Clinical CMN

Microbiology
Stay Current...
Stay Informed.

N e w s l e t t e r

CMN Digital PCR in the Clinical Microbiology Laboratory:

Vol. 40, No. 4
February 15, 2018
www.cmnewsletter.com
I n Th is Issu e
27 Digital PCR in the Clinical
Microbiology Laboratory:
Another Tool on the
Molecular Horizon
32 A Farewell Tribute to
Retiring CMN Editors and
their Contribution to
Clinical Microbiology
Another Tool on the Molecular Horizon
Eleanor A. Powell, Ph.D.1 and N. Esther Babady, Ph.D., D(ABMM),1,2 1Clinical Microbiology Service,
Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York,
2
Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York,
New York
Abstract
Digital PCR was first described in 1999. Improvements in instrumentation and reagents have increased
its routine use in molecular pathology laboratories. For clinical microbiology laboratories, digital
PCR applications could cover a broad range of purposes, from absolute pathogen quantification to
the detection of rare mutations that confer resistance to antibiotics. In this review, we describe several
potential applications of digital PCR in the clinical microbiology laboratory. As this method contin-
ues to evolve, digital PCR may become an important tool on the horizon for clinical and molecular
microbiology laboratories.

Introduction was first coined in a 1999 report by Vogelstein

Corresponding author: N. Esther
Babady, Ph.D., 327 East 64th
Street CLM 522, New York, NY
10065. Tel.: 212-639-8179.
E-mail: babadyn@mskcc.org
0196-4399/©2018 Elsevier Inc.
All rights reserved
The introduction of conventional endpoint poly-
merase chain reaction (PCR) and real-time PCR
in clinical microbiology laboratories was transfor-
mative, allowing the identification of pathogens
within a few hours when such tasks traditionally
took several days or weeks to complete. The
field of nucleic acid amplification technology
is continually evolving to improve the sensitiv-
ity of nucleic acid detection, reduce the risk of
cross-contamination, and allow accurate quan-
tification. Thus, digital PCR (dPCR) was born
out of a need to amplify and accurately quantify
a single molecule or a rare allele that is present
in excess of wild-type alleles. An allele is one of
several alternative forms of the same gene or the
same genetic locus.
Reports of single-molecule amplifications were
published as early as the late 1980s, a few years
after the description of PCR. Single-molecule
amplification was made possible by physically
separating single cells or by performing limit-
ing dilution of samples prior to amplification
and detection [1,2]. The term “digital” PCR
and Kinzler at Johns Hopkins [3, 4]. Building on
previous works that described limiting-dilution
and single-molecule amplification, Vogelstein
and Kinzler developed a method to amplify an
individual template DNA molecule in a separate
reaction and, with the use of fluorescent probes,
detect and count the amplified reactions, yield-
ing a “digital” signal (Fig. 1). They applied this
method to detect low concentrations of a rare
Ras oncogene mutation in a stool sample from a
patient with colorectal cancer. Since these early
reports, the number of publications that describe
the use of dPCR have increased significantly, with
most applications found in the fields of human
genetics and oncology. This increase was partly
due to improvements in a variety of instrument
microfluidics and partitioning chemistries that
resulted in simplification of the original proto-
col developed by Vogelstein and Kinzler. Most
recently, dPCR has provided value to an increas-
ing number of applications in clinical microbi-
ology (Table 1). This review summarizes and
discusses the applications of dPCR in the clinical
microbiology laboratory.

Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier 27

 Figure 1. dPCR workflow.
Sample (total nucleic acids
[NA]) is divided into multiple
partitions containing either 0
or 1 template NA. Amplification
of a single-molecule template
occurs within each partition.
If the assay is designed to
detect more than one target, a
different fluorescent probe (e.g.,
FAM or VIC) can be used for the
detection of amplified template.
The data are presented in
either a one-dimensional (1D)
scatterplot with all positive
partitions above an established
threshold or a 2D scatterplot
where the number of partitions
positive for one-target (a and
d) or two targets (c) or empty
partitions (b) can be visualized.

