ArticlePDF Available

A clinical specimen collection and transport medium for molecular diagnostic and genomic applications

Authors:

Abstract and Figures

Pathogen detection and genetic characterization has dramatically changed in recent years. Clinical laboratories are transitioning from traditional culture and primer-specific sequencing to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic characterization, respectively. Specimen collection is the first step in any downstream molecular diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be amplified using real-time RT-PCR for tracking the integrity of a specimen from the point of collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released RNA at ambient temperature for up to 30 days for downstream real-time and traditional RT-PCR detection and genetic characterization. PrimeStore MTM is also compatible with a variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has added utility for field collection in remote areas, triage centres, border crossings and during pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens are a concern.
Content may be subject to copyright.
A clinical specimen collection and transport medium for
molecular diagnostic and genomic applications
L. T. D AUM
1
*, S. A. W O RTH Y
1
,K.C.YIM
2
, M. NOGUERAS
3
,R.F.SCHUMAN
4
,
Y. W. C HOI
5
AND G. W. F ISC H ER
1
1
Longhorn Vaccines & Diagnostics, San Antonio, TX, USA
2
Virion Systems Inc., Bethesda, MD, USA
3
BioReliance Corp., Rockville, MD, USA
4
Antibody and Immunoassay Consultants, LLC, Rockville, MD, USA
5
Battelle Biomedical Research Center, Columbus, OH, USA
(Accepted 30 September 2010)
SUMMARY
Pathogen detection and genetic characterization has dramatically changed in recent years.
Clinical laboratories are transitioning from traditional culture and primer-specific sequencing
to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic
characterization, respectively. Specimen collection is the first step in any downstream molecular
diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of
nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be
amplified using real-time RT–PCR for tracking the integrity of a specimen from the point of
collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including
highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released
RNA at ambient temperature for up to 30 days for downstream real-time and traditional
RT–PCR detection and genetic characterization. PrimeStore MTM is also compatible with a
variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has
added utility for field collection in remote areas, triage centres, border crossings and during
pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens
are a concern.
Key words: Influenza detection, Longhorn Vaccines and Diagnostics, molecular diagnostics,
molecular transport medium, PrimeStore MTM, real-time RT–PCR, specimen collection and
storage, viral transport media.
INTRODUCTION
Emerging infectious respiratory diseases constitute
a continuing public health threat worldwide. Rapid
movement of people and goods around the world
increases the opportunity for local outbreaks to quickly
become worldwide pandemics. The 2009 H1N1 swine
influenza pandemic [1, 2] and continuing circulation
and evolution of H5N1 avian influenza isolated from
humans [3, 4] underscores a need for safe specimen
collection kits that : (1) are compatible with molecular
diagnostic and genomic analysis, (2) can effectively
inactivate potentially harmful microbes, (3) stabilize
and preserve nucleic acids including RNA, and (4) can
* Author for correspondence : L. T. Daum, Ph.D., Chief Scientific
Officer, Longhorn Vaccines & Diagnostics, 1747 Citadel Plaza,
Suite 206, San Antonio, Texas 78209, USA.
(Email : Longhorn-VandD@sbcglobal.net)
Epidemiol. Infect., Page 1 of 10. fCambridge University Press 2010
doi:10.1017/S0950268810002384
be easily used in the field and transported at ambient
temperatures while preserving microbial RNA.
Until recently, the majority of clinical diagnostic
laboratories employed traditional culture for patho-
gen identification that typically requires 3–7 days for
most viruses [5] and longer for some bacterial strains
[6]. Traditional culture requires specimen collection of
viable microbes, frozen transport, propagation and
handling of potentially infectious and often unknown
biological microbes. Furthermore, many infectious
agents, e.g. highly pathogenic avian influenza, SARS,
etc., are BSL-3 level pathogens that require specia-
lized facilities and precautions for analysis. There are
challenges in obtaining, shipping and maintaining
high-quality, viable biological specimens for culture.
Specimens must be shipped using a cold chain, most
often dry ice. Transporting potentially infectious
samples from remote sites or across international
borders using commercial transit can be costly and
tedious, particularly when specimens must be received
frozen.
The field of clinical molecular diagnostics changed
drastically with the advent of polymerase chain re-
action (PCR) [7], and subsequently, real-time PCR [8].
Real-time reverse transcription–PCR (rRT–PCR) can
deliver superior sensitivity and specificity results in
hours [9]. Thus, the majority of current diagnostic
laboratories have transitioned from traditional culture
to nucleic acid testing (NAT) such as real-time PCR.
Collection is the first step in diagnostic platforms or
molecular protocols requiring the detection of poten-
tially minute amounts of nucleic acids from microbes.
Regardless of the nucleic acid test used or the
RNA/DNA extraction protocol, specimen collection,
specifically the inactivation of potentially infectious
agents and the preservation and stability of pathogen
RNA/DNA remains a critical gap in clinical diag-
nostics, especially for use around the world.
