Clinical
Microbiology
N e w s l e t
Vol. 42, No. 7
April 1, 2020
www.cmnewsletter.com
I n Th is Issu e
53 Metagenomic
Next-Generation
Sequencing in Clinical
Microbiology
Corresponding author:
George S. Watts, Department
of Pharmacology, University of
Arizona Cancer Center, Room
3927, 3838 N Campbell Ave.,
Tucson, AZ 85719. Tel.:
520-626-4724. E-mail:
gwatts@email.arizona.edu
0196-4399/©2020 Elsevier Inc.
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Metagenomic Next-Generation Sequencing in
Clinical Microbiology
George S. Watts, Ph.D. and Bonnie L. Hurwitz, Ph.D., University of Arizona, Tucson Arizona
Abstract
Identification of causative pathogens in infectious disease is a critical component of health care, as
infectious diseases continue to be a leading cause of mortality and morbidity worldwide. In addition,
the detection of drug resistance by traditional and novel antimicrobial susceptibility testing methods
is becoming ever more important as antimicrobial resistance continues to emerge and spread. While
culture-based methods are the current reference standard for identifying microbes, the time required to
achieve results and the difficulty in culturing certain fastidious organisms have led to the development
of multiple alternatives. Alternatives to culture, such as polymerase chain reaction (PCR) assays, sero-
logic assays, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF
MS), and 16S rRNA gene sequencing have been used in clinical laboratories, and positive impacts on
patient care have been documented. Nonetheless, such methods require either presumption about the
type of microbes present in the sample or growth of the organism(s) before analysis. Subsequently, there
are some documented limitations in the routine detection methods for clinically relevant organisms.
In contrast to the routine alternatives listed above, modern nucleic acid sequencing platforms sup-
port sequencing of random DNA strands, an approach known as “shotgun” sequencing of the DNA,
also known as metagenomic next-generation sequencing (mNGS) present in the sample. The mNGS
approach offers an unbiased and hypothesis-free approach to pathogen identification with the future
potential to (i) achieve results in 12 to 24 hours; (ii) avoid the challenges associated with growth of
fastidious organisms; (iii) avoid the biased growth that occurs when only routine culture medium is
used; (iv) detect viral, fungal, and parasitic organisms in the same assay; and (v) detect the presence
of drug resistance genes. Advances in mNGS technology and data analysis have reduced testing costs
to the point where the potential advantages of mNGS over culture and other methods warrant its
development for use in clinical settings. In this review, the mNGS approach is discussed, along with a
comparison to other methods, limitations, and suggestions for further development and overcoming
hurdles to adoption in the clinical setting.
Background culture methods and a variety of alternative
methods, the causative agent in many infections
Identification of pathogens associated with infec-
often remains unknown. Routine culture can
tious diseases can provide valuable diagnostic
have a high false-negative rate in certain disease
information, which in turn can guide treatment.
Prompt and appropriate antimicrobial therapy states. The problem was highlighted in a study
is of great importance, particularly when the of 3,756 neutropenic patients that found only
patient is immunocompromised, whether due to 609 of all blood cultures collected (~20%) were
age, disease (e.g., infection with human immu- culture positive [1]. As one would expect, the risk
nodeficiency virus or diabetes), or medication of death associated with bloodstream infections
(e.g., chemotherapy or treatments for rheumatoid varied by the patient’s immune status and the
arthritis). Despite the widespread use of routine causative microorganism. Of note, the hazard
Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier 53
ratio of dying was nearly four-fold higher in culture-negative
patients than in patients for whom no blood culture was collected
and submitted, presumably due to lack of symptoms suggestive of
bacteremia or sepsis [1]. Since blood cultures are only capable of
detecting cultivatable bacteria and fungi, it is not surprising that
other sources of infection can be overlooked using current blood
culture-based technology.
Lacking knowledge of the identity and antimicrobial suscepti-
bility of the causative pathogen, clinicians use broad-spectrum
antibiotics as the first line of therapy, whether using blood cul-
tures or mNGS. Unfortunately, empiric therapy does not always
adequately treat the causative pathogen. Perhaps most notably
for immunocompromised patients, the higher incidence of fun-
gal and viral infection exacerbates the mismatch between therapy
and pathogen because the infection may be initially assumed to
be bacterial in origin.
Transitioning from culture to mNGS may improve the frequency
and speed with which pathogens are identified in patients with sus-
pected infections. By providing clinicians with evidence to inform
rational antimicrobial choices and guide treatment, mNGS could
benefit patients and society through improved patient outcomes,
reduced health care costs, and better antimicrobial stewardship.
