Comparison of fast-track diagnostics respiratory pathogens multiplex real-time RT-PCR assay with in-house singleplex assay for comprehensive detection of human respiratory viruses

Article (PDF Available)inJournal of virological methods 185(2):259-66 · July 2012with188 Reads
DOI: 10.1016/j.jviromet.2012.07.010 · Source: PubMed
Abstract
Fast-track Diagnostics respiratory pathogens (FTDRP) multiplex real-time RT-PCR assay was compared with in-house singleplex real-time RT-PCR assays for detection of 16 common respiratory viruses. The FTDRP assay correctly identified 26 diverse respiratory virus strains, 35 of 41 (85%) external quality assessment samples spiked with cultured virus and 232 of 263 (88%) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. Of 308 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 270 (87.7%) and 265 (86%) were positive by FTDRP and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (K=0.812, 95% CI=0.786-0.838). Individual FTDRP assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. The FTDRP enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. With the exceptions noted above, most FTDRP assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time PCR instrumentation.
Journal
of
Virological
Methods
185 (2012) 259–
266
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at
SciVerse
ScienceDirect
Journal
of
Virological
Methods
jou
rn
al
h
om
epage:
www.elsevier.com/locate/jviromet
Comparison
of
fast-track
diagnostics
respiratory
pathogens
multiplex
real-time
RT-PCR
assay
with
in-house
singleplex
assays
for
comprehensive
detection
of
human
respiratory
viruses
Senthilkumar
K.
Sakthivel
a,b
,
Brett
Whitaker
a,c
,
Xiaoyan
Lu
a
,
Danielle
B.L.
Oliveira
a,d
,
Lauren
J.
Stockman
a
, Shifaq
Kamili
a,c
,
M.
Steven
Oberste
a
,
Dean
D.
Erdman
a,
a
Division
of
Viral
Diseases,
Centers
for
Disease
Control
and
Prevention,
Atlanta,
GA,
United
States
b
Logistics
Health
Incorporated,
La
Crosse,
WI,
United
States
c
Atlanta
Research
and
Education
Foundation,
Atlanta,
GA,
United
States
d
Institute
of
Biomedical
Science,
University
of
São
Paulo,
São
Paulo,
Brazil
Article
history:
Received
16
February
2012
Received
in
revised
form
26
June
2012
Accepted
4
July
2012
Available online 11 July 2012
Keywords:
Respiratory
virus
Real-time
PCR
Multiplex
a
b
s
t
r
a
c
t
Fast-track
Diagnostics
respiratory
pathogens
(FTDRP)
multiplex
real-time
RT-PCR
assay
was
compared
with
in-house
singleplex
real-time
RT-PCR
assays
for
detection
of
16
common
respiratory
viruses.
The
FTDRP
assay
correctly
identified
26
diverse
respiratory
virus
strains,
35
of
41
(85%)
external
quality
assessment
samples
spiked
with
cultured
virus
and
232
of
263
(88%)
archived
respiratory
specimens
that
tested
positive
for
respiratory
viruses
by
in-house
assays.
Of
308
prospectively
tested
respiratory
speci-
mens
selected
from
children
hospitalized
with
acute
respiratory
illness,
270
(87.7%)
and
265
(86%)
were
positive
by
FTDRP
and
in-house
assays
for
one
or
more
viruses,
respectively,
with
combined
test
results
showing
good
concordance
(K
=
0.812,
95%
CI
=
0.786–0.838).
Individual
FTDRP
assays
for
adenovirus,
res-
piratory
syncytial
virus
and
rhinovirus
showed
the
lowest
comparative
sensitivities
with
in-house
assays,
with
most
discrepancies
occurring
with
specimens
containing
low
virus
loads
and
failed
to
detect
some
rhinovirus
strains,
even
when
abundant.
The
FTDRP
enterovirus
and
human
bocavirus
assays
appeared
to
be
more
sensitive
than
the
in-house
assays
with
some
specimens.
With
the
exceptions
noted
above,
most
FTDRP
assays
performed
comparably
with
in-house
assays
for
most
viruses
while
offering
enhanced
throughput
and
easy
integration
by
laboratories
using
conventional
real-time
PCR
instrumentation.
Published by Elsevier B.V.
1.
Introduction
Respiratory
viruses
are
among
the
most
important
causes
of
human
morbidity
and
mortality
worldwide
(Nair
et
al.,
2010;
Pavia,
2011).
Clinically
indistinguishable,
respiratory
virus
infections
require
accurate
laboratory
diagnosis
to
guide
treatment
effec-
tively
and
prevention
decisions.
Polymerase
chain
reaction
(PCR)
and
other
molecular
assays
are
now
routinely
used
for
diagnosis
The
contents
of
this
manuscript
are
solely
the
responsibility
of
the
authors
and
do
not
necessarily
represent
the
official
views
of
the
US
Centers
for
Disease
Con-
trol
and
Prevention
(CDC)
or
Department
of
Health
and
Human
Services
(DHHS).
Names
of
specific
vendors,
manufacturers,
or
products
are
included
for
public
health
and
informational
purposes;
inclusion
does
not
imply
endorsement
of
the
vendors,
manufacturers,
or
products
by
the
CDC
or
DHHS.
Corresponding
author
at:
Division
of
Viral
Diseases,
National
Center
for
Immu-
nization
and
Respiratory
Diseases,
Centers
for
Disease
Control
and
Prevention,
1600
Clifton
Road,
N.E.,
Atlanta,
GA
30333,
Mailstop
G-04,
United
States.
Tel.:
+1
404
639
3727;
fax:
+1
404
639
4416.
E-mail
address:
dde1@cdc.gov
(D.D.
Erdman).
of
respiratory
virus
infections
(Beck
and
Henrickson,
2010;
Kehl
and
Kumar,
2009),
but
the
large
and
increasing
number
of
viruses
makes
laboratory
testing
with
individual
(singleplex)
virus
assays
challenging.
Conversely,
multiplex
PCR
assays
that
combine
multi-
ple
individual
assays
in
a
single
reaction
facilitate
more
rapid,
high
throughput
and
cost-effective
testing
and
are
generally
preferred
in
the
clinical
setting
(Elnifro
et
al.,
2000;
Jansen
et
al.,
2011).
Numerous
laboratory-developed
and
commercial
multiplex
PCR
assays
using
different
amplification
platforms
have
been
described
for
respiratory
viruses
and
have
been
generally
shown
to
be
superior
to
traditional
diagnostic
methods,
such
as
virus
culture
and
antigen
detection
for
sensitive
and
specific
detection
of
respi-
ratory
viruses
(Arens
et
al.,
2010;
Bibby
et
al.,
2011;
Brittain-Long
et
al.,
2010;
Caliendo,
2011;
Gadsby
et
al.,
2010;
Kim
et
al.,
2009;
Lamson
et
al.,
2006;
Mahony
et
al.,
2007;
Raymond
et
al.,
2009).
