapy has been
Sepsityper®[13] are good examples of these innovations.
strongly associated with higher mortality [8]. Blindly targeting

CONTACT Kordo Saeed kordosaeed@nhs.net Consultant Microbiologist, Hampshire Hospitals NHS Foundation Trust, Basingstoke and Winchester, UK
*
Joint first authors.
© 2018 Informa UK Limited, trading as Taylor & Francis Group
544 S. POOLE ET AL.
Molecular
Diagnosis of
BSI
Whole Blood
Nucleic Acid Post-
Novel
Amplification amplification
Real-time PCR PCR + Array NMR
Figure 1. Molecular techniques for diagnosis of blood stream infection. BSI: Blood stream infection; BC: Blood culture; NMR: Nuclear magnetic resonance; PCR:
Polymerase chain reaction; VOC: Volatile organic compound: FISH: Fluorescent in situ hybridization.
Additionally, some of the traditional bench side rapid latex
agglutination tests have been updated for use with signal
positive blood cultures like the Wellcogen™ range from
Oxoid used in the rapid qualitative detection of antigens
from Group B Streptococci, Neisseria meningiditis A, B, C,
W135, Y, Haemophilius influenzae b, Escherichia coli K1.
Currently there are a number of novel diagnostic tests
and technologies at the disposal of clinical microbiologists
and diagnostic laboratories that can support clinicians in
better decision-making around diagnosis of infection
including BSI and antimicrobial stewardship. To our knowl-
edge this is the most up-to-date and broad ranging review
regarding these techniques and technologies (Table 1)
which adds novel developments and ideas to previous
work in the field, as well as our own local views based
on experience with several of these technologies [14–16].
We appreciate other diagnostic approaches are being
explored, in particular the field of differential gene expres-
sion profiles in sepsis [17,18]. The analysis of the host
immune response to infection will help to identify unique
biomarker signatures that could have diagnostic utility
when combined with pathogen detection strategies
[19,20]. This is an exciting field but does not fall within
the scope of this article.
2. Fluorescent in situ hybridization (FISH)
FISH is not dependent on amplification of pathogen mate-
rial making it less prone to inhibition and contamination
anomalies from the blood matrix and DNA from dead
microorganisms. Historically FISH was quite laborious and
time consuming; however rapid versions of these simple,
Signal Positive
BC
Nucleic Acid Post-
Hybridisation Novel
Amplification amplification
PCR + Array
Biosensors
FISH Real-time PCR PCR +
VOC analysis
Sequencing
robust, and reliable assays are now in use with no loss of
diagnostic performance versus historical versions.
2.1. AdvanDx – QuickFISH®
The QuickFISH® is the latest PNA-FISH (Peptide nucleic acid)
iteration from AdvanDx and can report a result within 30 min
using a portfolio of four assays. It uses PNA probes targeting
bacterial 16S rRNA genes to directly identify organisms. The
staphylococcal assay identifies and differentiates
Staphylococcus aureus from coagulase-negative staphylococci
with a sensitivity/specificity of 97.1–100%/89.5–100%, well
beyond that of tube coagulase tests [21,22] This technology
has also been applied in combination with the mecA
XpressFISH® probe to rapidly identify methicillin resistant S.
aureus (MRSA) direct from signal positive blood culture [23].
Assays are also available for Enterococcus species with a sensi-
tivity/specificity 97.0–98.3%/100% [24], and the Gram-negative
bacteria E. coli, Klebsiella pneumoniae and Pseudomonas aeru-
ginosa with sensitivities for each organism of 98.4–100%,
95–99.9%, and 85.8–99.5%, respectively, with an overall speci-
ficity of 94.8–99.6% [25]. Additionally there is a Candida spe-
cies assay with sensitivity/specificity 98.3–100%/99.4–100%
[26]. These assays are generally cheaper and require less
time to run than current real-time PCR assays.
Investigation into clinical applications has demonstrated a
significant improvement of patient outcomes, better antimi-
crobial stewardship and reduced length of stay between those
receiving rapid identification and those not [27–29]. However,
a study by Holtzman et al found no benefit to the instigation
of staphylococcal PNA FISH without accompanying active
reporting of results and antimicrobial stewardship advice [30]
reflecting findings with other rapid molecular tests[31].
Table 1. Summary of molecular methods for diagnosis of BSI discussed in this paper.