As such, mNGS of pathogens from whole blood and other sample
types have been referred to as the “holy grail” of infection diagno-
sis [2]. Not surprisingly, the development of mNGS is currently
being explored for used in clinical applications, with successes in
meningitis [3], culture-negative sepsis [4], febrile neutropenia [5],
and nosocomial transmission of drug-resistant Klebsiella pneumoniae
[6]. Further development has included the use of rapid, portable
sequencing technology [7]. In particular, the use of mNGS to
track a carbapenem-resistant Klebsiella sp. outbreak demonstrated
the epidemiological value of mNGS in tracking the sources of
outbreaks [6]. Lastly, the first prospective study of mNGS was
reported in meningitis, with positive results [3]. Adoption of
mNGS not only can improve individual patient outcomes, but
also enhances hospital and community antimicrobial stewardship
to protect the potency of currently available antimicrobials [8].
Several reviews were previously published on the topic of mNGS,
Figure 1. Schematic of mNGS workflow showing the three principal steps: (i) isolation of the DNA from the sample with or without enrichment for
microbial DNA, (ii) creation of a library and sequencing, and (iii) analysis of the sequence reads to determine the organisms present, their relative
abundances, and the presence of drug resistance genes.
54 Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier
with emphasis on the technology and applications [9], regulatory
and interpretation issues [10], and hurdles to implementation [11].
The mNGS Approach
Most commonly, mNGS analysis directly from clinical samples
involves isolating DNA; determining its sequence; and then ana-
lyzing that sequence in the context of a reference database to deter-
mine which organisms are present, and their potential phenotypes,
based on their genetic content (Fig. 1). Of note, if only DNA is
extracted, then many viral pathogens will not be detectable.
Immediately, several questions come to mind related to nucleic
acids, testing, and sequence reads (a sequence read is the string
of nucleotides identified from a fragment of the genomes pres-
ent in the sample). The following are examples of such questions.
How should the DNA be isolated? How many sequence reads
are needed? How should the sequence reads be analyzed? What
are the assay’s limits of detection? What is the false-positive rate?
Consideration of the goals of the analysis and the sample type
being analyzed allows some initial answers and becomes a starting
point for mNGS design and analysis. For example, the genome
sizes in question ranges from a few thousand base pairs in the case
of viruses to a few million for bacteria to approximately 15 mil-
lion base pairs for clinically relevant fungi (e.g., Candida albicans).
If we assume that 5 megabases is a reasonable genome size esti-
mate for bacteria and conservatively assume there will be 100-bp
read lengths, then 50,000 reads would provide 1× coverage of the
genome, meaning that on average each nucleotide in the genome
will have been sequenced once. If we prefer 10× coverage of the
genomes present in a sample that could contain 10 microbes, then
the number of reads required becomes 5 million—easily achiev-
able on several NGS platforms. Sequencing platform outputs have
been reviewed by Deurenberg et al. [9].
To date, most mNGS studies have selected an approach in which
the sample is not enriched for microbes, resulting in the great
majority (often >99%) of the sequence reads being derived from
the (human) host. With so much of the data generated being
uninformative from the standpoint of identifying microbes, the
approach is inefficient, increases costs, and reduces diagnostic pos-
sibilities [12], although it has been argued that some benefit can be
gained from insights into the host response available in the host
sequence [11]. Simple sample preparation techniques are available
to enrich samples for microbes; they include centrifugation, filtra-
tion, and chemical treatment (e.g., propidium monoazide [PMA])
of the sample to destroy cell-free DNA [13-15]. Consideration
of the sample type, the expected number of microbes, and the
microbial abundance can help to guide sample preparation and
the number of sequence reads required for the assay.
For example, a whole-blood sample from a suspected case of a
bloodstream infection would be expected to contain several sources
of DNA. There is significant host DNA in human white cells, as
well as cell-free circulating human DNA; in addition, there could
be microbial DNA inside white cells (due to phagocytosis), as well
as free circulating microbes. Plasma and serum samples represent
similar challenges, but to a lesser extent. In this circumstance,
centrifugation and filtration could be used to remove human cells,
along with PMA treatment to destroy cell-free DNA. For patients
with recent antibiotic therapy, PMA also has the potential benefit
of destroying DNA from dead bacteria, which could cause false-
positive results due to nonviable microbes.
The potential for the pathogen to be of fungal, viral, or parasitic
origin may also affect how the sample DNA may be isolated,
although protocols could be developed that cover all possibili-
ties. Establishing a validated sample preparation protocol that
can successfully isolate microbial DNA from diverse sample types
and microbes will be a significant step toward widespread mNGS
adoption in clinical settings.