This work describes the use of PrimeStore Mol-
ecular Transport Medium (MTM ; Longhorn Vaccines
& Diagnostics, USA), a clinical or environmental
sample collection system specifically formulated for
downstream molecular diagnostic testing. PrimeStore
MTM is an optimized blend of proprietary reagents
for RNA preservation that also includes the first non-
specific exogenous internal positive control (IPC) for
tracking the integrity of a specimen from the point
of collection to detection. PrimeStore MTM is shown
to efficiently: (1) lyse/inactivate potentially infectious
biological pathogens reducing infection risk so that
samples and can be handled, shipped, or transported
with minimal fear of pathogen release or contami-
nation, (2) stabilize and protect lysed ‘ naked’ RNA
polymers from hydrolysis, oxidative damage or
nuclease degradation, (3) preserve RNA for prolonged
periods at ambient temperature until they can be
processed using NAT, and (4) be compatible with
commercially available bench-top extraction kits.
MATERIALS AND METHODS
Microbe killing
Membrane filtration technique for bacterial and
fungal recovery
The membrane filtration method for bacterial and
fungal recovery was used to assess the killing ability
of PrimeStore MTM. Escherichia coli,Pseudomonas
aeruginosa,Staphylococcus aureus [non-methicillin-
resistant Staphylococcus aureus (MRSA)], Candida
albicans,Bacillus subtilis,andAspergillus brasiliensis
were used to determine whether PrimeStore MTM
could effectively kill and inactivate a panel of bacteria
and mould (yeast and filamentous fungi). Positive
controls incubated in a water matrix were performed
on day 0 only. A population of 1r10
6
c.f.u. for each
bacterial strain was inoculated into 0.5 ml PrimeStore
for each time-point and subsequently incubated
at 20–25 xC. The containers were enumerated and
evaluated at days 0, 1, 7, 14 and 28. The inoculum was
aseptically passed through a sterile filtration device
and subsequently rinsed three times with 100 ml
sterile neutralizing fluid D [1 g peptic digest of animal
tissue (peptone) and 1 ml polysorbate 80 dissolved
in 1.0 l of sterile water (final pH 7.1¡0.2)]. Where
necessary, dilutions of the inoculated test article were
performed to deliver a target count of 25–250 c.f.u.
per filter. For each time-point, inoculated negative
controls were processed in a similar fashion. Filters
inoculated with samples containing bacteria were
plated onto tryptic soy agar (TSAP) with lecithin and
polysorbate 80 and incubated at 30–35 xC for 72 h.
Filters inoculated with samples containing yeast or
mould were plated onto Sabouraud dextrose agar
(SAB) and incubated at 20–25 xC for no less than 72 h
but no more than 5 days. Colonies were counted to
calculate log
10
recoveries and percent (%) kill for each
organism used during microbial challenge.
Viral killing
Equal volumes of A/Wuhan/359/95 (10
8
TCID
50
/ml)
or adenovirus (type 5) (10
8
TCID
50
/ml) and
2 L. T. Daum and others
PrimeStore MTM were incubated for 10-, 30-, and
60-s intervals at ambient temperature. Influenza virus
only and PrimeStore MTM (minus virus) were also
tested as controls. After incubation, each solution was
subjected to a tenfold serial dilution, and inoculated
onto a 96-well plate confluent with MDCK cells
(influenza) or A549 (adenovirus). Prior to influenza
virus or adenovirus inoculation, 96-well plates
were washed 2rwith EMEM or EMEM containing
1mg/ml trypsin, respectively. All samples were pro-
cessed in quadruplicate. Plates were incubated at
37 xC with 5% CO
2
for 4 days, stained with Crystal
Violet reagent (0.06%) in 1% glutaraldehyde for 1 h
and rinsed with sterile water. Cells not stained ex-
hibited cytopathic effect (CPE) of virus. The titre of
the virus was recorded as the TCID
50
, i.e. the inverse
of the dilution that resulted in the CPE in 50 % of
the wells. Viral titres were calculated as geometric
means+standard error.
For testing against influenza A/Vietnam/1203/
04(H5N1) virus (10
7
TCID
50
/ml), a collection swab
was loaded with 0.1 ml H5N1 virus and placed in
1.5 ml PrimeStore MTM. Positive controls consisted
of virus-loaded swabs in the absence of PrimeStore
MTM, and swabs in PrimeStore MTM without
influenza virus were used as negative controls.
After 60-min incubation at ambient temperature, the
samples were vortexed and inoculated into 10-ml cell
culture media. Samples were serially diluted (tenfold)
and inoculated onto 96-well plates confluent with
MDCK cells. Plates were incubated at 37 xCwith5%
CO
2
for 4 days and assessed for viral CPE.
MRSA killing
A stock plate containing about 10
8
c.f.u. MRSA
(ATCC 33592) was transferred to TSB, vortexed
briefly and incubated at ambient temperature for
10 min. A total of 0.1 ml bacterial suspension was
transferred to 0.9 ml PrimeStore and vortexed for
60 s. A total of 0.1 ml suspension was transferred to
0.3 ml TSB (1 :4 dilution) and 100 ml was transferred
to blood agar plates (5 % sheep RBCs in TSA) after 0,
5 and 15 min. Positive controls included equivalent
volumes of MRSA and TSB. Plates were allowed to
dry, incubated overnight at 37 xC and analysed for
c.f.u./ml.
Nuclease digestion experiment
Stock influenza A(H3N2) virus (10
3
TCID
50
/ml)
spiked into human nasal washing was preserved in
PrimeStore MTM or other indicated media (0.3ml
solution to 0.1 ml virus) and incubated at 37 xCwith
RNase A (25 U), RNase T1 (125 U) and Turbo
DNase (2 U) for 1, 24 and 48 h (Life Technologies
Inc., USA). Reactions were inactivated at 75 xC for
2 min prior to RNA extraction.