Tested
Technology Principle matrix Targets Turnaround time Hands-on time Advantages Disadvantages
AdvanDx QuickFISH ® FISH BC (1) GN – 3 (E.coli, K. pneumoniae, P. aeruginosa) <30 min <5 min Cheaper than PCR Limited number of probes
(2) GP – 2 (CoNS, S. aureus) No amplification needed, No AST
(3) Enterococcal – E. faecalis, E. faecium or other less contamination
enterococcus
(4) FP – 3 C. albicans, C. parapsilosis, C. glabrata
Accelerate FISH and Morphokinetic BC GN – 8 (4 species) GP – 6 (4 species) 2 Candida spp. ID 90 min, 2 min Rapid phenotypic sensitivity Reliability in polymicrobial
PhenoTest ™ BC Cellular Analysis (C. albicans, C. glabrata) AST ~7 h Minimal hands on time infection not established
AST – MIC Broad panel
Not gram stain dependent
Xpert ® MRSA/SA BC Real-time PCR BC MSSA, MRSA (mecA/C) 62 min <1 min Minimal hands on time Expensive
Robust platform
BD MAX ™ MRSA Staph Real-time PCR BC MSSA, MRSA (mecA/C, MREJ) 2h <1 min More amenable to batch
SR/XT testing than GeneXpert
Cognitor ® Minus ETGA BC Registers nonspecific presence of bacteria/fungi *12 h after 12 h Not published Performed on signal negative Very labor intensive in current
BC incubation blood cultures form
VERIGENE® BC-GP + PCR + Microarray BC (1) GP – 13 (9 species), mecA, vanA/B 2.5 h 10 min Unreliable in polymicrobial
BC-GN (2) GN 9 – (5 species), CTX-M, IMP, KPC, NDM, OXA, VIM. infection
Biofire Filmarray® BC PCR + Microarray BC GP – 8 (5 species), GN – 11 (9 species.), 5 Candida spp. 1h 2 min Not gram stain dependent Limited microbial ID +
mecA, vanA/B, KPC resistance panel
ePLEX® BCID-GN + PCR + Microarray BC GN – 21 (17 species), CTX-M, KPC, NDM, OXA, VIM, IMP 1.5 h <2 min Fully automated
BCID-GP + BCID-FP + pan-GP, pan-Candida.
GP- 20 (13 species), mecA/C, vanA/B + pan-GN, pan-
Candida.
FP- 16 (13 species).
Curetis PCR + Microarray BC GP – 11 (9 species), GN – 15 (14 species), FP 8, 4h <2 min Largest panel which is not
Unyvero BCU ™ Mycobacterial spp., 16 resistance genes gram dependent
Master diagnóstica PCR + Microarray BC GP – 7, GN 10, Candida albicans, 20 resistance genes 3h Not reported Not gram stain dependent
sepsis flow chip Large resistance gene
panel
Fully automated platform
available
Bruker Sepsityper ® MALDI-ToF MS BC Database dependent 30 min 5 min
a
Lightcycler SeptiFast
® PCR + Microarray WB GP – 6 (4 species), GN – 10 (10 species), FP – 6 (6 6h Not reported Not culture dependent
species). Optional mecA.
a
SeeGene Magicplex ™ Endpoint & Real-time WB GP – 73, 3 resistance genes vanA/B, mecA 6h Not reported, 2.5 Not culture dependent Need separate instrument for
PCR GN – 12 (12 species), 6 fungal species h extraction extraction
T2Sepsis Solution™ NMR WB GP – 2 (2 species) + GN – 4 (4 species) on the same 3–5 h Not reported Not gram stain dependent
(T2Dx ) ® panel
FP (5 species)
Specific diagnostics VOC analysis BC Database dependent 5h Not reported Unknown Unknown
SpecID
a
™
Q-Linea ASTrID Padlock probe PCR + WB 10 Genus, 33 species, 11 resistance genes 4 h ID, 10 h AST Not reported No blood culture required:
® Microarray AST – MIC on whole bloodb
Suitable for POC use
Q-Linea ASTar™ BC AST – MIC 6h Not reported Requires independent
organism ID
PCR + Microarray WB Not published Not published Not published
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS

Qvella FAST™ ID
a
Does not require standard culture – see notes bas claimed by manufacturer, published data has undergone a 4 h “enrichment” culture prior to starting the assay. BC: Blood culture; WB: Whole blood; NMR: Nuclear magnetic
resonance; AST: Antimicrobial Susceptibility; MIC: Minimum inhibitory concentration; GP: Gram-positive; GN: Gram-negative; FP: Fungal panel; POC: Point of care; ETGA: Enzymatic template generation and amplification.
545
546 S. POOLE ET AL.

2.2. Accelerate Pheno™ System – PhenoTest™ BC Multiplexed assays are desirable diagnostic solutions
because multiple tests can be replaced by a single reaction
To have a dual rapid identification and phenotypic AST is a
which in turn reduces turnaround time and saves precious
diagnostic challenge and currently there is only one platform
patient specimen for further investigations. The post-amplifi-
that is both CE marked and FDA approved for in vitro diag-
cation stage analysis is achieved with microarrays which are
nostic use. The Accelerate Pheno™ System promises organism
less labor intensive and are conducive to automation, unlike
identification at 1.5 hours via gel-filtration, FISH, and generates
sequence analysis. Whereas broad-range assays can in theory
a quantitative phenotype a further 5.5 hours later via morpho-
detect the presence of any pathogen, multiplex assays are
kinetic cellular analysis [32,33]. A number of centers (including
limited by the size of the arrays typically 10–50 targets, but
ours) have evaluated the PhenoTest BC assay performance for
they still possess significantly more targets than pathogen
ID and AST versus our standard of care (SoC) and the results so
specific PCR assays. Multiplex assays also offer the ability to
far appear promising. From the studies published to date the
detect the presence of resistance genes which broad-range
Pheno™ System has a sensitivity/specificity of 88.7–100%/
assays cannot. When combining a procalcitonin (PCT) test
97.1–100, EA and CA of 94.3–97.9%/95.1–97.6% (Minor Error
and/or other suitable biomarkers with PCR diagnostics, clinical
(MiE) 0–4.8%, Major Error (ME) 0.3–2.3%, Very Major Error
utility of these assays greatly increases [43].