Once microbial DNA is isolated, sequencing libraries are prepared
using manufacturer-specific protocols. A collection of sequence-
able DNA fragments of uniform size is created and fragments are
then sequenced. DNA library technology uses several techniques,
in this case a “library” is a collection of DNA fragments generated
from the mNGS process. These libraries are tagged with a library-
specific DNA sequence, referred to as a molecular “barcode,” a
tag allowing multiple libraries to be pooled and sequenced simul-
taneously. Following sequencing, the reads are separated by their
barcodes into pools of sample-specific data for analysis. Barcod-
ing libraries serves two purposes; they allow optimal use of the
output capacity of high-throughput sequencers and increases the
number of samples that can be analyzed in a single sequencing
run. Improved data analysis protocols and reference databases are
needed to accommodate the diverse species that may be detected,
their genome sizes, and the number of species in the metagenome.
The Benefit of Using Bioinformatics Pipelines
Bioinformatics pipelines that streamline data analysis are in active
development, yet to date no clear methods for mNGS analysis
have emerged. Issues in analyses include removing contaminating
human host DNA reads without inadvertently removing micro-
bial sequences, identifying pathogens using incomplete genome
databases, detecting the abundance and prevalence of pathogens in
polymicrobial clinical samples, and differentiating pathogens from
commensals that may affect treatment decisions. The development
of rapid clinical diagnostics for rational therapy choices depends
on overcoming these bioinformatics challenges.
As a first step, an mNGS dataset must be compared to genetic
databases to detect host contamination and to determine the abun-
dance of known microbes. Recently, fast k-mer-based algorithms
have been used to classify metagenomic reads against known bacte-
rial genomes with remarkable throughput and speed. Specifically,
the CLARK (classifier based on reduced k-mers) [16], USEARCH
[17], KRAKEN [18], and NBC (naive Bayes classifier) [19] clas-
sifiers offer composition-based approaches to quickly identify
microbial species present in a metagenome. In each case, the
sequence reads are broken down into shorter fragments (typically
15 to 30 bp in length), and the frequency profiles of k-mers from
microbial genomes are analyzed to rapidly assign the read from
which the k-mers were derived to genomes in a reference database
[16,18,20,21]. Although these methods are exceptionally fast and
outperform alignment-based methods, building and using the fre-
quency profile for the reference database requires large amounts of
memory (>128 GB of RAM) [16]. Further, these methods are con-
strained by the representation of genomes and proteins in known
databases, making it difficult to illuminate “microbial dark matter.”
Microbial dark matter received its name because of the difficulty
in effectively studying it as a result of its inability to be cultured
by current methods. Although the inability to culture the species
included in microbial dark matter makes it difficult to estimate its
relative magnitude, the accepted gross estimate is that less than
1% of all microbial species in a given ecological niche are cultur-
able. In the clinical setting, the amount of microbial dark matter
is unknown but is likely to be far less due to the intense interest
in clinically relevant microbes over the past 100 years.
In addition to taxonomic identification, recently, approaches have
been developed for rapid annotation of short reads against pro-
tein reference databases that are orders of magnitude faster than
alignment-based (e.g. BLASTx) searches. Such options include
USEARCH, PAUDA, RAPSearch2, UProC, RAST, and DIA-
MOND [17,22-26]. These fast protein searches use either indexes
or suffix arrays for complete k-mer matching of a query to refer-
ence databases or match unique protein domains to find protein
families [23,26]. In either case, these algorithms are often con-
strained by system memory. For example, DIAMOND requires
~1.6 TB (~100 GB) of memory for the NCBI-nr database [22], and
UProc requires 16 GB (~4GB) of memory for the Pfam database
[23]. Thus, although these algorithms move us one step closer to
comparisons of a complete set of reference proteins, they are not
scalable and use computational resources that are not often acces-
sible to the clinical laboratory community. Further, these analyses
still neglect microbial dark matter and offer a limited perspective
of just the known cultivable microbes.
False-Positive and -Negative Results
As with all techniques, mNGS for pathogen identification has to
deal with false-positive and false-negative results. Some of the
sources of error could be overcome, while others are inherent to
sample collection. The first issue is contamination arising from
reagents, personnel, and the laboratory environment. Reagent con-
tamination with bacterial DNA is a well-known problem [27] that
is not surprising, since many molecular biology reagents used to
Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier 55
perform mNGS are isolated from microbes (e.g., polymerase and
ligases), making bacterial DNA contamination likely. In response,
commercial products that have been treated to remove bacterial
DNA have been developed, and such specialized reagents should
certainly be used in mNGS for clinical purposes. Other sources of
contamination can be reduced by creating dedicated workspaces
and following procedures to reduce microbes and microbial DNA
in the laboratory environment. These procedures are not particu-
larly different than those that one would use to prevent contami-
nation of cell cultures or PCR methods; for example, molecular
laboratory practices and cleaning protocols, such as wiping down
work surfaces with disinfectants, single-use gowns, hairnets and
face masks, pipette tips, and gloves are used along with molecular
laboratory unidirectional workflow.