Test samples and RNA extraction
For the RNA stabilization and preservation exper-
iments described in Figures 2 and 3, a human throat
swab containing a respiratory oral/pharyngeal matrix
was placed into collection tubes containing 1.5ml
PrimeStore MTM and viral transport medium (VTM ;
BD Universal Viral Transport Medium ; USA). An
influenza culture containing 10
2
TCID
50
/ml (Fig. 2)
or 10
4
TCID
50
/ml (Fig. 3) of A/California/04/2009
influenza A(H1N1) was spiked into each PrimeStore
MTM or VTM tube and incubated at the indicated
temperature (4, 25 or 37 xC).
RNA was extracted using the Ambion RNaqueous-
Micro kit (Life Technologies) according to the manu-
facturer’s protocol. For PrimeStore compatibility
experiment with commercial extraction kits (Fig. 4),
the Ambion RNAqeuous-Micro (Ambion cat. no.
1931), QIAamp Viral RNA Mini kit (cat. no. 52904),
Invitrogen Charge Switch Total RNA Cell kit
(cat. no. CS14010), and Ambion MagMAX Viral RNA
Isolation kit (cat no. 1928) were utilized according to
the manufacturer’s recommendations with 100 mlof
10
3
or 10
1
TCID
50
/ml A/California/04/2009 influenza
A(H1N1) stock culture. All time-point extractions
were frozen at x25 xC until analysis.
Real-time and traditional RT–PCR
For rRT–PCR, a 1rmaster-mix (PrimeMix) con-
taining real-time primers and probes specific for the
universal detection of influenza A or an IPC RNA
present within PrimeStore was utilized for detection
according to the manufacturer’s recommendations
(Longhorn Vaccines & Diagnostics, USA). Amplifi-
cation was performed using an ABI 7500 instrument
(Life Technologies). RT–PCR thermocycling con-
sisted of an RT incubation step at 50 xC for 20 min
followed by hot-start activation at 95 xC for 5 min
and 40 amplification cycles at 95 xC for 15 s and 60 xC
for 32 s.
Traditional RT–PCR was performed using the
SuperScript III High Fidelity One-Step kit (cat.
no. 12574-035, Invitrogen, USA) according to the
manufacturer’s recommendations. Two primer pairs
A molecular transport medium for nucleic acid testing 3
(forward 1204: 5k-TGTAAAACGACGGCCAGTAA-
GATGAAYACRCARTTCACAG-3kand reverse 1778:
5k-CAGGAAACAGCTATGACCGTGTCAGTAGA-
AACAAGGGTGTTT-3kand forward 379: 5k-TGT-
AAAACGACGGCCAGTACRTGTTACCCAGGR-
GATTTC-3kand reverse 1204 : 5k-CAGGAAACA-
GCTATGACCTCTTTACCYACTRCTGTGAA-3k)
described by the World Health Organization [10]
for amplification and sequencing influenza A(H1N1)
2009 field strains were used to generate a fragments
with base-pair (bp) lengths of exactly 574 and 825 bp,
respectively, from the H1N1 influenza haemag-
glutinin gene. Amplification was performed using
an Applied Biosystems 2720 instrument (Life Tech-
nologies). RT–PCR thermocycling consisted of an
RT incubation step at 50 xC for 30 min followed
by hot-start activation at 95 xC for 2 min and 40
amplification cycles at 95 xC for 15 s, 52 xC for 15 s,
and 68 xC for 1 min. A final incubation of 68 xC
for 5 min for extension was performed. Influenza
haemagglutinin gene fragments were visualized by
UV illumination using 1.2% pre-made agarose gels
stained with ethidium bromide (Invitrogen).
RESULTS
PrimeStore MTM microbial and viral inactivation
Microbial inactivation
PrimeStore MTM was shown to rapidly inactivate
microbes including fungi, Gram-positive/-negative
bacteria and viruses. Certified USP 32-NF 27<51>
Antimicrobial Effectiveness Testing was performed
using the membrane filtration technique for the
quantitation of bacteria and fungi. At the first test
period (24 h), 100 % of bacteria and fungi were killed
compared to the positive controls (Table 1 a). For
these microbes, PrimeStore MTM met the inacti-
vation criteria as described in USP Category 1 pro-
ducts (injections, emulsions, optic products, sterile
nasal products, and ophthalmic products made with
aqueous bases or vehicles). Additionally Bacillus sub-
tilis spores were challenged using the method descri-
bed in USP 51 to further evaluate PrimeStore MTM
inactivation of microbial populations. B. subtilis
spores were reduced by 99% within 24 h of exposure
(Table 1a). In a time-kill study of MRSA inoculated
into PrimeStore MTM, viable bacteria were not de-
tected (100% killing) at the earliest study time (5 min
post-inoculation) or at any of the later evaluation
times.
Viral inactivation
No viable virus influenza A(H3N2) or adenovirus
type 5 was detected within seconds after being placed
into PrimeStore MTM using viral loads as high as
10
7
–10
8
TCID
50
. The recovery of highly pathogenic
influenza A(A/Vietnam/1203/04) H5N1 virus at 10
8
TCID
50
/ml after incubation in PrimeStore MTM
was equivalent to negative controls, indicating com-
plete viral inactivation of H5N1 viruses (Table 1 b).