(VME) 0.5–1.0%) [34–37].
The rapid results that are generated by the Pheno™ System
versus SoC have seen a reported dramatic decrease in the 3.1. Laboratory validated assays
turnaround time (TaT) of results. Marschal et al reported a
Developing a molecular test can ensure you are specifically
mean decrease in ID reporting by 27.47 hours and mean
answering a local diagnostic need. The cost is often signifi-
decrease in AST by 40.39 hours versus SoC [35]. They also
cantly smaller than purchasing one from a vendor and ulti-
had success with a number of correctly reported E. coli
mately the control and target choice is yours. However, there
extended spectrum beta lactamases (ESßLs) and multi-drug
are a number of hurdles that have to be overcome to collect
resistant (MDR) P. aeruginosa. They also report that the
these benefits. Regulatory and quality requirements of a
Pheno™ System detected further pathogens that would other-
laboratory developed test (LDT) can be complex and time
wise have been reported as monomicrobial by SoC, this has
consuming and the initial expertise needed for development
been our experience as well.
is not always available in the clinical microbiology department.
To date there has been no published data relating to the
In the UK, Public Health England has developed a number of
improved stewardship or clinical outcomes of the Accelerate
algorithms and standards for microbiological investigations
Pheno™ System, but locally we have made a number of
(SMIs) including those involved in transitioning a LDT into
clinical decisions and antimicrobial interventions using the
clinical service [44].
system, especially in the context of resistant Gram-negative
Blood is a complex biological matrix and harbors many
pathogens. This has included de-escalation and patient speci-
components that are inhibitory to PCR including both haem
fic targeting of antimicrobial therapy in sepsis and reducing
(from lysed erythrocytes) and immunoglobulins. It is crucial
our burden of broad-spectrum antibiotics. Our manuscript for
the extraction, buffer and DNA polymerase utilized must be
the clinical impact analysis of the Pheno™ System is currently
robust enough to deliver an assay with reproducible perfor-
being prepared.
mance. The Phusion Blood Direct PCR Kit by ThermoFisher [45]
is a good example of such a development that could be
utilized by a LDT [46]. A second approach would be to validate
3. Nucleic acid amplification of signal positive blood a previously developed PCR for use on signal positive blood
culture – polymerase chain reaction (PCR) cultures that had not been initially indicated by the original
developers. This is an approach we at Hampshire Hospitals
The portfolio of diagnostic PCR strategies that are currently in (HHFT) are undertaking using the r-biopharm RIDA®GENE
use comprise pathogen specific, broad-range, and multiplex
MRSA [47] real-time PCR. This approach could open up avail-
assays [38]. The fundamental goal that each method shares is able diagnostic real-time PCR assays for use on signal positive
to amplify pathogen DNA against a background of host DNA.
blood cultures, but increased awareness of quality procedures
Pathogen specific assays employ genus/species level detection
is crucial to avoid issues with regulatory authorities.
for the likes of Candida spp. or MRSA but are traditionally
limited to <6 targets due to technical limitations of the ther-
mocycler optics. 3.2. Cepheid GeneXpert – Xpert® MRSA/SA BC
Universal broad-range assays amplify conserved regions of
The GeneXpert is a modular platform combining nucleic acid
the isolated microorganism; usually 16S/18S rRNA [39] or other
extraction and amplification that can be expanded to fit clin-
housekeeping genes which help broaden the scope of patho-
ical laboratory requirements [48]. This simple to answer solu-
gen detection. However, there is an associated post-amplifica-
tion provides the ability to detect pathogens within 1 hour
tion stage to complete the identification when using this
from sample preparation from numerous specimen types. The
technique that requires sequencing of amplicons [40];
Xpert® MRSA/SA BC real-time PCR assay requires minimal
Molzym have developed the SeptiTest™ [41,42] workflow as
hands-on-time, <5 mins, and can be operated by a non-labora-
an example. This approach can help to elucidate culture nega-
tory specialist directly on a signal positive blood culture fol-
tive/fastidious pathogens from negative blood cultures.
lowing an indicated Gram-stain [49–52]. It targets the Spa,

SSSmec cassette and mecA genes with a sensitivity/specificity
of 100%/98.6% for S. aureus and 98.3%/99.4% for MRSA,
respectively. The Xpert® MRSA/SA BC assay provides the infec-
tion specialist with a powerful tool for rapid antibiotic inter-
vention in areas with high prevalence of MRSA bacteremia or a
de-escalation stewardship tool if MRSA antibiotics constitute
part of empirical regimen.
3.3. BD MAX™ – StaphSR and the new MRSA XT assay
The BD MAX™ System is a fully-integrated and automated
platform that performs nucleic acid extraction and real-time
PCR with <5 min sample preparation time. The sensitivity and
specificity of the SR assay has been reported 100%/100% for S.
aureus, and 97.9%/98.1% and 99.0%/95.8% for MRSA and
coagulase-negative staphylococci, respectively [53,54].