dPCR Technology and Platforms manufacturers have instruments on the market (Table 2). Manu-
facturers of dPCR instruments vary primarily in their approaches
dPCR technology builds on methods that are well established for
to partitioning of the sample. The two main approaches are drop-
conventional and real-time PCR, including nucleic acid extraction,
let and chip partitioning. Of the systems commercially available
amplification of nucleic acids using specific primers, and detection
in the United States, the RainDrop (RainDance Technologies,
of amplified nucleic acids using fluorescent probes (e.g., TaqMan
recently acquired by Bio-Rad Laboratories, Hercules, CA) and
or Molecular Beacons) or DNA intercalating dyes (e.g., SYBR
QX100/200 (Bio-Rad Laboratories) systems use droplet-based
Green or EvaGreen). The workflow for dPCR can be divided into
partitioning, while the Biomark HD (Fluidigm) and QuantStudio
three steps that occur after DNA extraction: (i) sample partition-
3D (ThermoFisher Scientific, Waltham, MA) systems use chip-
ing, (ii) single-molecule amplification, and (iii) signal detection
based partitioning. Other systems available outside the U.S. have
by scatterplot (Fig. 1).
variations on the theme of the chip. For example, the Clarity sys-
Sample partitioning is the process by which a sample (e.g., tem (JNMedsys, Singapore) uses chip-based partitioning, but with
extracted DNA) is diluted and divided into thousands of partitions, a “chip-in-a-tube” design so that amplification can be performed
thereby dividing up the reaction into multiple smaller reactions. on any traditional thermal cycler, while the Naico Geode (Stilla
This step is critical to ensure that each partition ideally contains Technologies, France) uses the Sapphire chip to create crystal-like
either zero or one template molecule. Target amplification is then droplets. Depending on the manufacturer, amplification may be
performed inside each partition simultaneously, resulting in hun- carried out on a specialized thermocycler or on a standard thermo-
dreds to thousands of individual PCRs. Amplified templates are cycler. Detection is carried out on a manufacturer-specific reader.
detected using the fluorescent signal (e.g., 6-carboxyfluorescein
In addition to how partitions are generated, instruments also vary
[FAM] dye or VIC dye), with partitions that contain an amplified
in the number of partitions that are generated, ranging from a
template molecule counted as one (i.e., positive; a single count)
few thousand to several million. The maximum number of par-
and those without amplified template counted as zero (i.e., nega-
titions that can be generated is related to the dynamic range of
tive; no signal is detected). Hence, the total number of positive
the instrument; however, since one of the advantages of dPCR is
partitions reflects the absolute number of targets in the starting
the detection and quantitation of rare targets, a wide linear range
material (Figure 1). Data can be viewed as a single scatterplot, with
may not be necessary.
a threshold for positive set above the background fluorescence
of the negative, or empty, partitions. To account for instances in dPCR versus Real-Time PCR
which more than one template molecule may be present in a par-
dPCR offers several potential benefits over quantitative real-time
tition, a Poisson model mathematical correction is applied during
PCR (qPCR). First, quantification of nucleic acids is absolute,
the analysis. Additionally, using multiplexed dPCR with differ-
negating the need for an external standard curve. This is par-
ent probes targeting different templates, even rare events can be
ticularly useful for pathogens that do not have well-characterized
amplified and detected equally well, since no competition for PCR
reference materials (e.g., World Health Organization [WHO]
reagents with other targets occurs within each partition (Fig. 1).
standards) available. Additionally, with qPCR, differences in
The first commercially available dPCR instrument was released sequence between the materials used for the standard curve and
by Fluidigm (San Francisco, CA) in 2006. Today, several the sample can result in differing primer affinities and different