Unfortunately, even with the aforementioned precautions, contam-
ination of samples will occur by the very nature of sample collec-
tion. For example, any sample collected via a needle (e.g., blood or
cerebrospinal fluid) will always pose the risk of contamination from
skin commensal microbes, such as Cutibacterium acnes (formerly
Propionibacterium acnes), Staphylococcus epidermidis, and Corynebac-
terium spp. Unfortunately, these organisms can cause infectious
disease, especially in immunocompromised individuals, and their
presence cannot be disregarded as a false positive. The problem
of skin commensals will plague any technique, as it is inherent to
sample collection, but since it has always been a problem in the
clinical setting, standard procedures have been established to deal
with it, such as repeated assaying and careful cleaning of the skin
before sample collection. One way in which mNGS may be able
to overcome these challenges is that mNGS can provide a quanti-
tative abundance of the organism(s) detected, much like a routine
culture uses relative abundance to differentiate normal flora from
pathogens when some microbes can fall into both categories. It
may prove possible to set thresholds for the number of reads of an
organism that would be required to consider reporting its pres-
ence and thus ignore the false positives that can arise from needle
punctures and other types of sample collection.
Lastly, there is the challenge of an assay’s limit of detection. In a
scenario in which infection may be suspected but uncertain, the
question becomes, how many sequence reads from an organism
represent a true positive? The issue can be thought of in terms
of the DNA of the organism in competition with sources of con-
tamination. If no true infection is present, then contaminants can
amplify during the preparation of the mNGS library, leading to
false positives, so even if a pathogenic organism is present, its
abundance may be too low, and contaminants may out-compete it
in the PCR phase of the assay. As the abundance of the causative
organism increases, at some point, its presence will be detected
reliably—that point is called the assay’s limit of detection. The
effect of sample type on the limit of detection needs to be inves-
tigated in relation to the number of reads generated from the
sample for clinical implementation. Furthermore, a strategy in
which samples are spiked with a positive control may simultane-
ously provide a benchmark for monitoring the limit of detection
56 Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier
while suppressing stochastic false positives by providing a template
for library preparation.
Work in this area suggests that when a sample is truly negative,
false positives from contaminants and commensals arise, while
true infection appears to suppress background [15]. In the future,
providing a spiked positive control in the assay could provide the
same suppression of background, but studies will need to deter-
mine how the positive control affects the limit of detection while
seeking a balance with background suppression.
Standard of Care Techniques which Could be Supplemented
or Replaced by mNGS
The competitive landscape for mNGS consists of both compet-
ing technologies for identifying microbes by sequence analysis
and other traditional approaches to providing diagnostic infor-
mation. When laboratories consider the use of mNGS, careful
cost comparisons must be performed, as well as the impact to
workflow and productivity must be considered. Most importantly,
cost-benefit analysis is critical to assess which patient populations
or sample types would be most efficacious. For the sake of com-
parison, traditional technologies in use in clinical laboratories are
described here.
Conventional or routine culture
The first methods developed to identify microorganisms involved
growth in culture to isolate a pure sample, followed by phenotypic
analysis of features, such as microscopic morphology (e.g., cocci
versus rods and clusters versus chains); ability to grow on specific
culture media; stains (e.g., Gram stain); and biochemical attributes,
such as the ability to alter particular substrates (e.g., hemolysis or
enzyme presence, such as beta-galactosidase positive). The pri-
mary drawback of culture-based methods is that enrichment by
culture can be highly biased, since some organisms present in the
sample do not grow in the enrichment media and some organisms
grow more rapidly and can potentially obscure the presence and/
or the abundance of other organisms in the sample. In addition
to the bias inherent to culture, the method itself can take several
days, all while empiric antibiotic therapy is utilized until a specific
antimicrobial result can be attained. Finally, culture is not gener-
ally applicable to bacteria, fungi, viruses, and parasites using the
same culture media and methods, and is similar to metagenomics
in that regard. Thus, separate assays must be performed for each
class of organism, typically sequentially, with further delays and
costs for each one.
Fluorescence microscopy
A modern microscopy-based approach to microbial identification
has been developed that utilizes a short culture period followed by
fluorescence-based tagging of microbes with antibodies and DNA
probes. The sample then flows across the microscope field, with
automated computer-based identification of microbial shape and
automated fluorescence detection. Problems with this type of mod-
ern approach include reliance on culture to enrich the microbes,
bias in detection due to the need to pick antibodies and probes
to utilize, and difficulties in identifying non-bacterial microbes.