According to the manufacturers the new MRSA XT assay
has been designed to detect the novel MREJ types [55,56] not
detected by other molecular assays as well as MRSA strains
with the novel mecC gene which can account for 3–4% of all
cases [57]. It can also detect mecA dropouts which can make
up to 18% of all positive MRSA results reported by other
molecular assays which in fact are actually false positives
[58,59]. This platform lends itself to batch testing with its
medium throughput capacity compared to the GeneXpert
and can be operated by junior laboratory staff thus lowering
costs.
3.4. Momentum bioscience – Cognitor® Minus
Cognitor® Minus uses enzymatic template generation and
amplification (ETGA) technology [60,61] that has been devel-
oped to detect microorganisms by measuring nucleic acid
modifying enzymes present within their cells [62,63]. This
elegantly unique approach provides a rapid and sensitive
universal detection of all bacterial and fungal species present
in blood culture on any standard thermocycler likely to be
already present in a standard clinical laboratory. It does not
rely on a preselected panel of microorganisms so is free to
detect any viable pathogens present. Initial clinical evaluation
performed locally by Dryden et al has shown that this early
“rule out” of a BSI with a negative predictive value of 99.5%
versus 5 day negative blood cultures can be a powerful tool in
antimicrobial stewardship and patient management [64]. In its
current form this test is quite laborious and requires specialist
laboratory staff; however a rapid automated version of this
assay that can be used directly on whole blood (WB) without
the need for culture is imminent.
4. Post-nucleic acid amplification detection –
microarray and sequencing approaches
Microarrays offer the potential for a highly multiplexed diag-
nostic solution. They combine the utility of PCR amplification
and hybridization to oligonucleotide probe arrays of differing
sizes. The true microarrays were developed for the analysis of
differential gene expression [65,66] with probes numbering
tens of thousands, so in reality for the purist, the arrays used
in diagnostics should be referred to as macroarrays. The
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 547
solutions discussed offer differing amounts of automation
from very separate distinct stages present in the VERIGENE®
to the GenMark DX ePlex® fully automated sample-to-answer
solution.
The use of sequence analysis as a post-amplification tool
has been achieved and offers a very broad-range of pathogen
detection [14,32,42,67–69]. However, it is neither novel nor
rapid and only has a role in a reference laboratory in its
current format. Deurenberg et al review the potential future
impact of next generation sequencing (NGS) in clinical micro-
biology and how advancements in assay design and data
analysis improvements could one day translate to appropriate
diagnostic assays for clinical microbiology [70].
4.1. Luminex nanosphere VERIGENE® – BC-GP & BC-GN
The VERIGENE® combines automated nucleic acid extraction
and PCR amplification on the ProcessorSP followed by hybridi-
zation to Nanogrid arrays with gold nanoparticle detection.
The manufacturers claim that the gold nanoparticle is more
sensitive than fluorophore detection. The platform work flow
requires a Gram-stain to select the appropriate assays and is
not fully automated so would need to be placed within a
clinical laboratory [71].
This platform has a portfolio of different species and genus
probe targets for both Gram-positive organisms (92–99%
agreement with conventional methodology) [72–74] and
Gram-negative organisms [75–77]. The assay time is rapid,
taking approximately 10 min of hands on time followed by a
2.5 hour run. The VERIGENE also detects common resistance
genes with a high level of accuracy.
At this time there is limited published data about the
clinical impact of these platforms. Stewardship reviews of the
Gram-negative panel suggest optimization of antimicrobials
can occur considerably earlier [75,78]. This is reflected in stu-
dies with the Gram-positive portfolio [72] albeit in a demo-
graphic of patients with high rates of MRSA, fortunately
something which is less prevalent in our practice in the UK.
In high resource settings there has been demonstrated
improvement in length of ICU stay and 30 day mortality
when using these platforms [77]. However a study of 168
episodes of bacteremia in the US found no significant change
in clinical outcomes (mortality and length of stay) when using
the Gram-positive platform compared to standard of care,
albeit with significantly improved time to initiation of appro-
priate antibiotics with a 24 hour reduction [79]. This reflects
research on other rapid diagnostic platforms that suggest
rapid turnaround times are irrelevant if not coupled with
good, timely stewardship advice.
According to manufacturer’s information and published
data [72] these assays are unreliable in the detection of organ-
isms from polymicrobial cultures.
4.2. BioMeriéux – Biofire® FilmArray®
This platform provides a sample to answer solution with syn-
dromic panels that do not require a Gram-stain. The ease of
use and simple work flow lends itself to be used at point-of-
care and out-of-hours with extraction, PCR amplification and
548 S. POOLE ET AL.
array hybridization occurring within a sealed pouch. The run
time is 60 min and requires <5 min of assay pouch preparation
involving hydration of reagents and inoculation of signal posi-
tive blood culture [80].
The targets on the blood culture panel comprise 6 Gram-
positive (incl. Listeria monocytogenes, Streptococcus pyogenes,
Streptococcus pneumoniae), 10 Gram-negative (incl. H. influen-
zae, N. meningitidis, K. pneumoniae), and 5 Candida species,
plus mecA, vanA/B and KPC resistance genes. It is a well-
established assay and platform with many evaluations and
technology comparisons in the published literature [81–85]
reporting sensitivity and specificity of 89.4–91.6%/100%. In
comparison to the other array based assays the panel is lim-
ited in size but has more variety; with the likes of N. meningi-
tidis and L. monocytogenes included that are not often present
on similar platforms but is lacking with the selection of resis-
tance genes offered. It has seen clinical evaluations where
rapid ID has led to appropriate de-escalation of antibiotics
<14 hours faster [84]. However, having a full BC assay without
the need for Gram-stain driven selection of appropriate panels
is an advantage especially if the proposed use in outside of a
clinical microbiology laboratory like in ED/ITU or an acute
assessment ward (AAU). A new BCID panel is under develop-
ment and is undergoing beta-testing by Blaschke et al that
incorporates >25 microorganisms [86].