28 Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier

amplification efficiencies for the standard curve and sample (e.g.,
rhinoviruses) [5]. This circumstance can result in artificially high
or low viral loads (for higher affinities and lower affinities, respec-
tively). In contrast, dPCR is less affected by differences in sequence
variation, as partitions with any level of fluorescence are considered
positive [5]. Furthermore, compared to qPCR, dPCR is less sus-
ceptible to inhibitory substances, like those found in feces, heparin,
and SDS, which may allow direct-from-specimen testing or extrac-
tion methods that are more rapid and/or less labor-intensive [6,7].
Despite these advantages, compared to qPCR, dPCR has several
drawbacks. dPCR instruments and consumables are generally
more expensive than those used for qPCR. The workflow for
most dPCR platforms has, in some ways, taken a step backward
by requiring multiple steps (partitioning, amplification, and anal-
ysis of partitions), each with hands-on processing time. In addi-
tion, dPCR’s open system is more susceptible to contamination,
with risks similar to those with conventional endpoint PCR. The
throughput of dPCR instruments is also generally lower than that
of qPCR, particularly for droplet-based technologies. Finally, the
dynamic range of dPCR is usually less than that of qPCR, though
it does vary among instruments. The dynamic range is determined
by the number of partitions, which varies between platforms. The
RainDance platform is capable of 10 million partitions/reactions,
which achieves a 6-log-unit dynamic range [8]. This capability
approaches the 7-log-unit dynamic range that is typical of qPCR.
Other dPCR platforms have smaller dynamic ranges.
Clinical Microbiology Applications
dPCR has several potential applications for the clinical microbiol-
ogy laboratory, where qPCR (aka quantitative real-time PCR) is
typically used and nucleic acids are quantified by comparing signal
strength to that of an external standard curve. Although the avail-
ability of the WHO international standards and in vitro diagnostics
(IVD)-cleared qPCR assays has helped decrease the variability in
viral loads observed across laboratories, challenges with compara-
bility of viral load testing results still exist, especially when stan-
dards are not available. In light of qPCR variability, dPCR can be
used in pathogen quantification, with the ability to provide abso-
lute quantification instead of quantification relative to a standard
curve. In one study, quantifications of cytomegalovirus (CMV) by
dPCR and qPCR using standard reference materials across mul-
tiple laboratories and platforms were compared; although some
differences remained, dPCR demonstrated high reproducibility
and decreased inter-laboratory variations compared to qPCR [9].
Hayden and colleagues further investigated potential causes of dif-
ferences in absolute quantitation by comparing dPCR performance
across platforms (Bio-Rad QX200 and RainDance RainDrop)
using commercially available CMV analyte-specific reagents, spe-
cifically, Altona Diagnostics (Hamburg, Germany) RealStar CMV,
Abbott Laboratories (Abbott Park, IL) CMV ASR, and Focus
Diagnostics (Cypress, CA) CMV ASR [10]. The authors showed
good correlations across platforms and reagents when measuring
CMV standards, though increased variation was observed at low
viral loads. Differences in the lower limit of detection, linearity,
and overall agreement were both reagent and platform dependent.
Hence, interpretation of dPCR data needs to take into consider-
ation multiple variables.
Other studies have compared dPCR to qPCR, and in a recent
systematic literature review of the application of dPCR to HIV-1
quantification, dPCR was more accurate, precise, and reproduc-
ible than qPCR. Also, though the two methods had equivalent
sensitivity, the specificity of dPCR was lower due to the presence
of positive droplets in the negative-control partitions [11].
Several studies have reported increased sensitivity of dPCR com-
pared to qPCR for a wide range of viruses and sample types,
including parechovirus in cerebrospinal fluid (CSF), JC virus in
CSF and serum, hepatitis B virus, and H7N9 influenza virus [12-
15]. This increased sensitivity may be due to the partitioning,
which removes the competition between the genetic target and
other extraneous DNA by virtue of the low-copy-number target
in the partition [15]. The increased sensitivity of dPCR may allow
novel applications, like detecting human papillomavirus (HPV)
in liquid biopsy fluid. A study of patients with invasive carcinoma
showed 87% of the patients had detectable HPV using a dPCR
assay, while no patients with high-grade cervical intra-epithelial
neoplasia did [16].
Since dPCR is less susceptible to the effects of inhibitors than is
qPCR, it can be used to design sensitive assays in more difficult or
complex matrices. dPCR was used to detect CMV and Epstein-
Barr virus in formalin-fixed paraffin-embedded tissues from the
colonic mucosa of people with HIV [17] and Merkel cell polyoma-
virus, with higher sensitivity than qPCR [18]. dPCR was also able
to accurately quantify CMV in simple matrices, like Tris buffer and
cell lysate, without prior DNA extraction [19]. Avoiding a sepa-
rate DNA extraction step would simplify the workflow, remove
one possible source of cross-contamination, and remove a source
of inter-assay variability.