Mass spectrometry
Matrix-assisted laser desorption ionization–time of flight mass
spectrometry (MALDI-TOF MS) has been developed for the iden-
tification of bacteria and fungi. In this approach, a pure culture is
ionized, and the mass spectra of the resulting protein fragments
are compared to a reference database to match patterns of known
organisms. Recent advances in the method allow cruder prepara-
tions to be analyzed and even negate the need for culture. The
method is rapid and low cost once the initial equipment has been
installed. Drawbacks to the method include difficulty in detecting
drug resistance and virulence and in detecting viruses and parasites.
Lastly, complex mixtures of organisms present potentially unsolv-
able mass spectra making it best suited to scenarios in which only
a single organism is expected.
Polymerase chain reaction
Polymerase chain reaction (PCR) allows sensitive and rapid detec-
tion of specific nucleic acid sequences from even complex pools of
starting material. By targeting unique sequences in the genomes
or transcriptomes of organisms, the creation and detection of an
amplicon can be used to indicate the presence of the organisms in
a sample. Additional primers can be designed to indicate the pres-
ence of drug resistance and virulence gene sequences. Variations
of PCR have been developed since its inception several decades
ago, with modern approaches including droplet-based PCR, high
levels of automation, miniaturization, and rapid cycling times.
Drawbacks to this approach include the limited range of organ-
isms that can reasonably be identified, inability to identify organ-
isms that are not present in the panel design, the specificity of the
reaction, and the assumption that target sequences will always be
unique to one organism.
Microarray
Microarrays are a miniaturized and multiplexed version of a tra-
ditional membrane-based hybridization method, such as South-
ern blotting, with the additional distinction that the probes are
attached to the substrate while the targets are hybridized. Multiple
DNA probes are fixed to a solid surface and hybridized to sample
DNA fragments that are modified (typically with fluorescent tags
or a means to generate fluorescence following hybridization) to
allow detection of probes with hybridized sample DNA. The high
number of probes that can be put on a single microarray provides
the ability to detect and identify a broad range of organisms and
their drug resistance and virulence sequences in a single assay.
Furthermore, microarrays can be utilized to analyze mixtures of
organisms through careful choice of probes that distinguish one
organism from another. Drawbacks include the need to know the
sequences of the organisms being queried to design appropriate
probes, difficulties in distinguishing organisms at the strain level,
issues of specificity, and time required to perform the assay.
16S rRNA gene sequencing
The 16S rRNA gene sequencing approach takes advantage of the
alternating regions of high sequence conservation with interven-
ing regions of high sequence divergence found along the length
of the 16S rRNA gene. By designing PCR primers to conserved
regions, amplicons containing one or more divergent regions can
be created and sequenced. By comparing the sequence to a ref-
erence database of known organisms and their 16S rRNA gene
sequences, identifications can be made, often to the species level.
The approach allows the identification of fastidious organisms and
the analysis of samples containing many organisms, such as gut
microbiomes. While it is powerful, drawbacks arise when species’
sequences are not divergent enough to allow discrimination (e.g.,
Escherichia coli and Shigella spp.).
Serologic assays
With the development of molecular biologic techniques in the last
few decades, it has become possible to generate specific antibodies
to antigens present on the surfaces of microbes. These antibod-
ies can be utilized in various ways to detect the presence of and
even quantitate specific organisms or their products (e.g., toxins)
in a sample. Antibody-antigen complexes are detected in various
ways, including precipitation reactions, agglutination reactions,
complement fixation, labeling the antibody with fluorescent mol-
ecules, and immobilizing either the antigen or antibody, followed
by various enzyme-linked immunosorbent assay approaches. A
widely utilized example of antibody-based detection of a microbe
is the rapid strep test, which allows a clinician to confirm bacterial
pharyngitis caused by group A streptococci during a patient’s visit,
thus providing the information required to guide management of
the infection by prescription of antibiotics. Unlike the approaches
described above, antibody-based detection of specific microbes
can be performed within minutes without special equipment once
a specific organism is identified; however, clinical sensitivity and
specificity may not be optimal.
Antimicrobial drug resistance
Bacterial drug resistance is a serious worldwide human health
problem, and its detection in clinical samples can be more impor-
tant than the species-level identification possible from mNGS.