4.3. GenMark DX – ePlex® BCID
This is a highly multiplexed fully automated one-step single-
use cartridge assay system that won a silver medal at the 2017
Medical Design Excellence Awards [87]. Unlike the previous
two platforms the reaction cassette only requires inoculation
and placing in the control module. There are three options
available for signal positive blood cultures post-Gram stain:
BCID-GP, BCID-GN and BCID-FP. The Gram-negative assay has
21 targets, 6 resistance genes and a pan-GP and pan-Candida
target, the Gram-positive assay has 20 targets, 4 resistance
genes, pan-GN and pan-Candida target. The fungal assay has
16 targets. There only appears to be one published review of
the ePlex® system, and this details the BCID-FP assay [88].
Despite requiring a Gram-stain for selection of the correct
assay it does have the helpful feature of having pan-targets
present to capture a pathogen from a misread Gram-stain or
an unrecognized polymicrobial blood culture.
4.4. Curetis Unyvero™ – BCU
The Unyvero™ modular concept provides a sample to answer
solution in 4 hours using a cartridge that according to the manu-
facturer contains more components than any other on the market,
“eight labs on eight chips in a brick”. Targets include 10 Gram-
positive (including e.g. S. aureus, S. pyogenes), 15 Gram-negative
(e.g. E coli, K. pneumoniae), Mycobacterium tuberculosis, eight fun-
gal, and 16 resistance genes. This is the largest panel currently on
offer which can be used without the need for a Gram-stain making
it potentially more suited to a point-of-care application outside of
laboratory hours in ED/ITU or AAU. Currently clinical evaluations
are being undertaken in Germany and the US but there is no
published clinical application data at this time [89].
4.5. Master Diagnóstica – Sepsis Flow Chip assay
The Sepsis Flow Chip (SFC) assay comprises a multiplex PCR
and hybriSpot automated reverse hybridization to a low den-
sity DNA array [90]. It can detect seven Gram-positives (incl. S.
aureus, S. pyogenes, L. monocytogenes), 10 Gram-negatives
(incl. E. coli, P. aeruginosa, N. meningitidis) Candida albicans,
and 20 resistance genes. Galiana et al have done an early
evaluation of the assay reporting a sensitivity and specificity
of 93.3%/100% for ID and 93.6%/100% for the resistance
genes [91]. They conclude that the system is user friendly
and easy to interpret the results.
5. MALDI time-of-flight mass spectrometry
The use of MALDI-TOF MS for detection of bacteria and fungi
isolated from patient clinical specimens has been well docu-
mented and has revolutionized the work flow in clinical micro-
biology laboratories [92–96]. The most common MS solutions
in use are Bruker Daltonics Biotyper® (MBT) and bioMérieux
Vitek® MS which are becoming the workhorses of many clin-
ical microbiology laboratories after recent universal adoption
[93]. The protein spectral databases of microorganism are
increasing for each MS solution and the identification that
MALDI-TOF MS provides is as good as currently available
biochemical and nucleic amplification tests [92].
The desire for rapid diagnostics on signal positive blood
culture has seen attention focused on using MALDI-TOF MS.
The use of this technique direct on blood is more challenging
that on other less complex specimen types due to the complex
matrices contained within a blood culture bottle that interferes
with the generation of good quality spectra [97]. Numerous
groups are publishing peer-reviewed studies of their own labora-
tory developed methods to remove the need for traditional sub-
culture. The two most common methods are either lysis/extrac-
tion methods on a prepared bacterial/fungal pellet [98,99] or
from a novel rapid <6 hours subculture approach using pre-
warmed solid media first described by Idelevich et al [100–102].
Due to the time sensitive nature of a signal positive blood culture
report a direct subculture independent method is the most rapid
and desired approach. This method has been compared to post-
blood culture PCR assays with similar results at a fraction of the
cost [85]. The sensitivity of this direct blood culture MALDI-TOF
MS analysis has been established in the region of 80–90%
[103,104]. It must be noted that if you delve into the finer details
of these data Gram-negative sensitivity and concordance at
genus/species level is much higher. The main issues MALDI-
TOF MS has is distinguishing viridans streptococci from S. pneu-
moniae [105] which accounts for the Gram-positive scores
becoming significantly lower. These problems are compounded
by the background noise even the best sample preparation
method leaves behind. The quantity of lower m/z peaks causes
a lowering of the quality score because they are not present on
the corresponding microorganism spectra in the manufacturer’s
database. This problem is alleviated by the introduction of the
blood culture analysis software module on the Bruker Biotyper. It
must be noted that the analysis of the generated protein spectra
is only as good as the database that is interrogated.