The
US
Food
and
Drug
Administration
(FDA)
has
cleared
recently
two
commercial
assays,
the
xTAG
®
RVP
Fast
(Luminex
Molecular
Diagnostics,
Austin,
TX)
and
FilmArray
®
Respiratory
Panels
(Idaho
Technology
Inc.,
Salt
Lake
City,
Utah)
(Rand
et
al.,
2011),
for
in-
vitro
diagnostic
use
for
multiplex
detection
of
respiratory
viruses.
0166-0934/$
see
front
matter.
Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.jviromet.2012.07.010
260 S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266
However,
many
of
these
assays
are
costly,
require
specialized
lab-
oratory
equipment
and
use
highly
multiplexed
reactions
that
may
be
deficient
in
individual
assay
performance
and
can
be
difficult
to
modify
without
extensive
assay
reoptimization
(Gunson
et
al.,
2008).
The
FTD
respiratory
pathogens
(FTDRP)
multiplex
assay
kit
(Fast-track
Diagnostics,
Luxembourg)
uses
standard
commercial
one-step
reverse
transcription
(RT)-PCR
hydrolysis
probe
chem-
istry
and
common
real-time
PCR
instrumentation.
The
FTDRP
assay
consists
of
5
discrete
primer/probe
mixes
that
together
cover
16
common
human
respiratory
viruses.
This
study
reports
the
results
of
a
comparison
of
the
FTDRP
multiplex
assay
with
a
panel
of
val-
idated
in-house
singleplex
real-time
RT-PCR
assays
developed
at
the
Centers
for
Disease
Control
and
Prevention
(CDC).
2.
Materials
and
methods
2.1.
Viruses
and
specimens
Virus
isolates
and
archived
clinical
specimens
were
obtained
from
CDC
collections
acquired
during
routine
surveillance
and
out-
break
investigations.
These
included
26
laboratory
reference
virus
strains
and
field
isolates
and
265
geographically
(U.S.,
Central
and
South
America
and
Africa)
and
compositionally
diverse
specimens
[nasopharyngeal
and
oropharyngeal
swabs
(223),
nasal
washes
and
aspirates
(21),
sputum
(1),
lung
autopsy
tissue
(1)
and
unidenti-
fied
(19)]
collected
from
children
and
adults
with
acute
respiratory
illnesses
(ARIs)
acquired
between
2008
and
2011
and
previously
testing
positive
for
respiratory
viruses
by
the
in-house
singleplex
assays.
All
residual
samples
and
extracts
were
stored
at
70
C.
Whenever
possible,
archived
specimens
were
selected
to
achieve
a
proportional
representation
of
viral
loads.
Forty-six
mock
human
specimens
spiked
with
moderate-to-low
concentrations
of
virus
were
available
from
the
2010
Quality
Control
for
Molecular
Diag-
nostics
(QCMD,
Glasgow,
Scotland)
external
quality
assessment
(EQA)
programs
for
rhinovirus/coronavirus,
adenovirus,
parain-
fluenza
viruses,
human
metapneumovirus/respiratory
syncytial
virus,
and
influenza
A
&
B
viruses
(Wallace,
2003).
Pooled
nasal
wash
specimens
from
20
consenting
healthy
new
military
recruits
was
kindly
provided
by
Dr.
Lisa
Lott,
Eagle
Applied
Sciences,
L.L.C.,
San
Antonio,
TX.
Finally,
a
subset
of
308
nasopharyngeal
aspirates
(NPAs)
from
an
etiologic
study
of
1162
children
<
2
years
of
age
hospitalized
with
ARI
at
a
tertiary
hospital
in
São
Paulo,
Brazil,
between
March
2008
and
September
2010,
were
selected
from
the
seasonal
peaks
of
respiratory
virus
circulation
for
each
of
the
study
years
based
on
local
surveillance
data.
The
NPAs
were
collected
directly
into
liquid
nitrogen,
aliquoted
and
transferred
to
70
C
and
retained
until
retrieved
for
this
study.
This
study
was
approved
by
institutional
review
boards
at
the
University
of
São
Paulo
and
Santa
Casa
de
Misericórdia
de
São
Paulo
Hospital,
Brazil,
and
CDC.
2.2.
Total
nucleic
acid
extraction
Total
nucleic
acid
(TNA)
extracts
were
prepared
from
samples
using
the
NucliSENS
®
easyMAG
®
(bioMérieux).
Because
of
their
multiple
study
origins
and
testing
histories,
residual
archived
spec-
imen
extraction
volumes
varied
from
100
to
300
L
and
TNA
elution
volumes
from
55
to
100
L.
RNase-free
water
was
added
to
a
few
archived
extracts
(2-fold
dilution)
to
obtain
sufficient
volume
for
comparison
testing.
For
prospectively
tested
nasal
aspirate
speci-
mens,
300
L
of
each
sample
was
extracted
and
the
TNA
recovered
in
210
L
of
elution
buffer
which
was
then
split
into
3
aliquots
and
frozen
at
70
C
until
testing.
All
extracts
were
subjected
to
iden-
tical
freeze-thaw
cycles
for
comparison
testing.
All
extracts
were
Table
1
Comparison
of
FTDRP
and
in-house
assays
with
26
virus
isolates.
Virus
(strain)
In-house
(Ct)
FTDRP
(Ct)
a
AdV
C1
(Ad.71)
Pos
(13.7)
Pos
(16.7)
AdV
C5
(Ad.75) Pos
(18.4) Pos
(20.1)
AdV
B7
(SA-104) Pos
(13.8)
Pos
(18.8)
AdV
B14
(deWit)
Pos
(20.8)
Pos
(23.3)
AdV
E4
(RI-67)
Pos
(15.2)
Pos
(18.2)
CoV
229E
Pos
(10.3)
Pos
(13.2)
CoV
OC43
Pos
(13.0)
Pos
(14.9)
CoV
SARS
(Urbani) Pos
(19.2) n/a
c
EV,
echovirus
6
b
Pos
(21.3) Pos
(15.8)
EV,
echovirus
11
b
Pos
(16.3)
Pos
(15.5)
EV,
enterovirus
68
b
Pos
(21.3)
Pos
(24.6)
HMPV
A
(CAN
97-83)
Pos
(15.0)
Pos
(17.0)
HMPV
B
(CAN
98-75)
Pos
(18.3)
Pos
(21.2)
Inf
A
H1N1
(A/California/09) Pos
(14.7) Pos
(14.8)
Inf
A
H2N1
(A/Japan/57)
Pos
(27.3)
Pos
(24.4)
Inf
B
(B/Shanghai/99) Pos
(14.6)
Pos
(15.1)
PIV
1
(C35)
Pos
(16.6)
Pos
(19.1)
PIV
2
(Greer) Pos
(16.9) Pos
(15.8)
PIV
3
(C-43)
Pos
(15.2)
Pos
(16.3)
PIV
4a
(M-25) Pos
(16.7)
Pos
(19.5)
PIV
4b
(CH
19503)
Pos
(21.5)
Pos
(21.1)
PeV
1
b
Pos
(16.0)
Pos
(16.4)
RSV
A
(Long)
Pos
(15.0)
Pos
(15.7)
RSV
B
(CH
93-18B)
Pos
(15.1)
Pos
(16.7)
RV
A1a Pos
(13.4) Pos
(15.3)
RV
B14
Pos
(15.7)
Pos
(32.0)
a
Unless
otherwise
indicated,
all
other
FTDRP
assays
were
negative.
b
FTDRP
EV/PeV
assay
does
not
distinguish
between
EV
and
PeV.
c
FTDRP
SARS
CoV
assay
not
available
(n/a).
confirmed
positive
for
human
RNase
P
gene
by
real-time
RT-PCR
before
inclusion
in
the
study.