Table 1. Applications of dPCR in clinical microbiology

Application Example Reference
Detection Cell-free human papillomavirus DNA in plasma 16
Absolute quantitation Cytomegalovirus quantitation 10
Rare genotype identification Heteroresistance in Mycobacterium tuberculosis 24
Detection of viral genome integration in host genome Chromosomally integrated human herpesvirus 6 20
Reference materials and standards characterization Diversity of viral subpopulations in WHO BK virus standards 33

Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier 29

Current methods to differentiate between cell-free human herpes-
virus 6 (HHV-6) and chromosomally integrated HHV-6 include
fluorescent in-situ hybridization and testing of hair follicles for
HHV-6. A group at the University of Washington recently devel-
oped a multiplexed dPCR to measure HHV-6 integration by
quantifying both HHV-6 and a cellular (host) housekeeping gene,
calculating the ratio of virus to the cellular housekeeping gene.
Ratios of 1 HHV-6 copy to 2 cellular gene copies (as the cells are
diploid) were consistent with HHV-6 integration [20]. This assay
has been used to show reactivation of HHV-6 in 25% of hema-
topoietic stem cell recipients with donor- or recipient-derived
chromosomal HHV-6 [21].
Quantification assays based on dPCR have also been developed for
other parts of the clinical microbiology laboratory besides virology.
dPCR was used to detect Mycobacterium tuberculosis DNA in the
plasma of people with pulmonary tuberculosis (Tb) [22]. Though
the assay was relatively insensitive (65%), it represented a novel
and less invasive option for the diagnosis of Tb. The partitioning
of dPCR also makes it ideal to detect low numbers of resistant bac-
teria and viruses among a large population of sensitive organisms.
dPCR detected Legionella pneumophila with gyrA mutations, which
mediate fluoroquinolone resistance, at a ratio of 1 mutated allele
to 1,000 wild-type alleles [23]. Similar ratios of mutant to wild-
type alleles were identified in a study that investigated the use of
dPCR to detect hetero-resistant Mycobacterium tuberculosis [24]. In
that study, dPCR was more sensitive than both qPCR and Sanger
sequencing, which could detect resistance only at 1:10 and 1:1
mutant–to–wild-type allele ratios, respectively. Another reported
application of dPCR is the detection of single-nucleotide poly-
morphisms associated with antiviral resistance in influenza A virus,
found in only 0.1% of the population tested, making dPCR 50
times more sensitive than the currently available clinical test [25].
dPCR may also be used to provide rapid antimicrobial susceptibil-
ity results. For these assays, bacteria are exposed to an antibiotic
for 15 minutes, and then, the cells are lysed and the bacterial chro-
mosomal DNA is partitioned in dPCR, which quantifies bacte-
rial resistance gene replication [26]. While this method has been
demonstrated only in a proof-of-concept study, it could eventually
be used for rapid phenotypic antimicrobial susceptibility testing.
dPCR has also been used in the detection of parasites. In one study
using both genus- and species-specific assays, dPCR was used to
detect Plasmodium species from whole blood, demonstrating a high
sensitivity of 11 parasites/ml of blood [27]. The inhibitor-resistant
nature of dPCR also makes it ideal for stool parasite quantification,
as shown by its ability to detect Cryptosporidium oocytes directly
from fecal samples [28]. Assays are currently being developed to
detect Schistosoma japonicum in urine, saliva, and stool but have
thus far been tested only in a mouse model [29].
Another application of dPCR that is important for clinical micro-
biology laboratories is its use for the characterization and standard-
ization of reference materials. dPCR can provide accurate, absolute
quantification of reference materials and other control materials
that are later used for qPCR. Researchers from the U.S. National
Institute of Standards and Technology used dPCR on the BioMark
HD instruments (Fluidigm) to quantify CMV DNA concentra-
tion of the CMV standard reference material (SRM2366) they
produce for use in calibration of CMV quantitative assays [30].
Similarly, dPCR was used to quantify plasmid DNA standards for
Shiga toxin-producing Escherichia coli with accuracy similar to that
of ultraviolet spectrophotometry and high-resolution inductively
coupled plasma mass spectrometry [31]. Though the required dilu-
tion and fragmentation steps resulted in higher uncertainty in the
quantifications for dPCR, the method is much more accessible for
laboratories that need to make their own control material.