Because PrimeStore MTM is designed to destroy
membranes of cells and microbes, both MDCK and
A549 cell lines were destroyed until the test samples
were diluted 1 :1000, LOD 10
3
. Therefore, >4 log re-
ductions (>99.99% killing) were documented in all
spiked samples placed in PrimeStore MTM (Table 1b).
Table 1. PrimeStore Molecular Transport Medium
inactivates : (a)bacteria, fungi and (b)viruses
(a) Bacteria and fungi
Organism*
Positive
control
(c.f.u./ml)
PrimeStore+
organism
(% killed)
E. coli 6.4r10
7
100
P. aeruginosa 3.6r10
7
100
S. aureus 6.0r10
7
100
C. albicans 5.7r10
7
100
A. brasiliensis 1.9r10
6
100
B. subtilis (spores) 3.7r10
6
99.9
MRSA 4.7r10
8
100
(b) Viruses
Organism#
Positive
control
(TCID
50
/ml)
PrimeStore+
organism
(% killed)$
Influenza A(H3N2) 7.5r10
8
>99.99
Adenovirus type 5 7.7r10
8
>99.99
Influenza A(H5N1) 1.0r10
7
>99.99
* All bacteria with the exception of MRSA were tested
using the Membrane Filtration Technique. MRSA killing
was performed using standard c.f.u. plate enumeration
method.
#Influenza A(H3N2) inactivation testing performed by
Virion Systems Inc. Highly pathogenic influenza A(H5N1)
inactivation testing performed at Battelle Biomedical
Research Center.
$Initial 4-log dilutions of PrimeStore MTM+viruses were
required prior to inoculation into tissue culture lines due to
PrimeStore MTM lysis of the MDCK tissue culture cells.
Therefore, only a minimum 4-log TCID
50
/ml reduction was
observable.
4 L. T. Daum and others
PrimeStore MTM inactivates RNA/DNA nucleases
Nuclease digestion with RNase A, RNase T1, and
DNase was used as a simple and effective method to
examine PrimeStore protection of whole viral and
single-stranded (ss)RNA. Influenza A virus inocu-
lated in PrimeStore MTM was protected from exo-
genous addition of nucleases for 2 days at 37 xC
compared to commercial VTM, ethanol and other
lysis buffers (Fig. 1). Assuming 100 % PCR assay
efficiency, rRT–PCR cycle threshold (C
T
) differences
of 3.3 equate to an approximate tenfold change in
initial template RNA, with a C
T
of 40 representing
no detection [11]. RNA from influenza A virus
(Fig. 1a) and naked IPC ssRNA stored in Prime-
Store MTM and incubated in a pool of nucleases
(Fig. 1b) exhibited a C
T
reduction of only 5.7and1
.0,
respectively, compared to almost complete digestion
(C
T
values at or near 40) for other media after 48 h.
This equates to preservation of about 10
4
viral copies
compared to other media (Fig. 1).
PrimeStore MTM enhances RNA stability and
preservation
An influenza A(H1N1) reference strain (A/California/
04/2009) was spiked into PrimeStore MTM contain-
ing a human clinical throat swab and incubated at
25 xC (77 F) for 30 days. Influenza RNA from whole
virus was highly preserved, with an average rRT–
PCR C
T
reduction of 2.73 from day 0 baseline average
at 25 xC (Fig. 2).
Degradation of sample nucleic acids in PrimeStore
MTM can be tracked through a non-specific, IPC
(a) Influenza A virus
0
5
10
15
20
25
30
35
40
12448
Time (h)
Cycle threshold (CT)
(b) Internal positive control (IPC) ssRNA
0
5
10
15
20
25
30
35
40
124 48
Time (h)
Cycle threshold (CT)
FluA Control (–nuc)
ZR Viral RNA buffer
VTM
Ethanol
AVL lysis
PrimeStore MTM
Water + IPC Control RNA
(+nuc)
PrimeStore MTM (+ nuc)
PrimeStore MTM (–nuc)
Fig. 1. PrimeStore MTM inactivates nucleases and preserves (a) influenza A viral RNA and (b) single-stranded internal
positive control (IPC) RNA. Real-time RT–PCR analysis was performed using : 10
3
TCID
50
/ml whole influenza A(H3N2)
virus preserved in PrimeStore MTM and other media* (a), and IPC RNA (b) after incubation in RNA/DNA nucleases
(Ambion RNase A, T1, and Turbo DNase) at 37 xC for 48 h. Nucleic acid extraction at 1, 24 and 48 h was performed and
PCR amplified using PrimeMix Universal Influenza A and PrimeMix IPC assays (Longhorn Vaccines & Diagnostics). Mean
and standard error bars for duplicate extractions are shown. A C
T
reading of 40 (C
T
=40) is no amplification. [* Viral
transport media (VTM) is BD Universal Viral Transport Medium for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas
(BD, USA); AVL Lysis Buffer is Buffer AVL viral lysis buffer (Qiagen, cat. no. 1014777; USA). ZR Viral RNA Buffer is from
Zymo Research (cat. no. R1034-1-50).]
A molecular transport medium for nucleic acid testing 5
ssRNA that is included in PrimeStore MTM at an
initial concentration of 0.02 pg/ml. IPC RNA was
preserved and stable at 25 xC according to rRT–PCR
amplification using a PrimeMix IPC rRT–PCR assay
specific for the IPC RNA fragment (Fig. 2).