2.3.
FTDRP
multiplex
assay
The
FTDRP
multiplex
real-time
RT-PCR
assay
(ver.5,
cat.
no.
FTD
2-96/12)
consists
of
5
separate
primer/probe
mixes
covering
16
human
respiratory
viruses
and
brome
mosaic
virus
(BMV),
an
RNA
plant
virus
that
serves
as
an
internal
extraction
control
when
spiked
into
the
sample
(virus
provided);
mix
#1:
influenza
A
virus
(Inf
A),
influenza
B
virus
(Inf
B),
BMV;
mix
#2:
coronavirus
(CoV)
NL63,
229E
and
OC43
and
enterovirus/parechovirus
(EV/PeV);
mix
#3:
parainfluenza
virus
(PIV)
2,
3
and
4;
mix
#4:
PIV
1,
human
metapneumovirus
(HMPV)
and
human
bocavirus
(HBoV);
mix
#5:
rhinovirus
(RV),
respiratory
syncytial
virus
(RSV)
and
adenovirus
(AdV).
Individual
assays
within
each
pool
are
distinguished
by
use
of
different
probe
fluorophores,
with
the
exception
of
the
EV
and
PeV
assays,
where
both
probes
are
ROX-labeled
and
therefore
can-
not
be
distinguished.
Each
kit
also
contains
a
positive
plasmid
control
pool
and
detailed
instructions
on
test
performance.
The
FTDRP
assay
was
performed
following
the
manufacturer’s
recom-
mendations.
Briefly,
194
L
of
2×
RT-PCR
buffer
was
combined
with
23.3
L
of
each
primer/probe
pool
and
15.5
L
of
25×
enzyme
mix
(AgPath-ID
TM
One-Step
RT-PCR
Kit,
Applied
Biosystems),
and
15
L
of
each
mixture
was
then
added
to
14
wells
of
a
PCR
plate
(12
sample
reactions
plus
one
positive
and
one
negative
virus
control).
Ten
L
of
sample
TNA
extract
or
controls
were
then
added
to
the
respective
wells
of
each
primer/probe
pool.
The
following
cycling
conditions
were
performed
on
a
7500
Fast
Dx
Real-Time
PCR
Instru-
ment
(Applied
Biosystems):
15
min
at
50
C,
10
min
at
95
C
and
40
cycles
of
8
s
at
95
C
and
34
s
at
60
C.
Threshold
cycle
(Ct)
values
were
determined
by
manually
adjusting
the
fluorescence
baseline
to
fall
within
the
exponential
phase
of
the
amplification
curves
and
above
any
background
signal.
A
positive
test
result
was
consid-
ered
a
well-defined
curve
that
crossed
the
threshold
cycle
within
40
cycles.
Positive
and
negative
virus
plasmid
controls
provided
in
the
kit
were
included
in
all
runs
to
monitor
assay
performance.
The
S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266 261
Table
2
Comparison
of
FTDRP
and
in-house
assays
with
46
samples
from
5
QCMD
EQA
programs.
QCMD
EQA
a
Virus
QCMD
Key
b
In-house
(Ct)
FTDRP
(Ct)
Adenovirus
(AdV)
ADV10-02 AdV
F41 Pos
(113) Neg/Pos
(39.6)
c
Pos
(39.4)
ADV10-03 AdV
C1
Pos
(64121)
Pos
(30.9)
Pos
(29.2)
ADV10-04
AdV
E4
Pos
(767)
Pos
(36.7)
Pos
(35.1)
ADV10-06 AdV
C1
Pos
(4055)
Pos
(34.0)
Pos
(32.0)
ADV10-08
AdV
B34
Pos
(1225)
Pos
(34.0)
Neg/Neg
c
ADV10-07
No
virus
Neg
Neg
Neg
Influenza
virus
(Inf)
INFRNA
09-01 Inf
A
subtype
H1 Pos
(29.4)
Pos
(29.4)
Pos
(28.9)
INFRNA
09-02 Inf
A
subtype
H3
Pos
(31.4)
Pos
(28.8)
Pos
(29.7)
INFRNA
09-03
Inf
B
Pos
(39.2)
Pos
(38.3)
Pos
(38.2)
INFRNA
09-04
Inf
A
subtype
H1v
d
Pos
(28.7)
Pos
(28.7)
Pos
(25.7)
INFRNA
09-06
Inf
A
subtype
H1
Pos
(27.9)
Pos
(28.7)
Pos
(27.8)
INFRNA
09-07 Inf
B Pos
(32.1) Pos
(30.3) Pos
(27.5)
INFRNA
09-09
Inf
A
subtype
H1v
d
Pos
(32.1)
Pos
(28.8)
Pos
(28.7)
INFRNA
09-10 Inf
A
subtype
H1
Pos
(29.4)
Pos
(29.9)
Pos
(29.1)
INFRNA
09-11 Inf
A
subtype
H1 Pos
(33.1) Pos
(33.5) Pos
(33.0)
INFRNA
09-12
Inf
A
subtype
H3
Pos
(35.6)
Pos
(33.3)
Pos
(33.0)
INFRNA
09-05 No
virus Neg
Neg
Neg
Parainfluezavirus
(PIV)
PINF10-01
PIV
1
Pos
(33.1)
Pos
(32.7)
Pos
(38.1)
PINF10-02
PIV
4
Pos
(31.9)
Pos
(35.2)
Pos
(33.5)
PINF10-03 PIV
1 Pos
(31.0) Pos
(31.1) Pos
(33.8)
PINF10-06
PIV
3
Pos
(34.5)
Pos
(25.7)
Pos
(23.7)
PINF10-07 PIV
2
Pos
(28.2)
Pos
(24.0)
Pos
(21.3)
PINF10-08
No
virus
Neg
Neg
Neg
Respiratory
syncytial
virus
(RSV)
&
Human
metapneumovirus
(HMPV)
MPV.RSV10-01
RSV
A
Pos
(38.4)
Pos
(36.7)
Neg/Pos
(36.8)
c
MPV.RSV10-02 RSV
B Pos
(37.1) Pos
(31.7) Neg/Pos
(33.1)
c
MPV.RSV10-04
RSV
A
Pos
(33.4)
Pos
(31.1)
Pos
(30.7)
MPV.RSV10-09 RSV
B Pos
(39.9)
Pos
(34.8)
Neg/Pos
(37.3)
c
MPV.RSV10-10
RSV
B
Pos
(32.4)
Pos
(24.6)
Pos
(25.4)
MPV.RSV10-11 RSV
A
Pos
(37.3)
Pos
(33.7)
Pos
(36.5)
MPV.RSV10-03
HMPV
B2
Pos
(35.5)
Pos
(29.7)
Pos(32.5)
MPV.RSV10-05
HMPV
B2
Pos
(38.5)
Pos
(32.8)
Pos
(34.2)
MPV.RSV10-07 HMPV
A1 Pos
(39.3)
Pos
(34.9)
Neg/Pos
(39)
c
MPV.RSV10-08
HMPV
A1
Pos
(33.2)
Pos
(29.1)
Pos
(33.2)
MPV.RSV10-12 HMPV