For example, detection of “methicillin-resistant Gram-positive
cocci” by culture or mNGS is more relevant to a clinical decision-
making and therapy than a result simply describing detection of
“Staphylococcus aureus.” If mNGS that misses the presence of the
mecA gene conferring methicillin resistance, a significant gap to the
result relevance occurs. There are definite limits to the detection
of drug resistance by mNGS due to the diversity of drug resis-
tance mechanisms; multidrug resistance; and limitations arising
from incomplete coverage, leading to insufficient sequence reads
to detect the presence of drug resistance genes. Currently, mNGS
is considered too immature for inferring drug susceptibility clini-
cally [28]. Nonetheless, there are numerous examples of success-
ful identification of drug resistance in samples analyzed by mNGS
[3,29,30]. Consideration of the sample type and the number of
organisms expected can assist in ensuring enough sequence reads
are generated to detect resistance genes.
These considerations are demonstrated by work to identify the
mecA sequence in three samples from two ventilator-acquired
pneumonia patients that were culture-positive for methicillin-
resistant S. aureus. Low coverage in two of the samples prevented
Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier 57
detection of the mecA gene, but the third had enough reads so that
the entirety of the mecA gene was covered between 2× and 17×. In
the sample in which mecA was detected, 619,389 sequence reads
were classified as S. aureus from a total of 2.7 million. In contrast,
just 472 and 962 sequence reads were classified as S. aureus in the
samples in which mecA was not detected [15]. Given the critical
importance of drug resistance to the success of antibiotic therapy,
the interplay between the total number of reads obtained, the
number of reads classified to a pathogen, the number of reads clas-
sified to the host genome, and the relative genome sizes involved
will need to be carefully explored for successful clinical transla-
tion of mNGS.
While mNGS can detect the presence of DNA coding for drug
resistance genes, there is no guarantee that a gene is active. While
there is evidence that genotype accurately predicts phenotype, at
least in S. aureus [29], there are scenarios in which this is not the
case, such as S. aureus carrying the mecA gene but with a mutation
in the promoter that greatly alters the phenotype [31]. This sce-
nario led to suggestions that a more relevant material for mNGS
is the RNA of the sample, which could provide information on
what proteins the microbes are synthesizing and thus whether drug
resistance genes are active, along with a more accurate picture of
the active metabolic pathways. Such an approach would come at
the cost of reducing discriminatory sequences or polymorphisms
that could allow accurate discrimination between closely related
species or track specific strains in an outbreak in which just a few
nucleotides per genome may be informative. Here again, consid-
eration of the setting and questions at hand could help guide the
choice of DNA versus RNA.
Hurdles to mNGS Adoption
While mNGS has clear potential to provide diagnostic informa-
tion, several hurdles to widespread adoption exist. Regulatory
hurdles and issues of reimbursement certainly limit the wholesale
replacement of culture, but as with other alternatives to culture,
mNGS will likely find use where culture has already failed or
cannot provide the necessary information (e.g., strain outbreaks).
While cost is of concern, it is not unreasonable that the cost of
sequencing a sample could drop to a few hundred dollars soon,
which would bring mNGS into the same cost range as targeted
PCR or some high-end cultures. Despite the concerns of the cost
of mNGS, it should be kept in mind that while the cost of the
assay may never be as low as that of alternatives, such as routine
culture, overall health care costs may be reduced by increasing
the rate of diagnoses with mNGS, leading to improved therapy
and patient outcomes and reduced hospitalizations, morbidity,
and mortality. Of more immediate concern will be establishing
standard protocols for sample preparation, sequencing, and data
analysis. Furthermore, spiked controls need to be developed that
can simultaneously suppress background and allow absolute quan-
titation, which could allow clinicians to differentiate between true
positives and commensals. Lastly, the limits of detection need to
be determined for each clinical setting in which mNGS will be
applied. Once these technical details are in place, the pipelines
will have to demonstrate not just clinical validity, but clinical
58 Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier
utility, as alluded to above (lowered mortality, morbidity, health
care costs, etc.). Demonstrating clinical validity can only be done
by comparison to culture or other alternatives in prospective
studies. Aside from the difficulty of getting funding for testing
and the challenging issues with this type of study design, only
samples that are positive by the standard can be fully compared.
The design ignores samples in which mNGS detects a pathogen
that was not identified by culture or whichever assay is used as the
reference standard. This drawback could be overcome eventually
by studies to demonstrate clinical utility. While we may never be
able to assess the validity of mNGS in culture-negative samples,
if treatment decisions are made based on the diagnostic informa-
tion provided and patient outcomes are better than with empiri-
cal antimicrobial therapy, then the evidence to support the use of
mNGS clinically is obtainable. Of course, the adoption of mNGS
will be far less black and white than this. The recent publication of
a prospective trial of mNGS in meningitis demonstrated various
ways in which mNGS could be used as an adjunct or supplement
to culture rather than outright replacement. A final consideration
for the adoption of mNGS is the costs associated with establishing
a capable laboratory and the personnel to perform it and interpret
the results. Given the complexities, the temptation would be to
establish a few specialized laboratories to which samples could be
sent for analysis. However, this approach would add at least 24
hours to the turnaround time as the samples are mailed overnight
from their collection point to the laboratory. Instead, it seems
more likely that as medical personnel become more familiar with
the methods, standardized protocols become established, costs
come down, and evidence of clinical utility accumulates, mNGS
will be adopted in regional centers with dedicated microbiology
laboratories with active research efforts.