To demonstrate the preservation and stability of
RNA polymers in PrimeStore MTM for downstream
DNA sequencing, a stock strain (10
4
TCID
50
/ml) of
A/California/04/2009 influenza A(H1N1) 2009 was
spiked into a PrimeStore MTM and VTM and sub-
sequently incubated at 38 xC. Triplicate extractions
at days 0, 2, 7, and 14 were performed for each time-
point and subjected to standard and rRT–PCR
amplification. Traditional RT–PCR was performed
using primers to generate 574 bp and 825 bp products
from the viral segment encoding the haemagglutinin
protein (segment 4, y1795 RNA bases) (Fig. 3 a). The
574 bp amplicon from influenza A H1N1 2009 virus
preserved in PrimeStore MTM was visualized by
agarose gel electrophoresis at all time-points compared
to no amplification bands observed in extractions
from influenza virus samples stored in standard VTM
40
35
30
25
20
15
Cycle threshold (CT)
123456815192330
Time (days)
Influenza A (H1N1) virus
IPC ssRNA
Fig. 2. PrimeStore stabilizes and preserves influenza
A(H1N1) 2009 virus. A human clinical swab spiked with
5.3r10
2
TCID
50
/ml of influenza A(H1N1) A/California/04/
2009 reference virus was placed into PrimeStore MTM and
incubated at 25 xC. Triplicate samples were extracted and
analysed by real-time RT–PCR at the indicated time-point
using an assay specific for influenza A virus and the internal
positive control (IPC) single-stranded RNA piece present at
fixed concentration (i.e. 0.02 pg) within PrimeStore MTM.
Influenza H1N1-09 virus and single-stranded IPC RNA
were preserved in PrimeStore MTM for 30 days with only
minimal degradation (average 2.73 C
T
reduction) noted by
day 30 for influenza virus at 25 xC. Triplicate averages and
standard error bars are shown.
(a) Traditional RT–PCR of 574 bp and 825 bp amplicons
PS VTM PS VTM PS VTM PS VTM Pos+ Neg– Ladder
PS VTM PS VTM PS VTM PS VTM Pos+ Neg– Ladder
Day 0 Day 2 Day 7 Day 14 Controls
Day 0 Day 2 Day 7 Day 14 Controls
574 bp
825 bp
(b) Real-time RT–PCR of 194 bp amplicon
40
35
30
25
20
15
10
5
002714
Cycle threshold (C
T
)
Days at 38 °C
PrimeStore MTM
VTM
Fig. 3. RT–PCR amplification of influenza A(H1N1) 2009
stock virus in PrimeStore MTM and commercial VTM at
38 xC using: (a) standard RT–PCR with primers to amplify
574-bp and 825-bp fragments and (b) real-time RT–PCR
with primers to amplify a 194-bp fragment. A stock strain
(10
4
TCID
50
/ml) of A/California/04/2009 was placed into
PrimeStore MTM and VTM and incubated at 38 xC for 0, 2,
7 and 14 days. Traditional RT–PCR amplification of 574 bp
and 835 bp amplicons (using gel electrophoresis and UV
visualization) were observed in PrimeStore preserved
samples at days 14 and 7, respectively, compared to no
amplification in VTM-preserved samples after day 0 (a).
PrimeStore-preserved virus samples exhibited considerably
less RNA degradation (average C
T
reduction=4.0) com-
pared to no detection in VTM-preserved samples at day 14
(b). Reactions are average of triplicate with standard error
shown. Commercial VTM is Copan UTM medium for
Viruses, Chylaymida, Mycoplasma & Ureaplasma (Copan
Diagnostics, Italy).
6 L. T. Daum and others
after day 0 (Fig. 3 a). The PCR band corresponding to
the 825 bp amplification was evident to day 7 in virus
stored in PrimeStore MTM
TM
,comparedtonoam-
plification in virus stored in VTM after day 0 (Fig. 3a).
Similar results were observed using rRT–PCR
for amplification of a 194-bp fragment specific for a
conserved region of influenza A Matrix gene. Influ-
enza virus preserved in PrimeStore showed consider-
ably less degradation according to rRT–PCR (average
C
T
reduction over 14 days=4.0) compared to no de-
tection observed in virus stored in VTM (Fig. 3 b).
PrimeStore is compatible with commercial silica- and
bead-based extraction kits
Commercial extraction kits are available in a variety
of formats with most exploiting the basic principle of
nucleic acid affinity for glass silicon dioxide particles
present on filters or beads. To demonstrate Prime-
Store MTM compatibility with commercial kits, refer-
ence influenza A(H1N1) virus (A/California/04/2009)
was inoculated into PrimeStore containing a human
throat swab specimen at two concentrations, 10
3
TCID
50
/ml and 10
1
TCID
50
/ml, and extracted using
two commercial spin-column and two bead-based kits
(Fig. 4). Viral influenza RNA in PrimeStore MTM
was detected by rRT–PCR after extraction in all four
commercial kits at high (10
3
TCID
50
/ml) and low (10
1
TCID
50
/ml) viral loads. Additionally, the IPC RNA
(0.02 pg/ml) present in PrimeStore MTM was also
detected by rRT–PCR after extraction using all four
kits at levels consistent with the manufacturer’s rec-
ommendations [12].
DISCUSSION
Several companies such as Becton Dickinson, and
Copan offer viral collection media for preserving
viable organism for culture analysis. Unknown speci-
mens collected in these media remain viable and are
typically transported frozen to the laboratory and
treated as biohazardous and potentially infectious.