B2
Pos
(35.6)
Pos
(30.0)
Pos
(32.2)
MPV.RSV10-06
No
virus
Neg
Neg
Neg
Rhinovirus
(RV)
&
Coronavirus
(CoV)
RV.CV10-01
RV
B42
Pos
(29.6)
Pos
(26.9)
Pos
(36.7)
RV.CV10-02 RV
A8 Pos
(25.8) Pos
(22.5) Pos
(24.0)
RV.CV10-03
RV
B72
Pos
(22.9)
Pos
(21.5)
Neg/Neg
c
RV.CV10-05 RV
A90
Pos
(32.6)
Pos
(28.7)
Pos
(31.6)
RV.CV10-07
RV
A16
Pos
(30.5)
Pos
(27.5)
Pos
(30.3)
RV.CV10-09
RV
A16
Pos
(34.1)
Pos
(30.9)
Pos
(33.3)
RV.CV10-04
CoV
229E
Pos
(28.5)
Pos
(27.9)
Pos
(26.6)
RV.CV10-08
CoV
229E
Pos
(35.0)
Pos
(34.0)
Pos
(32.5)
RV.CV10-06
CoV
OC43
Pos
(31.1)
Pos
(32.6)
Pos
(32.2)
RV.CV10-10
CoV
NL63
Pos
(26.9)
Pos
(25.7)
Pos
(24.1)
RV.CV10-11
EV
e
Neg
Neg
Neg
a
QCMD
EQA,
2010
Quality
Control
for
Molecular
Diagnostics
External
Quality
Assessment
program
samples.
b
QCMD
test
results;
Ct
values
(RV/CoV,
PIV,
RSV/HMPV)
and
genome
copies/mL
(AdV).
QCMD
Ct
values
should
not
be
used
for
method
comparison
or
as
a
target
for
individual
laboratory
assessment.
c
Original
and
repeat
result.
d
Inf
A
subtype
H1v
=
new
variant
pandemic
H1N1
strain.
e
QCMD
EQA
negative
RV
control
sample
contained
coxsackievirus
A1.
BMV
internal
control
was
spiked
into
clinical
specimens
to
monitor
sample
extraction
and
reverse
transcription.
Previously
extracted
TNA
samples
were
evaluated
for
RNase
P
only.
2.4.
In-house
singleplex
assays
In-house
singleplex
real-time
RT-PCR
assays
for
RSV,
HMPV,
PIV1-4,
RV,
AdV,
HBoV
and
CoVs
(229E,
OC43,
NL63,
HKU1,
SARS-
CoV)
as
previously
described
(Dare
et
al.,
2007;
Emery
et
al.,
2004;
Fry
et
al.,
2010;
Heim
et
al.,
2003;
Kodani
et
al.,
2011;
Lu
et
al.,
2006,
2008;
Morgan
et
al.,
2012)
were
performed
on
a
MX3000P
QPCR
System
(Agilent
Technologies)
using
AgPath-ID
TM
One-Step
RT-PCR
reagents
(Applied
Biosystems)
with
the
following
cycling
condi-
tions:
45
C
for
10
min,
95
C
for
10
min
and
45
cycles
of
95
C
for
15
s
and
55
C
for
1
min.
Primer/probe
sequences
are
available
from
D.E.
on
request.
The
in-house
EV
and
PeV
assays
as
modified
from
previ-
ous
reports
(Kilpatrick
et
al.,
2009;
Nix
et
al.,
2008)
were
performed
on
a
MX3000P
QPCR
System
using
the
SuperScript
III
Platinum
®
One-Step
Quantitative
RT-PCR
System
reagents
(Invitrogen)
with
the
following
cycling
conditions:
50
C
for
30
min,
95
C
for
5
min
262 S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266
and
45
cycles
of
95
C
for
15
s,
55
C
(EV)
or
58
C
(PeV)
for
45
s
and
72
C
for
10
s.
Universal
Inf
A
and
Inf
B
assays
were
performed
on
a
7500
Fast
Dx
Real-Time
PCR
Instrument
with
SDS
software
ver.
1.4
(Applied
Biosystems)
using
the
SuperScript
III
Platinum
®
One-Step
Quantitative
RT-PCR
System
with
the
following
cycling
conditions:
50
C
for
30
min,
95
C
for
2
min
and
45
cycles
of
95
C
for
15
s
and
55
C
for
30
s
(Stephen
Lindstrom,
CDC,
personal
communication).
Following
standard
operating
procedures,
all
in-house
assays
were
performed
in
25
L
final
reaction
volumes
containing
5
L
of
sample
TNA
extract.
A
positive
test
result
was
considered
a
well-
defined
curve
that
crossed
the
threshold
cycle
within
40
cycles.
Positive
and
negative
virus
RNA
transcript
or
whole
virus
extract
controls
were
included
in
all
runs
to
monitor
assay
performance.
2.5.
Statistics
Percent
sensitivity
and
specificity
of
the
FTDRP
assay
for
prospectively
collected
specimens
were
calculated
using
the
in-
house
assays
as
the
reference
standard.
Agreement
between
assays
was
measured
using
the
Kappa
statistic
(Cohen,
1960)
where
0
indicates
no
agreement
and
1
indicates
perfect
agreement.
3.
Results
3.1.
Virus
isolates
The
FTDRP
assay
was
first
evaluated
with
undiluted
TNA
from
cultures
of
26
respiratory
virus
strains
corresponding
to
most
assays
in
the
multiplex
to
assess
assay
specificity
and
virus
strain
inclusivity
(Table
1).
Although
no
FTDRP
assay
for
SARS-CoV
was
available,
this
virus
was
tested
to
assess
the
specificity
of
the
other
FTDRP
CoV
assays.
HBoV
and
CoV
NL63
and
HKU1
isolates
were
not
available
for
testing.
Positive
results
were
obtained
with
both
assays
for
all
viruses
with
no
cross-reactions
detected.
FTDRP
and
in-house
assay
results
were
within
3Ct
values
for
22
(88%)
of
the
viruses
tested.
Notably,
the
FTDRP
RV
assay
gave
a
substantially
higher
Ct
value
(
16.3Ct)
with
one
RV
isolate
(RV-B14).