Table 2. dPCR instruments and characteristics

Instruments
Partition No. of
Manufacturer type partitions Partitioning Amplification Detection Detection channels
Bio-Rad Droplet 20,000 QX200 droplet Thermal cycler QX200 droplet FAM/EvaGreen, VIC/HEX
Laboratories generator reader
RainDance Droplet 10,000,000 RainDrop source Thermal cycler RainDrop sense FAM/EvaGreen, VIC
Technologiesa

Life Technologies Chip 20,000 QuantStudio Dual flat block QuantStudio FAM/SYBR green, VIC,
(ThermoFisher) 3D chip loader thermocycler 3D reader ROX
and sealer
Fluidigm Chip 37,000 Biomark HD Thermocycler EP1 reader 2; FAM, VIC

Stilla Chip 30,000 Naica Geode Naica Geode Naica Prism 3 3; FAM, Cy3/VIC/Hex,
Cy5/Quasar 705
Formulatrix Chip 8,000- Constellation Constellation Constellation 5; FAM, VIC/HEX, and
12,000 digital PCR system digital PCR system digital PCR system user defined
jnmedsys Chip in 10,000 Clarity autoloader Thermocycler Clarity reader 2: FAM, VIC/HEX
a tube and sealer
a
Acquired by Bio-Rad Laboratories in 2017.