Therefore, there is considerable expense and risk of
infection associated with the collection and transport
of clinical specimens to reference laboratories.
Krafft et al. [13] evaluated the use of respiratory
samples fixed in ethanol as an alternative to com-
mercial transport media using rRT–PCR. While
ethanol may provide a suitable medium for influenza
and other common upper respiratory pathogens,
its use presents several other concerns including
alcohol-shipping restrictions in many countries, high
flammability, and the potential for incomplete lyses of
certain microbes.
In a study by Blow et al. [14], viral RNA from field-
collected arboviruses was stabilized at 32 xC for 48 h
using the AVL lysis buffer from a commercially
available Qiagen extraction kit. While the samples are
lysed and presumably non-infectious, the short, 2-day
window of RNA preservation in Qiagen AVL makes
this method unrealistic, even for routine pathogen
surveillance.
The use of RNALater
TM
(Ambion, USA) has been
reported in the literature as a solution for the preser-
vation of collected specimens for molecular analysis
[15, 16]. RNALater is a commercially available pro-
duct designed for the preservation and maintenance
of whole tissues for histology and microscopy of cells
and microbes [17], but its use has also included the
collection of field specimens for molecular analysis
[18–20]. RNALater contains a high concentration
of ammonium sulfate for maintaining phospholipid
integrity and intact cells. Several reports indicate
that tissues and viruses from samples preserved in
RNALater remain viable for extended periods of
time and at a variety of temperatures [21, 22].
40
35
30
25
20
15
10
5
0103 TCID50/ml
Cycle threshold (CT)
101 TCID50/ml ssRNA IPC
Ambion RNAqueous-Micro
Qiagen Viral Mini
Invitrogen ChargeSwitch
Ambion MagMax
Fig. 4. PrimeStore molecular transport solution is compat-
ible with commercial bench-top nucleic acid extraction kits
including silica- and bead-based varieties. Two concen-
trations (10
3
TCID
50
/ml and 10
1
TCID
50
/ml), representing
clinically relevant viral loads, i.e. a high and low, of stock
H1N1 virus (A/California/04/2009) were inoculated into
PrimeStore containing a clinical throat swab. Extractions
were performed using two common silica- and bead-based
nucleic acid kits (according to manufacturer’s recommend-
ations) and subjected to real-time RT–PCR analysis using
an ABI 7500. Mean and standard error for triplicate ex-
tractions are shown.
A molecular transport medium for nucleic acid testing 7
Maintaining viability of viruses and other microbes
can be dangerous for many groups interested in safe,
non-infectious transport of collected specimens for
molecular-based diagnostics.
PrimeStore MTM was developed specifically to
overcome these limitations in clinically collected
specimens earmarked for routine downstream mol-
ecular diagnostics. Since PrimeStore MTM inacti-
vates and kills a wide range of microbes (Table 1) it is
well-suited for field collection in remote areas, triage
centres, border crossings and during pandemics where
cold-chain, transport and dissemination of potentially
infectious pathogens are a concern. PrimeStore MTM
lyses and inactivates microbial pathogens so they are
non-viable; thus an alternative transport media is re-
quired for traditional culture. Preliminary data from a
study in progress has demonstrated that PrimeStore
MTM rapidly kills M. tuberculosis from clinical
sputum samples (Dr Nazir Ahmed Ismail, Medical
Microbiologist, University of Pretoria and NHLS,
Pretoria, South Africa, personal communication).
PrimeStore MTM is an ambient thermostable, pro-
prietary molecular transport medium that rapidly
inactivates nucleases (Fig. 1) and stabilizes and pre-
serves released nucleic acids including labile RNA
according to rRT–PCR analysis (Fig. 2). Further-
more, PrimeStore MTM stabilizes and preserves
RNA polymers from hydrolysis and oxidation, and
promotes thermostability at temperatures as high as
37 xC for at least 14 days (Fig. 3).
A unique IPC ssRNA that is pre-mixed (3r10
5
target copies/ml) into PrimeStore MTM (and is thus
stabilized) can be amplified by rRT–PCR to verify
sample stability from the time of sample collection
through extraction and detection (Fig. 2). The IPC
RNA serves as a carrier species for low-level samples,
controls for nucleic acid extraction and monitors
sample integrity from the point of collection to de-
tection. The RNA IPC in PrimeStore MTM is a syn-
thetically produced (in vitro) 114-bp ssRNA polymer
that is non-homologous by BLAST analysis to com-
mon upper respiratory pathogens and normal flora. A
developed rRT–PCR assay (PrimeMix IPC, Longhorn
Vaccines & Diagnostics) that targets this synthetic
IPC RNA polymer is used as a uniplex rRT–PCR
assay. A clinical collection medium containing exo-
genous IPC RNA fragments is important for ensuring
integrity of transported or stored specimens for a wide
variety of molecular-based detection systems.
PrimeStore MTM will also facilitate standard
sequencing and meta-genomic analysis of original
clinical samples by improving the quality of microbial
nucleic acids in original specimens when they finally
arrive in the laboratory. Recovery of RT–PCR ampli-
fication fragments over 1400 bases (data not shown)
has been observed from viral RNA preserved and
shipped in PrimeStore MTM at ambient temperature
for several weeks. In harsh conditions, i.e. 38 xC
incubation, RT–PCR amplification of 574-bp and
825-bp fragments were observed from PrimeStore
preserved virus where no amplification was observed
from stock virus in commercial VTM (Fig. 3 a).