The availability of mNGS sequencing platforms presents and
important challenge for medical laboratory scientists — know-
ing how to make sense of the massive sequence data generated
by new sequencing technologies. When performing testing on
site, suitable IT infrastructure and well-trained bioinformatics
scientists are required prior to adopting the mNGS for clinical
use — this infrastructure is expensive and suitable trained staff
can be hard to find. Even when sending samples to reference
or for-profit boutique laboratories, the clinical laboratory must
understand the technology well enough to select a suitable labo-
ratory. Simple reports and simple and user-friendly bioinformat-
ics work-flow is mandatory, but there are currently few products
that meet the needs of non-research-based, non-reference-based
and non-profit clinical laboratories. Reference databases, gene
calling software, protocols for microbial identification and cost-
effective technology utilization are all key challenges that require
cost-effective solutions. Currently, mNGS sequencing quality
is a topic being addressed by the Division of Laboratory Sys-
tems, at the Center for Disease Control and Prevention (https://
www.cdc.gov/cliac/docs/fall-2019/6_Fitzgerald_NGS-Quality-
CLIAC_Nov_2019_FINAL.pdf ). The American Public Health
Association posts additional guidance about mNGS implementa-
tion (https://www.aphl.org/aboutAPHL/publications/Documents/
ID-NGS-Implementation-Guide102016.pdf ). The Association
for Molecular Pathology provides support to assess cost-benefit
of mNGS ((https://www.amp.org/advocacy/advocacy-resources/
coverage-and-reimbursement/ngs-pricing-project/).
Summary
Although there are limitations and challenges, the mNGS
approach offers an unbiased and hypothesis-free approach to
pathogen identification with the potential to detect uncultivable
bacterial, viral, fungal, and parasitic organisms in the same assay.
Advances in mNGS technology and data analysis will likely reduce
testing costs to the point where the advantages of mNGS over
culture warrant its use in some clinical settings.
References
[1] van Walraven C, Wong J. Independent influence of negative blood
cultures and bloodstream infections on in-hospital mortality. BMC
Infect Dis 2014;14:36.
[2] Ecker DJ, Sampath R, Li H, Massire C, Matthews HE, Toleno D,
et al. New technology for rapid molecular diagnosis of bloodstream
infections. Expert Rev Mol Diagn 2010;10:399-415.
[3] Wilson MR, Sample HA, Zorn KC, Arevalo S, Yu G, Neuhaus J, et
al. Clinical metagenomic sequencing for diagnosis of meningitis and
encephalitis. N Engl J Med 2019;380:2327-40.
[4] Dai Y, Chen L, Chang W, Lu H, Cui P, Ma X. Culture-negative
Streptococcus suis infection diagnosed by metagenomic next-generation
sequencing. Front Public Health 2019;7:379.
[5] Parize P, Muth E, Richaud C, Gratigny M, Pilmis B, Lamamy A, et
al. Untargeted next-generation sequencing-based first-line diagnosis
of infection in immunocompromised adults: a multicentre, blinded,
prospective study. Clin Microbiol Infect 2017;23:574.e1-e6.
[6] Snitkin ES, Zelazny AM, Thomas PJ, Stock F, NISC Compara-
tive Sequencing Program Group, Henderson DK, et al. Tracking a
hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with
whole-genome sequencing. Sci Transl Med 2012;4:148ra116.
[7] Nakagawa S, Inoue S, Kryukov K, Yamagishi J, Ohno A, Hayashida
K, et al. Rapid sequencing‐based diagnosis of infectious bacterial
species from meningitis patients in Zambia. Clin Transl Immunol
2019;8:e0202049.
[8] Ventola CL. The antibiotic resistance crisis: part 2: management
strategies and new agents. P T 2015;40:344-52.
[9] Deurenberg RH, Bathoorn E, Chlebowicz MA, Couto N, Ferdous
M, García-Cobos S, et al. Application of next generation sequenc-
ing in clinical microbiology and infection prevention. J Biotechnol
2017;243:16-24.