Serial
dilutions
of
RV-B14
TNA
showed
the
FTDRP
assay
to
be
>1000-fold
less
sensitive
than
the
corresponding
in-house
assay
with
this
virus
strain
(data
not
shown).
3.2.
Pooled
human
respiratory
specimens
The
specificity
of
the
FTDRP
assay
was
further
evaluated
with
pooled
nasal
wash
samples
from
20
consenting
normal
healthy
adults
to
represent
diverse
microbial
flora
in
the
human
respiratory
tract.
Positive
results
were
obtained
with
the
in-house
assays
for
RV
(Ct
25.0),
CoV
229E
(Ct
35.1)
and
AdV
(Ct
39.3)
which
were
confirmed
by
alternate
RT-PCR
assays
and
sequencing.
The
FTDRP
assay
was
positive
for
RV
(Ct
28.3)
and
CoV
229E
(Ct
34.8),
but
did
not
detect
the
AdV
on
initial
or
repeat
testing.
All
other
in-house
and
FTDRP
assays
were
negative.
3.3.
QCMD
EQA
program
samples
Forty-one
mock
respiratory
samples
spiked
with
low
to
mod-
erate
levels
of
different
viruses
and
5
negative
control
samples
selected
from
2010
QCMD
EQA
programs
for
HRV/CoV,
AdV,
PIV,
RSV/HMPV
and
Inf
A/B,
were
tested
to
assess
assay
performance
against
the
reference
QCMD
assays
(Table
2).
Overall,
expected
results
were
obtained
with
40
(98%)
and
35
(85%)
of
positive
EQA
program
samples
with
the
in-house
and
FTDRP
assays,
respectively.
All
program
negative
control
samples
were
negative
by
both
assays.
One
sample
(AdV10-02),
with
low
concentration
AdV-F41,
was
ini-
tially
negative
by
the
in-house
assay,
but
positive
on
repeat
testing.
The
FTDRP
assay
gave
expected
results
with
all
PIV
(5),
CoV
(4)
Inf
Table
3
Comparison
of
FTDRP
and
in-house
assays
with
263
archived
respiratory
specimens
previously
positive
for
respiratory
viruses.
Virus
a
In-house
+ FTDRP
+
FTDRP
%
+
Ct
<30
b
Ct
30
to
37
b
Ct
>37
to
<40
b
Total
+ FTDRP
+ FTDRP
FTDRP
%
+ Total
+ FTDRP
+ FTDRP
FTDRP
%
+ Total
+ FTDRP
+ FTDRP
FTDRP
%
+
AdV 25 17
68%
8
8
0
100%
11
8
3
73%
6
1
5
17%
CoV
229E 5 5 100%
3 3 0 100%
2 2 0 100%
0
CoV
OC43
7
7
100%
5
5
0
100%
2
2
0
100%
0
CoV
NL63 8 8 100%
7 7 0 100%
1
1
0
100%
0
EV/PeV
c
8 8 100%
4 4 0 100%
4 4 0 100%
0
HBoV 2 2
100%
2
2
0
100%
0
0
HMPV 26 26 100%
20 20 0 100%
6 6 0 100%
0
Inf
A
17
17
100%
12
12
0
100%
5
5
0
100%
0
Inf
B 11 11 100%
10 10 0 100%
1
1
0
100%
0
PIV
1 20 17 85%
9 9 0 100%
11 8 3 73%
0
PIV
2
13
12
92%
6
6
0
100%
6
5
1
83%
1
1
0
100%
PIV
3 31 30 97%
15 15 0 100%
16 15 1 94%
0
PIV
4
12
12
100%
9
9
0
100%
3
3
0
100%
0
RSV 31 23 74%
12 12
0
100%
11
10
1
91%
8
1
7
14%
RV 47 37 79%
38 33 5 87%
9 4 5 44%
0
Total 263
232
88%
160
155
5
97%
88
74
14
84%
15
3
12
20%
a
Virus
co-detections
not
included
in
the
analysis.
b
In-house
assay
results
classified
as
strong
(Ct
<30),
moderate
(Ct
30
to
37)
or
weak
(Ct
>37)
positive.
c
FTDRP
EV/PeV
assay
does
not
distinguish
between
EV
and
PeV.
Eight
specimens
separately
tested
positive
for
EV
(3Ct
<30;
2Ct
30
to
37)
and
PeV
(1Ct
<30;
2Ct
30
to
37)
by
in-house
assays.
S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266 263
Table
4
Comparison
of
FTDRP
and
in-house
assays
with
32
archived
respiratory
specimens
with
sequence
confirmed
rhinovirus
(RV)
or
enterovirus
(EV).
Virus
a
RV
EV
PeV
EV/PeV
In-house
(Ct)
FTDRP
(Ct)
In-house
(Ct)
In-house
(Ct)
FTDRP
(Ct)
b
EV,
enterovirus
68
Neg
Neg
Pos
(31.1)
Neg
Pos
(30.7)
EV,
enterovirus
68
Neg
Neg
Pos
(28.9)
Neg
Pos
(24.4)
EV,
echovirus
9 Neg Neg Pos
(23.5) Neg Pos
(23.4)
EV,
coxsackievirus
B4 Neg Neg
Pos
(22.6)
Neg
Pos
(21.6)
EV,
coxsackievirus
B5 Neg
Neg
Pos
(31.0)
Neg
Pos
(27.1)
RV
A18
Pos
(17.7)
Pos
(23.1)
Neg
Neg
Neg
RV
A19
Pos
(25.1)
Pos
(23.5)
Neg
Neg
Neg
RV
A22
Pos
(19.1)
Pos
(19.6)
Neg
Neg
Neg
RV
A30 Pos
(16.7) Pos
(16.7) Neg Neg Neg
RV
A30 Pos
(20.3) Pos
(23.6) Neg Neg Neg
RV
A33 Pos
(22.3)
Pos
(27.7)
Neg
Neg
Neg
RV
A38
Pos
(18.8)
Pos
(22.6)
Neg
Neg
Neg
RV
A38 Pos
(21.1)
Pos
(24.6)
Neg
Neg
Pos
(36.1)
RV
A49
Pos
(19.5)
Pos
(18.1)
Neg
Neg
Neg
RV
A58
Pos
(17.7)
Pos
(20.2)
Neg
Neg
Neg
RV
A76
Pos
(23.2)
Pos
(29.6)
Neg
Neg
Neg
RV
A68
Pos
(22.8)
Pos
(22.6)
Neg
Neg
Pos
(31.4)
RV
A96
Pos
(25.9)
Pos
(26.3)
Neg
Neg
Neg
RV
B6 Pos
(12.9) Pos
(24.1) Neg Neg
Neg
RV
B6
Pos
(27.8)
Neg
Neg
Neg
Neg
RV
B6 Pos
(25.1)
Neg
Neg
Neg
Neg
RV
B48
Pos
(21.5)
Neg
Neg
Neg
Neg
RV
B97
+
C
Pos
(28.3)
Pos
(29.5)
Neg
Neg
Neg
RV
C
Pos
(23.2)
Pos
(22.9)
Neg
Neg
Neg
RV
C
Pos
(26.8)
Neg
Neg
Neg
Neg
RV
C Pos
(20.6) Pos
(20.8)
Neg
Neg
Neg
RV
C
Pos
(23.1)
Pos
(29.7)
Neg
Neg
Neg
RV
C Pos
(17.3)
Pos
(19.9)
Neg
c
Neg
Pos
(28.3)
RV
C
Pos
(20.0)
Pos
(21.5)
Neg
Neg
Neg
RV
C
Pos
(18.7)
Pos
(28.7)
Neg
Neg
Neg
RV
C Pos
(18.6) Pos
(25.5) Pos
(31.6) Neg Pos
(34.6)
RV
C
Pos
(23.4)
Pos
(28.2)
Neg
Neg
Neg
a
RV
species
A,
B,
C;
no
serotype-specific
determination
for
RV
species
C.