The primers used to generate the 825-bp fragment
represent the largest haemagglutinin gene fragment
described by the WHO for influenza A(H1N1) se-
quencing [10]. Ambient temperature collection and
preservation of large RNA fragments from clinical
samples in PrimeStore MTM may be highly useful to
groups who wish to perform direct DNA sequencing,
genotyping, meta-genomic analysis and other NAT
directly from original specimens.
In a 2007–2008 prospective paediatric clinical
study, nasal washings preserved in PrimeStore
MTM were subsequently analysed for influenza
by rRT–PCR [23] and DNA sequencing of viral
surface genes (Daum et al., unpublished ob-
servations). During the 2009 swine pandemic cli-
nical throat swabs preserved in PrimeStore MTM
were transported at ambient temperature and sub-
jected to entire genome sequencing of swine influenza
A(H1N1) virus using a Roche 454 system (Haung
et al., unpublished data). In this work Haung
et al. characterized four complete influenza genomes
from the Dominican Republic, Columbia, Nicaragua,
and Russia from swabs in PrimeStore MTM
[GenBank accession numbers CY0Y3099-CY0Y3106,
CY049833-CY049839, CY044152-CY044159, CY049896-
CY049903; Haung et al., unpublished data].
PrimeStore MTM is compatible with many com-
mercial extraction kits (Fig. 4). Nucleic acids are ex-
tracted directly from PrimeStore MTM according to
standard manufacturer’s protocol with only minor
differences noted in C
T
values between column- and
bead-based kits. Previous studies have shown that
spin-column kits are slightly more robust compared
to bead-based kits with certain sample types [24, 25],
but bead-based kits may be better when certain
sample matrices, i.e. faecal and environmental are
analysed. Additional studies are currently underway
to evaluate the utility of PrimeStore MTM for use
with other samples matrices such as faeces, blood,
environmental samples and veterinary specimens.
8 L. T. Daum and others
A 2010 influenza study that compared swabs in liquid
Stuart’s transport media (RTLS) and PrimeStore
MTM from pigs experimentally infected with H1N1
influenza A virus found that virus from swabs in
PrimeStore MTM and not Stuart’s medium was de-
tected at 5 days post-infection, a time when shedding
is typically decreased or non-detectable [26].
While PrimeStore MTM effectively lyses microbal
pathogens, it is more than simply a lysis buffer as
evident by comparison to typical lysis buffers from
commercial extraction kits (Fig. 1). PrimeStore MTM
is a blend of eight reagents that have been optimized
to enhance RNA/DNA preservation after cellular
lysis. PrimeStore MTM does not exhibit inhibition
during nucleic acid extraction and PCR testing
and has been shown to improve optimal recovery of
influenza RNA (Fig. 3).
Notable reductions (4–5 C
T
s) in initial rRT–PCR
C
T
values are routinely observed from virus in-
oculated in commercial VTM compared to equal
amounts of virus in PrimeStore MTM (Fig. 3 b). This
suggests that extraction and/or PCR inhibitors are
present in commercial VTM that effect RNA detec-
tion during rRT–PCR. This may be problematical
for groups who routinely extract RNA from original
clinical samples collected in commercial VTMs for
rRT–PCR or gene sequencing analysis. Accordingly,
there is a need for an effective clinical VTM that
serves the dual role of ensuring the viability of col-
lected microorganisms for subsequent culture and
propagation procedures and does not substantially
interfere with downstream NAT processes.
In February 2010, PrimeStore MTM received
FDA-Emergency Use Authorization as part of the
complete Longhorn Influenza A/H1N1-09 Prime
RRT–PCR Assay
TM
[12]. PrimeStore is the first
molecular transport medium to receive EUA FDA
approval, and the first to contain an IPC to con-
trol for specimen degradation from collection to
detection.
ACKNOWLEDGEMENTS
We acknowledge the countless hours of effort
from scientists, physicians and students alike who
contributed to the development and clinical utility
of PrimeStore MTM in molecular diagnostics.
Specifically, Deena E. Sutter, M.D., Marty Ottolini,
M.D., Aryeneesh K. Dotiwala, John Rodriquez,
Demetra Kelenis, Jeff Fischer and Lou Schriefer.
DECLARATION OF INTEREST
Drs Luke T. Daum and Gerald W. Fischer and
Ms. Susan A. Worthy are employees of Longhorn
Vaccines & Diagnostics.
REFERENCES
1. Garten RJ, et al. Antigenic and genetic characteristics
of swine-origin 2009 A (H1N1) influenza viruses circu-
lating in humans. Science 2009 ; 325: 197–201.
2. Novel Swine-Origin Influenza A (H1N1) Virus
Investigation Team, et al.Emergence of a novel swine-
origin influenza A (H1N1) virus in humans. New
England Journal of Medicine 2009 ; 360: 2605–2615.
3. Buchy P, et al. Molecular epidemiology of clade
1 influenza A viruses (H5N1), southern Indochina
peninsula, 2004–2007. Emerging Infectious Diseases
2009; 15: 1641–1644.
4. Webster RG, Govorkova EA. H5N1 influenza – con-
tinuing evolution and spread. New England Journal of
Medicine 2006; 355: 2174–2177.