[10] Goldberg B, Sichtig H, Geyer C, Ledeboer N, Weinstock GM.
Making the leap from research laboratory to clinic: challenges and
opportunities for next-generation sequencing in infectious disease
diagnostics. MBio 2015;6:e01888-15.
[11] Simner PJ, Miller S, Carroll KC. Understanding the promises and
hurdles of metagenomic next-generation sequencing as a diagnostic
tool for infectious diseases. Clin Infect Dis 2018;66:778-88.
[12] Frey KG, Herrera-Galeano JE, Redden CL, Luu TV, Servetas SL,
Mateczun AJ, et al. Comparison of three next-generation sequencing
platforms for metagenomic sequencing and identification of patho-
gens in blood. BMC Genomics 2014;15:96.
[13] Marotz CA, Sanders JG, Zuniga C, Zaramela LS, Knight R, Zengler
K. Improving saliva shotgun metagenomics by chemical host DNA
depletion. Microbiome 2018;6.
[14] Watanabe N, Kryukov K, Nakagawa S, Takeuchi JS, Takeshita M,
Kirimura Y, et al. Detection of pathogenic bacteria in the blood from
sepsis patients using 16S rRNA gene amplicon sequencing analysis.
PLoS One 2018;13:e0202049.
[15] Watts GS, Thornton JE, Jr, Youens-Clark K, Ponsero AJ, Slepian
MJ, Menashi E, et al. Identification and quantitation of clinically
relevant microbes in patient samples: comparison of three k-mer
based classifiers for speed, accuracy, and sensitivity. PLoS Comput
Biol 2019;15:e1006863.
[16] Ounit R, Wanamaker S, Close TJ, Lonardi S. CLARK: fast and
accurate classification of metagenomic and genomic sequences using
discriminative k-mers. BMC Genomics 2015;16:236.
[17] Edgar RC. Search and clustering orders of magnitude faster than
BLAST. Bioinformatics 2010;26:2460-1.
[18] Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence
classification using exact alignments. Genome Biol 2014;15:R46.
[19] Rosen G, Garbarine E, Caseiro D, Polikar R, Sokhansanj B. Metage-
nome fragment classification using n-mer frequency profiles. Adv
Bioinform 2008;2008.
[20] Rosen GL, Reichenberger ER, Rosenfeld AM. NBC: the Naive
Bayes Classification tool webserver for taxonomic classification of
metagenomic reads. Bioinformatics 2011;27:127-9.
[21] Bazinet AL, Cummings MP. A comparative evaluation of sequence
classification programs. BMC Bioinform 2012;13:92.
[22] Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment
using DIAMOND. Nat Methods 2015;12:59-60.
[23] Meinicke P. UProC: tools for ultra-fast protein domain classification.
Bioinformatics 2015;31:1382-8.
[24] Zhao Y, Tang H, Ye Y. RAPSearch2: a fast and memory-efficient
protein similarity search tool for next-generation sequencing data.
Bioinformatics 2012;28:125-6.
[25] Huson DH, Xie C. A poor man’s BLASTX—high-throughput
metagenomic protein database search using PAUDA. Bioinformatics
2014;30:38-9.
[26] Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al.
The RAST Server: rapid annotations using subsystems technology.
BMC Genomics 2008;9:75.
[27] Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF,
et al. Reagent and laboratory contamination can critically impact
sequence-based microbiome analyses. BMC Biol 2014;12:87.
[28] Ellington MJ, Ekelund O, Aarestrup FM, Canton R, Doumith M,
Giske C, et al. The role of whole genome sequencing in antimicrobial
susceptibility testing of bacteria: report from the EUCAST Subcom-
mittee. Clin Microbiol Infect 2017;23:2-22.
[29] Stoesser N, Batty EM, Eyre DW, Morgan M, Wyllie DH, Del Ojo
Elias C, et al. Predicting antimicrobial susceptibilities for Escherichia
coli and Klebsiella pneumoniae isolates using whole genomic sequence
data. J Antimicrob Chemother 2013;68:2234-44.
[30] Aanensen DM, Feil EJ, Holden MTG, Dordel J, Yeats CA, Fedosejev
A, et al. Whole-genome sequencing for routine pathogen surveillance
in public health: a population snapshot of invasive Staphylococcus
aureus in Europe. MBio 2016;7.
[31] Chen F-J, Wang C-H, Chen C-Y, Hsu Y-C, Wang K-T. Role of
the mecA gene in oxacillin resistance in a Staphylococcus aureus clini-
cal strain with a pvl-positive ST59 genetic background. Antimicrob
Agents Chemother 2014;58:1047-54.
Clinical Microbiology Newsletter 42:7,2020 | ©2020 Elsevier 59