30 Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier

dPCR has also been used to characterize commercially available
reference materials. Three commercial standards for CMV quan-
titation were tested using dPCR and qPCR, and both methods
demonstrated deviations from the reference CMV concentration
[32]. The two dPCR systems used showed no significant difference
in their quantifications, while the results from the qPCR systems
varied with the assay utilized. dPCR was also used to characterize
the BK virus standards from the WHO and commercial provid-
ers [33]. While there was good agreement with the reference viral
load for all providers, the WHO standard had a sub-population
of viruses with deletions in the T region that could lead to under-
quantitation for PCR designed to target this region of the genome.
Similarly, dPCR was used to confirm next-generation sequencing
findings that the WHO standard for JC polyomavirus had 4- to
8-fold variation in the copy numbers of different genes, which
would result in large variations in viral load in assays targeting
different regions of the genome [34].
Conclusions
Although real-time PCR is a mature and well-established meth-
odology in clinical laboratories, some of its shortcomings have
increased the recent interest in dPCR. The potential applications
of dPCR in the clinical microbiology laboratory cover a broad
range of purposes, targets, and specimen types. The applications
are likely to continue to expand dramatically as the availability and
use of dPCR increases. Furthermore, variations of dPCR, includ-
ing digital loop-mediated isothermal amplification, have been pub-
lished (e.g. H5N1 quantification), with sensitivity comparable to
that of qPCR and dPCR [35]. As the technology is used more in
clinical laboratories, variations of the amplification method used
will likely occur. For dPCR to supplant qPCR as the method of
choice for quantification in clinical laboratories, several factors
(cost, workflow, and batch capacity) will need to be addressed and
optimized. More immediately, dPCR can be used as an additional
tool in the molecular arsenal. As molecular methods continue to
develop at a rapid pace, dPCR will be an important tool in the
clinical microbiology laboratory.
References
[1] Li HH, Gyllensten UB, Cui XF, Saiki RK, Erlich HA, Arnheim N.
Amplification and analysis of DNA sequences in single human sperm
and diploid cells. Nature 1988;335:414-7.
[2] Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT,
et al. Primer-directed enzymatic amplification of DNA with a ther-
mostable DNA polymerase. Science 1988;239:487-91.
[3] Morley AA. Digital PCR: a brief history. Biomol Detect Quant
2014;1:1-2.
[4] Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci
1999;96:9236-41.
[5] Sedlak RH, Nguyen T, Palileo I, Jerome KR, Kuypers J. Superiority
of digital reverse rranscription-PCR (RT-PCR) over real-time RT-
PCR for quantitation of highly divergent human rhinoviruses. J Clin
Microbiol 2017;55:442-9.
[6] Dingle TC, Sedlak RH, Cook L, Jerome KR. Tolerance of droplet-
digital PCR vs real-time quantitative PCR to inhibitory substances.
Clin Chem 2013;59:1670-2.
[7] Sedlak RH, Kuypers J, Jerome KR. A multiplexed droplet digital
PCR assay performs better than qPCR on inhibition prone samples.
Diagn Microbiol Infect Dis 2014;80:285-6.
[8] Jones GM, Busby E, Garson JA, Grant PR, Nastouli E, Devonshire
AS, et al. Digital PCR dynamic range is approaching that of real-time
quantitative PCR. Biomol Detect Quant 2016;10:31-3.
[9] Pavsic J, Devonshire A, Blejec A, Foy CA, Van Heuverswyn F, Jones
GM, et al. Inter-laboratory assessment of different digital PCR
platforms for quantification of human cytomegalovirus DNA. Anal
Bioanal Chem 2017;409:2601-14.
[10] Hayden RT, Gu Z, Sam SS, Sun Y, Tang L, Pounds S, et al. Compar-
ative performance of reagents and platforms for quantitation of cyto-
megalovirus DNA by digital PCR. J Clin Microbiol 2016;54:2602-8.
[11] Trypsteen W, Kiselinova M, Vandekerckhove L, De Spiegelaere W.
Diagnostic utility of droplet digital PCR for HIV reservoir quanti-
fication. J Virus Erad 2016;2:162-9.
[12] Aizawa Y, Koyama A, Ishihara T, Onodera O, Saitoh A. Performance
of a real-time PCR-based approach and droplet digital PCR in
detecting human parechovirus type 3 RNA. J Clin Virol 2016;84:27-
31.
[13] Tang H, Cai Q, Li H, Hu P. Comparison of droplet digital PCR to
real-time PCR for quantification of hepatitis B virus DNA. Biosci
Biotechnol Biochem 2016:1-6.
[14] Yan Y, Jia XJ, Wang HH, Fu XF, Ji JM, He PY, et al. Dynamic
quantification of avian influenza H7N9(A) virus in a human infection
during clinical treatment using droplet digital PCR. J Virol Methods
2016;234:22-7.
[15] Giovannelli I, Ciccone N, Vaggelli G, Malva ND, Torricelli F,