5.1. Bruker Biotyper – Sepsityper® kit
This is essentially a sample processing kit to enable the stan-
dardization of the sample preparation process associated with
direct analysis of signal positive blood cultures. There are
numerous laboratory developed methods but this has led to
inconsistent sensitivity and specificities reported and most are
no more than research tools. When compared to general
standard of care of sub-culture and classical phenotypic iden-
tification methods the Sepsityper® kit generates a genus/spe-
cies concordance with SoC of 94.7–100%/94.7–100% for Gram-
negatives and 91.7–93.3%/70–73.1%- for Gram-positives when
using the blood culture analysis software module [106].
Morgenthaler et al have completed a comprehensive meta-
analysis of studies involving the Sepsityper® kit [107] which
makes good reading. They conclude having a standardized CE-
IVD marked kit for processing of signal positive blood cultures
does remove the regulatory requirements associated with
using a laboratory developed method despite the cost impli-
cations involved.
5.2. MALDI-TOF MS and antimicrobial susceptibility
testing (AST)
It is not surprising that the use of MALDI-TOF MS is being
explored for the generation of a phenotypic AST. We have
discussed the positive impact it has had in routine clinical
microbiology and now more so in processing of signal posi-
tive blood cultures. The majority of the methods that are
being developed are very much in their infancy and are
presented as research tools but represent some exciting
uses of MALDI-TOF MS. DeMarco et al, Faron et aland Vella
et al provide well written reviews on the potential
approaches the MALDI-TOF MS could use in generating a
phenotypic AST [108–110]. There are four avenues in devel-
opment that appear to be the most popular [92] (i) Detection
of the modification of an antibiotic i.e. the hydrolysis product
of a ß-lactam demonstrated by the MBT-STAR assay [111] and
the newly CE-IVD marked MBT-STAR Carba assay from Bruker
for rapid detection of Class A, B or D carbapenemase activity
in Gram-negatives [112] (ii) Detecting the evidence of meta-
bolism of a pathogen in the presence of an antibiotic, dead
(S) or alive (R), MBT-RESIST assay [113], (iii) Detection of an
antibiotic action on an antibiotic target site [114], (iv) Semi-
quantification of the presence of intracellular or extracellular
antibiotic, the MBT-ASTRA assay has been shown to produce
equivalent MICs to ETEST® MICs on the antibiotics that were
tested by Sparbier et al [115].
Whichever further methods progress through standardiza-
tion and clinical evaluation they would need to be comparable
to the ‘gold standard’ broth microdilution (BMD) method to
pass the regulatory requirements set out by EUCAST and CLSI
like the MBT-STAR Carba assay.
6. Direct – blood culture independent (whole blood)
Traditional blood culture processing ‘the gold standard’ is
widely used in routine diagnostic laboratories, however patho-
gen identification can take 24–48 hours or even longer, with
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 549
recommended 5 days incubation to report a negative.
Sensitivity for acutely ill patients is ~70% and even lower for
fastidious microorganisms [116]. Analysis of patient whole
blood without the need for blood culture has the potential
to dramatically decrease the time to pathogen identification
versus blood culture. These would be used on patients pre-
senting in the emergency department or symptomatic inpati-
ents. The amplification of the pathogen signal during real-time
PCR is crucially important owing to the low numbers of circu-
lating bacteria, 10 CFUs/ml or fungi, 1–10 CFU/ml, reported in
adult sepsis. Direct molecular assays also facilitate detection of
fastidious, difficult-to-culture organisms such as fungi and
common intracellular bacteria.
6.1. Roche LightCycler® – SeptiFast test
There are many studies evaluating its diagnostic performance
and clinical impact owing to the many years it has been
available [117–122]. However, in the UK it was the subject of
a recent Health Technology Assessment study [43] commis-
sioned by the UK’s National Institute for Health Research
(NIHR). The sensitivity and specificity from this study was
50%/85.8%, which was lower than the pooled data from the
meta-analysis however this was a larger well planned multi-
center prospective evaluation than those previously under-
taken. As a result its recommendation was the SeptiFast was
not suitable to replace blood culture, but could be used to
augment current SoC in place within clinical microbiology. A
positive result could be acted upon without waiting for blood
culture to signal positive but a negative result did not indicate
an absence of pathogen.
6.2. SeeGene – Magicplex™ sepsis
SeeGene have created a full process for analysis of whole
blood incorporating pathogen DNA extraction with the
SelectNA blood pathogen kit automated on the SEEPrep12.
The extracted DNA is amplified using the novel combination
of endpoint PCR and real-time PCR assay that offers a highly
multiplexed assay without the need for blood culture. The
pathogen identification and possible presence of resistance
genes is reported at 6 hours. The panel consists of 73 Gram-
positives, 12 Gram-negatives, mecA, vanA/B and six fungi,
providing 90% coverage of sepsis related pathogens. An
early studies show that it can applied in hospital adult and a
pediatric populations but as yet there are no further published
clinical evaluations [123,124].