b
FTDRP
EV/PeV
assay
does
not
distinguish
between
EV
and
PeV.
c
Ct
(41.4)
above
assay
cutoff.
A
(8)
and
Inf
B
(2)
positive
samples,
and
3
of
6
(50%)
RSV,
4
of
5
(80%)
HMPV,
5
of
6
(83%)
RV
and
4
of
5
(80%)
AdV
positive
samples.
RSV
(MPV.RSV10-01,
MPV.RSV10-02,
MPV.RSV10-09)
and
HMPV
(MPV.RSV10-07)
positive
samples
that
were
negative
by
FTDRP
assay
had
generally
lower
virus
loads
and
were
positive
on
repeat
testing.
In
contrast,
EQA
samples
spiked
with
RV-B72
(RV.CV10-03)
and
AdV-B34
(ADV10-08)
were
consistently
negative
and
RV-B42
(RV.CV10-01)
showed
substantially
higher
Ct
values
(
9.8Ct)
by
the
FTDRP
RV
assay.
3.4.
Archived
clinical
specimens
Two
hundred
sixty-five
diverse
respiratory
specimens
that
pre-
viously
tested
positive
for
respiratory
viruses
by
in-house
assays
were
selected
for
comparison
with
the
FTDRP
assay.
Of
these,
263
were
positive
for
at
least
one
of
the
16
assays
available
in
the
FTDRP
multiplex;
two
specimens
positive
for
CoV
HKU1
for
which
there
was
no
corresponding
FTDRP
assay
were
also
tested
to
assess
the
specificity
of
the
other
FTDRP
CoV
assays
(Table
3).
Because
of
limited
available
sample
volume,
only
FTDRP
multiplex
mixes
containing
the
virus-specific
assay
were
performed
and
virus
co-
detections
by
the
other
assays
in
each
multiplex
mix
were
not
included
in
the
analysis.
All
specimens
were
confirmed
positive
by
in-house
singleplex
assays
on
retesting.
The
FTDRP
assay
iden-
tified
all
specimens
that
were
positive
for
HBoV
(2),
CoV
NL63
(8),
Inf
A
(17),
Inf
B
(11),
HMPV
(26),
PIV4
(12)
and
EV/PeV
(5
EV
and
3
PeV);
>90%
for
PIV2
(12/13)
and
PIV3
(30/31);
85%
for
PIV1
(17/20);
79%
for
RV
(37/47);
74%
for
RSV
(23/31);
and
68%
for
AdV
(17/25).
Overall,
the
FTDRP
assay
identified
correctly
88%
of
the
archived
specimens
positive
for
respiratory
viruses
by
the
in-house
assays
and
97%
of
specimens
with
lower
Ct
values
(<30).
Two
specimens
positive
for
CoV
HKU1
by
in-house
singleplex
assay
were
negative
by
the
FTDRP
CoV
229E,
OC43
and
NL63
assays.
The
FTDRP
AdV,
RSV
and
RV
assays
gave
the
lowest
relative
sensitivities
with
the
archived
specimens
at
68%,
74%
and
79%,
respectively.
With
the
exception
of
RV,
most
discrepancies
occurred
with
samples
containing
low
levels
of
viral
target.
For
example,
most
FTDRP
AdV
false-negatives
occurred
with
moderate
to
high
Ct
value
specimens
(mean
Ct
37.3;
range
33.0–39.5),
but
this
did
not
appear
to
be
associated
with
any
particular
AdV
type.
A
wide
range
of
sequence-confirmed
AdV
types
were
represented
among
the
archived
specimens,
including
species
B
(types
3,
7
and
50),
C
(types
2,
5,
6
and
untyped)
and
F
(types
40
and
41),
suggesting
that
the
FTDRP
AdV
assay
is
inclusive
for
all
recognized
human
AdV
types.
In
contrast,
FTDRP
RV
assay
failed
to
detect
5
RV
positive
samples
with
low
Ct
values
by
the
corresponding
in-house
assay.
To
further
assess
the
FTDRP
RV
and
EV/PeV
assays
for
virus
type/strain
inclusivity
and
group
exclusivity,
32
archived
samples
with
high
RV
(27)
or
EV
(5)
loads
and
typed
by
partial
VP1
and/or
VP4/2
RT-PCR
and
sequencing
(protocols
available
from
X.L.
on
request)
were
retested
(Table
4).
The
FTDRP
RV
assay
gave
negative
results
with
4
samples
and
was
10Ct
values
higher
than
the
in-
house
assay
with
2
others,
all
species
B
or
C
RVs.
The
FTDRP
EV/PeV
assay
was
also
positive
with
4
sequence-confirmed
RV
positive
specimens
of
which
1
was
also
positive
by
the
in-house
EV
assay;
a
second
sample
also
gave
an
exponential
fluorescence
amplifi-
cation
curve
with
the
in-house
EV
assay,
but
with
a
>40Ct
value
and
was
therefore
classified
as
EV-negative
based
on
test
cutoff
criteria.
Although
EV
was
not
detected
in
2
of
these
samples
by
VP4/2
RT-PCR,
and
PeV
was
not
detected
by
the
in-house
assay,
the
264 S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266
presence
of
these
viruses
could
not
be
ruled
out
definitively.
Never-
theless,
the
most
probable
explanation
for
these
results
is
that
the
FTDRP
and
in-house
EV
assays
cross-react
with
some
RV
strains.
Five
sequence-confirmed
EV-positive
samples
were
positive
by
the
FTDRP
EV/PeV
assay
with
no
evidence
of
cross-reactions
with
the
RV
and
PeV
assays.
3.5.
Prospectively
tested
clinical
specimens
Three
hundred-eight
nasopharyngeal
aspirates
selected
from
a
study
of
infants
and
young
children
hospitalized
with
acute
res-
piratory
infection
were
tested
prospectively
by
both
in-house
and
FTDRP
assays.