Rossolini GM, et al. Utility of droplet digital PCR for the quantita-
tive detection of polyomavirus JC in clinical samples. J Clin Virol.
2016;82:70-5.
[16] Jeannot E, Becette V, Campitelli M, Calmejane MA, Lappartient E,
Ruff E, et al. Circulating human papillomavirus DNA detected using
droplet digital PCR in the serum of patients diagnosed with early
stage human papillomavirus-associated invasive carcinoma. J Pathol
Clin Res. 2016;2:201-9.
[17] Gianella S, Chaillon A, Mutlu EA, Engen PA, Voigt RM, Keshavar-
zian A, et al. Effect of CMV and EBV replication on intestinal muco-
sal gene expression and microbiome composition of HIV-infected
and uninfected individuals. AIDS 2017; doi: 10.1089/aid.2017.0145.
[18] Arvia R, Sollai M, Pierucci F, Urso C, Massi D, Zakrzewska K.
Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR)
approach for detection and quantification of Merkel cell polyoma-
virus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE)
cutaneous biopsies. J Virol Method. 2017;246:15-20.
[19] Pavsic J, Zel J, Milavec M. Digital PCR for direct quantification of
viruses without DNA extraction. Anal Bioanal Chem 2016;408:67-75.
[20] Sedlak RH, Cook L, Huang ML, Magaret A, Zerr DM, Boeckh M,
et al. Identification of chromosomally integrated human herpesvirus
6 by droplet digital PCR. Clin Chem 2014;60:765-72.
[21] Sedlak RH, Hill JA, Nguyen T, Cho M, Levin G, Cook L, et al.
Detection of human herpesvirus 6B (HHV-6B) reactivation in
hematopoietic cell transplant recipients with inherited chromosom-
ally integrated HHV-6A by droplet digital PCR. J Clin Microbiol
2016;54:1223-7.
[22] Ushio R, Yamamoto M, Nakashima K, Watanabe H, Nagai K,
Shibata Y, et al. Digital PCR assay detection of circulating Mycobac-
terium tuberculosis DNA in pulmonary tuberculosis patient plasma.
Tuberculosis 2016;99:47-53.
[23] Hennebique A, Bidart M, Jarraud S, Beraud L, Schwebel C, Maurin
M, et al. Digital PCR for detection and quantification of fluoro-
quinolone resistance in Legionella pneumophila. Antimicrob Agents
Chemother 2017;61:e00628-17.
Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier 31
[24] Pholwat S, Stroup S, Foongladda S, Houpt E. Digital PCR to detect
and quantify heteroresistance in drug resistant Mycobacterium tuber-
culosis. PLoS One 2013;8:e57238.
[25] Whale AS, Bushell CA, Grant PR, Cowen S, Gutierrez-Aguirre I,
O’Sullivan DM, et al. Detection of rare drug resistance mutations by
digital PCR in a human influenza A virus model system and clinical
samples. J Clin Microbiol 2016;54:392-400.
[26] Schoepp NG, Khorosheva EM, Schlappi TS, Curtis MS, Humphries
RM, Hindler JA, et al. Digital quantification of DNA replication and
chromosome segregation enables determination of antimicrobial
susceptibility after only 15 minutes of antibiotic exposure. Angew
Chem 2016;55:9557-61.
[27] Srisutham S, Saralamba N, Malleret B, Renia L, Dondorp AM,
Imwong M. Four human Plasmodium species quantification using
droplet digital PCR. PLoS One 2017;12:e0175771.
[28] Yang R, Paparini A, Monis P, Ryan U. Comparison of next-gener-
ation droplet digital PCR (ddPCR) with quantitative PCR (qPCR)
for enumeration of Cryptosporidium oocysts in faecal samples. Int J
Parasitol 2014;44:1105-13.
[29] Weerakoon KG, Gordon CA, Cai P, Gobert GN, Duke M, Wil-
liams GM, et al. A novel duplex ddPCR assay for the diagnosis of
Schistosomiasis japonica: proof of concept in an experimental mouse
model. Parasitology 2017;144:1005-15.
32 Clinical Microbiology Newsletter 40:4,2018 | ©2018 Elsevier
[30] Haynes RJ, Kline MC, Toman B, Scott C, Wallace P, Butler JM,
et al. Standard reference material 2366 for measurement of human
cytomegalovirus DNA. J Mol Diagn 2013;15:177-85.
[31] Liang W, Xu L, Sui Z, Li Y, Li L, Wen Y, et al. Quantification
of plasmid DNA reference materials for Shiga toxin-producing
Escherichia coli based on UV, HR-ICP-MS and digital PCR. Chem
Central J 2016;10:55.
[32] Hayden RT, Gu Z, Sam SS, Sun Y, Tang L, Pounds S, et al. Com-
parative evaluation of three commercial quantitative cytomegalovirus
standards by use of digital and real-time PCR. J Clin Microbiol
2015;53:1500-5.
[33] Bateman AC, Greninger AL, Atienza EE, Limaye AP, Jerome KR,
Cook L. Quantification of BK virus standards by quantitative real-
time PCR and droplet digital PCR is confounded by multiple virus
populations in the WHO BKV international standard. Clin Chem
2017;63:761-9.
[34] Greninger AL, Bateman AC, Atienza EE, Wendt S, Makhsous N,
Jerome KR, et al. Copy number heterogeneity of JC virus standards.
J Clin Microbiol 2017;55:824-31.
[35] Hu Y, Xu P, Luo J, He H, Du W. Absolute quantification of H5-sub-
type avian influenza viruses using droplet digital loop-mediated
isothermal amplification. Anal Chem 2017;89:745-50.