6.3. T2 biosystems – T2Dx® T2Candida® & T2Bacteria®
The T2Dx platform is a novel diagnostic technology that uti-
lizes nuclear magnetic resonance as the detection mechanism
with a run time of 3 hours [125]. Two assays are available on
the T2Dx: T2Bacteria®, and T2Candida®. The T2Bacteria® iden-
tifies six bacterial species (E. coli, K. pneumoniae, P. aeruginosa,
Acinetobacter baumannii, S. aureus, and Enterococcus faecium)
with at least two of these often not covered by empirical
therapy. The T2Candida® identifies five Candida species (C.
albicans, Candida parasilosis, Candida krusei, Candida tropicalis
550 S. POOLE ET AL.
and Candida glabrata). Together both panels cover 90% of
Gram-negative and 70% community acquired infections
encountered in the emergency department (ED) [126] where
we believe this would be the perfect placement of the system.
At this time only the T2Candida® has been the subject of
analytical and clinical evaluations [127–130]. The large pro-
spective study involving the T2Candida® conducted by
Clancy et al [131] reported a clinical sensitivity of 89% com-
pared to blood culture and that T2 positivity was associated
with current antifungal therapy, neutropenia and C. albicans
BSI. The T2Bacteria® assay has yet to have any performance
data published but having both assays in use outside of the
clinical microbiology laboratory in ED/ITU operated by non-
specialized staff on whole blood would dramatically decrease
diagnostic uncertainty and help facilitate rapid targeted
treatment.
7. Novel diagnostic solutions and future directions
When reading published literature there are many novel and
ingenious proposed solutions to improve diagnostics of BSI.
For example, Templer et al describe a proof of concept micro-
array coupled with a Surface Plasmon Resonance Imager (SPRi)
to detect bacteria in clinical specimens [132] with a limit of
detection of 1 CFU/ml of blood. This would make it suitable for
detection of a BSI on whole blood without the need for
culture. Another on the horizon is the utilty of NGS within
clinical microbiology, moving out of the reference laboratory
and closer to the patient. However, there are a number of
hurdles to overcome first like the adoption of a universal
library preparation method to facilitate standardization and
creation of a user friendly data analysis interface for a start.
Resources will undoubtedly be directed to streamline NGS
where the unbiased nature of metagenomic analysis will
enhance the breadth of pathogen detection, especially help-
ing in cases where conventional diagnostics have not suc-
ceeded [133,134]. We have seen targeted NGS used in near
real-time during the Ebolavirus pandemic 2014–2016 when
the Oxford Nanopore MinION was used in the field to
sequence clinical specimens for Ebolavirus surveillance toward
the end of the outbreak with great success [135].
Many of these novel approaches are not yet close to com-
mercial development and therefore not ready for a routine
clinical diagnostic use. Here we discuss a few exciting plat-
forms that have made it to early analytical and clinical
evaluation.
7.1. Headspace volatile organic compound (VOC)
analysis – SpecID™
As bacteria and fungi respire in blood culture they inevitably
emit the waste products of this process as they divide and
multiply [136–138]. The headspace present in the blood culture
bottle has been analyzed [139] for unique signatures of these
microorganisms and data has shown that this signal can be
diagnostic [140,141]. This technology has been adapted by
Specific Technologies to a laboratory based diagnostic test
called SpecID™ that promises pathogen identification from sig-
nal positive blood culture in 5 hours. It is based on detection of
VOCs via a colorimetric array [142,143]. Full clinical diagnostic
performance has yet to be established but this represents an
exciting development in clinical laboratory medicine.
7.2. Q-Linea- ASTrID® & ASTar™
Q-Linea are offering two novel diagnostic and quantitative
AST phenotypic solutions, one from whole blood, ASTrID®
and one from signal positive blood cultures, ASTar™. The
ASTrID® generates pathogen identification using highly spe-
cific and selective padlock probes [144,145] forming circular-
ized DNA strands[146] that are amplified via rolling-circle
amplification (RCA) [147] and subsequent circle-to-circle
amplification (C2CA) [148]. The amplified products are fluores-
cently labelled and detected on a microarray. A poster at the
ASM 2017 highlighted an evaluation of the ASTrID® by Morck
et al in Sweden [149]. Identification was performed on 4 hours
incubated blood cultures. Sensitivity and specificity versus
blood cultures taken at the same time from patients attending
an infectious disease ED in Örebro, Sweden was 97%/99.6%.
The AST was performed on signal positive spiked blood cul-
tures and reported a 96% essential agreement (EA) and 95%
categorical agreement (CA) compared to reference broth
microdilution (BMD).
7.3. Qvella – FAST™ ID
This is a novel way of addressing the sensitivity issues surround-
ing direct PCR on whole blood. Qvella has developed the FAST™
ID technology to rapidly concentrate bloodstream pathogens
and release their nucleic acids electrically. While groups focus
on developing more sensitive detection methods to enhance the
performance of their assays, Qvella has focused on optimizing
sample preparation via automation of isolation, concentration
and lysis of a small number of pathogens in a whole blood
sample. This unique approach is designed to achieve rapid and
automated pathogen identification in minutes, not hours, and is
expected to play a key role in enabling earlier tailored antimicro-
bial treatment. This system is currently not available to purchase
but could provide an exciting diagnostic option if placed in the
emergency department to help stratify patient management and
flow [150].
8. Expert commentary
Here we present a number of novel technologies and techniques
that are either available or close to market. Clinical microbiology
aims to provide rapid and accurate diagnostics to clinicians to
improve patient outcome. This is particularly relevant in BSI in
order to assist targeted antibiotic therapy and reduce the use of
unnecessary broad-spectrum antibiotics.