Of
these,
277
(89.9%)
were
positive
for
one
or
more
of
the
16
viruses
by
either
the
in-house
singleplex
or
FTDRP
mul-
tiplex
assays,
with
270
(87.7%)
positive
by
the
in-house
assay
and
265
(86%)
positive
by
FTDRP
assay
alone
(Table
5).
Overall,
the
in-
house
and
FTDRP
assays
showed
good
concordance
(K
=
0.812,
95%
CI
=
0.786–0.838)
(Table
6).
As
seen
with
the
archived
specimens,
however,
the
FTDRP
AdV,
RSV
and
RV
assays
gave
consistently
lower
detection
rates
than
the
corresponding
in-house
assays,
at
43.7%,
72.5%
and
75.5%,
respectively,
and
missed
some
specimens
with
high
virus
loads.
Coincidently,
these
three
assays
are
com-
bined
in
the
same
reaction
mix
(mix
#5)
and
had
the
highest
co-detection
rate
for
these
viruses
by
in-house
assays
at
41.9%;
fol-
lowed
by
mix
#4
(PIV1,
HBoV,
HMPV)
at
23.8%;
mix
#2
(CoV
229E,
CoV
OC43,
CoV
NL63,
EV/PeV)
at
16.3%;
mix
#3
(PIV2,
PIV3,
PIV4)
at
7.3%;
and
mix
#1
(Inf
A,
Inf
B)
at
2.2%.
Simultaneous
presence
of
multiple
targets
in
the
same
specimen
may
have
led
to
compet-
itive
inhibition
of
amplification
of
less
abundant
targets
and
may
explain
some
loss
of
assay
sensitivity.
The
FTDRP
HBoV
assay
appeared
to
be
more
sensitive
than
the
corresponding
in-house
assay
(Lu
et
al.,
2006)
with
specimens
containing
low
levels
of
HBoV.
To
further
investigate
this
finding,
limited
sequencing
studies
were
performed
using
a
newly
devel-
oped
semi-nested
PCR
assay
specific
for
the
HBoV
NS1
gene
that
amplifies
all
4
recognized
HBoV
types
(protocol
available
from
X.L.
upon
request).
Of
the
49
specimens
positive
for
HBoV
by
both
in-
house
and
FTDRP
assays,
36
of
37
(mean
in-house
Ct
26.6;
range
13.3–37.3)
were
successfully
sequenced
(all
HBoV
type
1).
In
con-
trast,
only
2
of
4
in-house
assay
positive
(mean
Ct
37.7;
range
Ct
37.3–38.4)/FTDRP
negative
and
none
of
the
20
FTDRP
positive
(mean
Ct
38.2;
range
Ct
36.3–39.9)/in-house
negative
specimens
could
be
confirmed
by
NS1
PCR
and
sequencing.
Failure
to
resolve
these
discrepancies
may
be
due
to
(i)
a
higher
sensitivity
of
the
FTDRP
assay
with
specimens
containing
low
levels
of
HBoV
DNA,
possibly
attributable
to
the
larger
volume
of
TNA
extract
used
in
the
FTDRP
assay
(10
L
vs.
5
L),
(ii)
failure
of
both
assays
to
detect
some
variant
HBoV
strains
and/or
(iii)
non-specific
amplification
or
amplicon
contamination
in
these
samples.
The
FTDRP
EV/PeV
assay
also
appeared
to
be
more
sensitive
and
specific
than
the
corresponding
in-house
EV
assay
with
some
spec-
imens.
Of
18
specimens
positive
by
the
FTDRP
EV/PeV
assay
(mean
Ct
34.6;
range
29.9–38.4),
and
negative
by
in-house
EV
and
PeV
assays,
9
had
recoverable
VP1
and/or
VP4/2
sequences
represent-
ing
8
different
EVs
(echovirus
6,
24,
30;
enterovirus
68;
poliovirus
1;
coxsackievirus
A4,
B1,
B4);
3,
that
were
also
positive
by
in-house
and
FTDRP
RV
assays
(Ct
<
30),
had
sequence-confirmed
species
A
RV
of
which
2
also
had
type-indeterminate
EV
sequences
present;
1
gave
a
fluorescence
amplification
curve
with
the
in-house
PeV
assay,
but
with
a
Ct
value
>40
and
therefore
was
classified
as
PeV
negative;
and
5
could
not
be
sequenced.
Of
9
samples
positive
by
the
in-house
EV
assay
and
negative
by
the
FTDRP
EV/PeV
assay,
all
were
strongly
positive
for
RV
(Ct
<
30)
by
both
in-house
and
FTDRP
RV
assays
and
were
confirmed
positive
for
RV
species
A
or
C
by
VP1
and/or
VP4/2
sequences.
All
12
specimens
positive
by
the
in-house
Table
5
Comparison
of
FTDRP
and
in-house
assays
with
308
prospectively
tested
respiratory
specimens.
Virus
a
In-house
+
FTDRP
+
In-house
+
FTDRP
In-house
FTDRP
+
c
In-house
FTDRP
Ct
<30
b
Ct
30
to
37
b
Ct
>37
to
<40
b
Total
+
FTDRP
+
FTDRP
FTDRP
%
+
Total
+
FTDRP
+
FTDRP
FTDRP
%
+
Total
+
FTDRP
+
FTDRP
FTDRP
%
+
AdV
38
49
0
221
32
29
3
91%
47
9
38
19%
8
0
8
0%
CoV
229E
6
0
1
301
4
4
0
100%
2
2
0
100%
0
CoV
OC43
19
4
0
285
14
14
0
100%
6
5
1
83%
3
0
3
0%
CoV
NL63
19
0
0
289
9
9
0
100%
10
9
1
90%
0
EV/PeV
d
36
9
18
245
19
16
3
84%
23
17
6
74%
3
3
0
100%
HBoV
49
4
20
235
25
25
0
100%
17
17
0
100%
11
7
4
64%
HMPV
50
17
0
241
39
39
0
100%
19
10
9
53%
9
1
8
11%
Inf
A
29
1
0
278
17
17
0
100%
10
10
0
100%
3
2
1
67%
Inf
B
14
1
0
293
9
9
0
100%
5
5
0
100%
1
0
1
0%
PIV
1
9
4
0
295
7
7
0
100%
5
2
3
40%
1
0
1
0%
PIV
2
1
0
0
307
1
1
0
100%
0
0
PIV
3
51
2
0
255
35
35
0
100%
13
12
1
92%
3
2
1
67%
PIV
4
8
0
2
298
2
2
0
100%
3
3
0
100%
3
3
0
100%
RSV
74
28
0
206
80
68
12
85%
15
6
9
40%
7
0
7
0%
RV
80
26
3
199
90
73
17
81%
15
7
8
47%
1
0
1
0%
Total
483
145
44
3948
383
348
35
91%
190
114
76
60%
53
18
35
34%
a
Virus
co-detections
included
in
the
analysis.
b
In-house
assay
results
classified
as
strong
(Ct
<30),
moderate
(Ct
30
to
37)
or
weak
(Ct
>37
to
<40)
positive.
c
FTDRP
assay
Ct
values,
median
(range):
HBoV,
38.3
(36.3–39.9);
CoV
229E,
38.3;
PIV
4,
39.4
(38.9,
39.9);
RV,
34.8
(34.8–37.0);
EV/PeV
34.7
(30.0–38.4).
d
FTDRP
EV/PeV
assay
does
not
distinguish
between
EV
and
PeV.
Twelve
samples
positive
by
in-house
PeV
assay
were
also
positive
by
FTDRP
EV/PeV
assay.
S.K.
Sakthivel
et
al.
/
Journal
of
Virological
Methods
185 (2012) 259–
266 265
Table
6
FTDRP
and
in-house
assay
sensitivity,
specificity
and
Kappa
values
with
308
prospectively
tested
respiratory
specimens.
Virus
a
FTDRP
b
In-house
b
Kappa
statistic
c
(95%
CI)
Sensitivity
Specificity
Sensitivity
Specificity
AdV
43.7
100.0
100.0
82.0
0.527
(0.405–0.648)
CoV
229E
100.0
99.7
85.7
100.0
0.921
(0.767–1)
CoV
OC43 82.6
100.0
100.0
98.6
0.898
(0.798–0.997)
CoV
NL63 100.0
100.0
100.0
100.0
1.(1–1)
EV/PeV
d
80.0
93.2
66.7
96.5
0.676
(0.559–0.793)
HBoV
92.5
92.2
71.0
98.3
0.756
(0.662–0.85)
HMPV
74.6
100.0
100.0
93.4
0.822
(0.739–0.904)
Inf
A
96.7
100.0
100.0
99.6
0.981
(0.946–1)
Inf
B 93.3
100.0
100.0
100.0
0.964
(0.893–1)
PIV
1 69.2
100.0
100.0
98.7
0.812
(0.628–0.995)
PIV
2 100.0
100.0
100.0
100.0
1.(1–1)
PIV
3
96.2
100.0
100.0
99.2
0.977
(0.945–1)
PIV
4 100.0
99.3
80.0
100.0
0.886
(0.728–1)
RSV
72.5
100.0
100.0
88.0
0.780
(0.702–0.857)
RV
75.5
98.5
96.4
88.4
0.780
(0.704–0.856)
All
assays
77.0
98.8
91.3
96.5
0.812
(0.786–0.838)
a
Virus
co-detections
included
in
the
analysis.
b
Referenced
to
FTDRP
or
in-house
assay.
c
Kappa
statistic:
<0–0.2
=
poor;
0.21–0.4
=
fair;
0.41–0.6
=
moderate;
0.61–0.8
=
good;
and
0.81–1
=
very
good.
CI,
confidence
interval.
d
FTDRP
EV/PeV
assay
does
not
distinguish
between
EV
and
PeV.
PeV
assay
(mean
Ct
33.8;
range
27.7–38.4)
were
also
positive
by
the
FTDRP
EV/PeV
assay
with
similar
Ct
values.
4.
Discussion
Diagnosis
of
ARI
in
both
clinical
care
and
public
health
settings
has
greatly
advanced
in
recent
years
with
the
increased
availability
of
rapid,
sensitive
and
specific
molecular
tests
for
the
simultane-
ous
detection
of
multiple
respiratory
pathogens.
Some
commercial
assays
in
particular
that
have
received
FDA
510(k)
clearance
have
made
substantial
inroads
into
the
diagnostic
laboratory
(Rand
et
al.,
2011).
However,
these
assays
are
often
costly,
require
dedicated
laboratory
equipment,
use
highly
multiplexed
reactions
where
individual
assay
performance
may
be
compromised,
and
can
be
difficult
to
modify
quickly
in
response
to
the
emergence
of
new
medically
important
virus
strains,
as
occurred
during
the
2009
H1N1
influenza
pandemic.
The
commercial
multiplex
FTDRP
real-time
RT-PCR
assay
addresses
some
of
these
limitations
by
offering
a
complete
kit
with
moderate
throughput
for
detection
of
16
respiratory
viruses
that
could
be
easily
integrated
into
the
workflow
of
laboratories
using
conventional
real-time
PCR
platforms.
The
FTDRP
assay
setup
and
runtime
requires
approximately
2.5
h
for
12
samples
and
controls
(assay
reagents
are
aliquoted
in
12
sample
test
units),
excluding
sample
extraction,
and
with
a
kit
list
price
of
$27.34/sample
(PCR
enzyme
kit
costs
not
included).
By
combining
assays
into
5
multi-
plex
reaction
mixes,
individual
mixes
could
more
easily
modified
if
needed
without
impacting
the
other
mixes
and
could
allow
for
more
efficient
targeted
testing
based
on
epidemiologic
findings.
In
this
study,
the
FTDRP
multiplex
assay
was
compared
with
in-
house
singleplex
assays
corresponding
to
each
of
the
test
viruses.
Overall,
the
FTDRP
and
in-house
assays
performed
comparably
for
most
viruses
tested,
particularly
when
the
virus
was
abundant
in
the
sample
(low
Ct
values).
With
exceptions
noted
below,
most
discordant
results
were
seen
with
samples
containing
lower
con-
centrations
of
virus
(high
Ct
values),
suggesting
that
differences
in
assay
sensitivity
near
their
detection
limits
was
responsible
for
these
discrepancies
rather
than
failure
of
primer/probe
hybridiza-
tion
due
to
critical
target
sequence
mismatches.
FTDRP
assays
for
RSV,
RV
and
AdV
in
particular
showed
lower
relative
sensitivities
than
the
corresponding
in-house
assays
with
some
clinical
specimens.
The
FTDRP
RV
assay
showed
clear
evi-
dence
of
dropouts
with
some
RV
strains
(see
further
discussion
below),
and
some
prospectively
tested
specimens
were
negative
for
RSV
and
AdV,
even
when
the
viruses
were
abundant.
It
is
notable
that
these
three
FTDRP
assays
are
combined
in
the
same
reac-
tion
mix
and
these
three
viruses
showed
the
highest
co-detection
rates
by
singleplex
in-house
in
these
specimens.
It
is
possible
that
competing
amplification
reactions
in
some
specimens
containing
multiple
virus
targets
may
have
reduced
the
sensitivity
of
some
assays
for
low
abundant
targets.
This
may
have
had
a
more
notice-
able
impact
on
detection
of
AdV,
where
a
disproportionate
number
of
AdV
positive
specimens
had
lower
virus
loads.
This
would
be
expected
in
a
population
comprised
of
infants
and
young
children
where
persistent
low
level
AdV
shedding
is
common.
Development
of
real-time
RT-PCR
assays
that
can
detect
all
RV
and
EV
strains
and
distinguish
between
both
groups
is
challeng-
ing
due
to
the
extensive
sequence
diversity
within
each
group
and
sequence
similarity
between
some
EV
and
RV
strains.
These
data
confirmed
previous
experience
with
the
in-house
EV
and
RV
assays:
both