In light of the pressures associated with continual improve-
ment within financially challenged healthcare systems it is
vital each new iteration of a diagnostics test be faster, easier
to use, more sensitive/specific and affordable. If it cannot
provide all of these features it must at least have a comparable
diagnostic performance, be more rapid and demonstrate an
economical benefit otherwise laboratories will have major
challenges to adopt them. This has been a particular hurdle

in the adoption of molecular assays with are generally con-
siderably more expensive than traditional methods. Culbreath
et al [151] propose a six-step model for test adoption that
appraises a whole process approach from ordering of the test
to reporting of the actionable result and shows much merit.
The performance of blood culture as a diagnostic test is
made up of multiple components extending well beyond the
diagnostic assays including pre-analytical (arm to incubation),
analytical (incubation to result) and post-analytical (commu-
nication of result) stages. It is important in an era of revolu-
tionary molecular diagnostics in BSI that we do not lose sight
of the need for robust pre- and post-analytical systems. The
UK SMI [152] recommended a 4-hour target for incubating
collected blood cultures which is often breached out of nor-
mal working hours. Optimization of the pre-analytical stage
has been shown to improve turnaround time [153,154]. This
can be achieved by either having a 24 hour service in the
hospital with a pan-pathology approach [155] or by placing a
satellite incubator in ED or ITU [156], especially effective at
sites that operate a “hub and spoke” clinical laboratory system.
Quality is an integral part of clinical microbiology and numer-
ous schemes are in place to ensure a fit for purpose service is
being delivered. External quality schemes (EQA) are one of the
tools employed in this process. However, the EQA schemes can be
slow to adapt to novel technologies and so this important quality
tool is not always available. Local laboratories could initiate inter-
lab exchange schemes in lieu of an EQA and offer to collaborate
with national schemes and manufacturers to set one up.
During the review preparation it has become apparent that the
standard of the evaluations of new assays and platforms are of
differing quality. This makes it problematic to compare the studies
to present fair comparisons. Despite the Standards for Reporting
of Diagnostic Accuracy Studies (STARD) guideline being updated
in 2015 to harmonize the reporting of such studies there appears a
lack of uptake. The guidelines were written to allow the readers to
judge the trustworthiness and applicability of study findings [157]
and to facilitate easier comparisons. Blood culture is universally
accepted to be insensitive which can be affected by the presence
of fastidious organisms or antibiotic pre-treatment. Novel mole-
cular tests are designed to be more sensitive, herein lies a problem
for designing a robust technology evaluation. The published stu-
dies we have reviewed have often reported a molecular test as
positive versus a negative blood culture. These are difficult to
interpret, where dead bacterial DNA may be muddying the waters.
The increased quality standard would encourage better designed
studies where the imperfect “gold standard” comparator of blood
culture could be augmented with biomarkers and clinical details
and a third test, like sequencing would be used to help interpret
the discrepant results. We hope the diagnostic community uni-
versally adopts STARD to ensure future research and technology
evaluations are of a higher quality.
9. Five-year view
The landscape of traditional diagnostic laboratories is chan-
ging; there is a major emphasis in the UK and elsewhere in the
developed world on laboratory mergers and consolidations.
Parallel to this there is advance in diagnostic technology,
devices and innovative point-of-care products which are
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 551
aimed to complement clinical decision-making at the patients’
bed side. These devices do not require operation by laboratory
professionals, but by staff that are trained and in direct con-
tact with the patients. We see molecular testing moving more
toward near-point testing where clinical decisions can be
influenced in real time.
All of the reviewed molecular tests dramatically improve the
turnaround time of results with high levels of sensitivity and
specificity versus traditional culture methods. In spite of this,
evidence showing improvement in clinical outcomes is sparse
and there is evidence that this impact is minimized without
adequate support of clinicians from antimicrobial stewardship
teams. Moving forward infection specialists must develop robust
systems to communicate the significance of results to clinical
teams so that the impact can be maximized. Further studies are
needed to shed more light on the effectiveness of these tests,
both in financial terms and clinical outcomes. We hope collea-
gues who work on these technologies and or evaluate them
clinically are able to publish and share these data as we aim to
do with our Accelerate Pheno™ System clinical evaluation.
Direct testing of whole blood is the most exciting develop-
ment on the horizon. If reliable pathogen identification and a
quantitative phenotype AST is achieved this will have an
undoubted impact in patient outcomes in sepsis. Currently
developments have not replaced blood culture but will aug-
ment the portfolio of tests at the infection specialist disposal
to diagnose and treat BSI. The reduction in diagnostic uncer-
tainty in sepsis and BSI is the key for all.
Key issues
● There is an unmet need to reduce diagnostic uncertainty in
sepsis and BSI.
● The use of novel molecular technologies offers solutions to
augment patient care and reduce mortality.
● The increased speed and sensitivity offered by this technol-
ogy requires robust post-analytical systems in order for
clinical and economic impacts to be maximized.
● There is a need for higher quality, standardized appraisal
studies evaluating new diagnostic technologies.
● Novel diagnostics direct on whole blood is becoming an
exciting field.
Funding
This paper was not funded.
Declaration of Interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties. Peer
reviewers on this manuscript have no relevant financial or other relation-
ships to disclose.
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