5. Daum LT, et al. Genetic and antigenic analysis of the
first A/New Caledonia/20/99-like H1N1 influenza iso-
lates reported in the Americas. Emerging Infectious
Diseases 2002; 8: 408–412.
6. Zoetendal EG, Vaughan EE, de Vos WM. A microbial
world within us. Molecular Microbiology 2006 ; 59:
1639–1650.
7. Saiki RK, et al. Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for
diagnosis of sickle cell anemia. Science 1985 ; 230:
1350–1354.
8. Higuchi R, et al. Kinetic PCR analysis : real-time
monitoring of DNA amplification reactions. Bio-
technology 1993; 11: 1026–1030.
9. Daum LT, et al. Real-time RT-PCR assays for type
and subtype detection of influenza A and B viruses.
Influenza and Other Respiratory Viruses 2007 ; 1:
167–175.
10. World Health Organization. Sequencing primers and
protocol. (http://www.who.int/csr/resources/publications/
swineflu/sequencing_primers/en/index.html). Accessed
12 May 2009.
11. Applied Biosystems Incorporated. Real-Time PCR :
Understanding C
T.
Application Note. May, 2008.
Publication Number 136AP01-01.
12. U.S. Food and Drug Administration (FDA) website.
Influenza A/H1N1-09 prime RRT-PCR assay. Instruc-
tions for use for detection of 2009 H1N1 influenza virus.
Version 1, 2010. Longhorn Vaccines & Diagnostics
(http://www.fda.gov/MedicalDevices/Safety/Emergency
Situations/ucm161496.htm).
13. Krafft AE, et al. Evaluation of PCR testing of ethanol-
fixed nasal swab specimens as an augmented surveil-
lance strategy for influenza virus and adenovirus
identification. Journal of Clinical Microbiology 2005 ;
43: 1768–1775.
A molecular transport medium for nucleic acid testing 9
14. Blow JA, et al. Viral nucleic acid stabilization by RNA
extraction reagent. Journal of Virological Methods
2008; 150: 41–44.
15. Mutter GL, et al. Comparison of frozen and RNAlater
solid tissue storage methods for use in RNA expression
microarrays. BMC Genomics 2004; 10: 88.
16. Florell SR, et al. Preservation of RNA for functional
genomic studies : a multidisciplinary tumor bank pro-
tocol. Modern Pathology 2001; 14: 116–128.
17. Ambion. RNAlater
1
tissue collection: RNA Stabiliza-
tion Solution. Handbook, Part Numbers AM7020
(100 ml), AM7024 (250 ml), AM7021 (500 ml), AM7022
(50r1.5ml), AM7023(20r5 ml). (http://www.ambion.
com/techlib/prot/bp_7020.pdf).
18. Nsubuga AM, et al. Factors affecting the amount of
genomic DNA extracted from ape faeces and the
identification of an improved sample storage method.
Molecular Ecology 2004; 13: 2089–2094.
19. Webster BL. Isolation and preservation of schistosome
eggs and larvae in RNAlater(R) facilitates genetic
profiling of individuals. Parasites & Vectors 2009 ;
23: 50.
20. McClure C, et al. Evaluation of a reverse transcriptase
polymerase chain reaction test and virus isolation on
field samples collected for the diagnosis of infectious
hematopoietic necrosis virus in cultured Atlantic
salmon in British Columbia. Journal of Aquatic Animal
Health 2008; 20: 12–18.
21. Kurth A. Possible biohazard risk from infectious tissue
and culture cells preserved with RNAlater. Clinical
Chemistry 2007; 53: 1389–1390.
22. Uhlenhaut C, Kracht M. Viral infectivity is maintained
by an RNA protection buffer. Journal of Virological
Methods 2005; 128: 189–191.
23. Daum LT, et al. Comparison of influenza virus
detection methods from pediatric patients and
household contacts. Journal of Medical Virology
(in press).
24. Cler L, et al. A comparison of five methods
for extracting DNA from paucicellular clinical
samples. Molecular and Cellular Probes 2006 ; 20:
191–196.
25. Dauphin LA, et al. Comparison of five commercial
DNA extraction kits for the recovery of Yersinia pestis
DNA from bacterial suspensions and spiked environ-
mental samples. Journal of Applied Microbiology 2010 ;
108: 163–172.
26. Gramer M, et al. Effect of swab type, collection media,
and storage on the detection of influenza A virus
in porcine nasal secretions. American Association of
Veterinary Laboratory Diagnosticians (AAVLD). 11–17
November 2010 (Abstract).
10 L. T. Daum and others
... Following these observations, we investigated if stabilization of SS samples using a commercially available kit would preserve sputum sample quality for eventual gene expression analysis. To this purpose we used PrimeStore ® MTM a proprietary blend of reagents that has been shown to efficiently lyse biological pathogens, stabilize and protect lysed RNA polymers from hydrolysis, oxidative damage or nuclease degradation and preserve RNA at ambient temperature for prolonged periods (Daum et al., 2011). We also evaluated if isolation of sputum plugs from saliva and other residues would significantly improve the transcriptomic outcomes. ...
Article
Full-text available
Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV 200 ) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV 200 higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.
... The viral RNA in the control sample was not subjected to any stress before its extraction and was used to determine the recovery of the virus after elution. PrimeStore MTM media (Longhorn Vaccines and Diagnostics, San Antonio, TX) was the elution liquid of choice since it is known to inactivate viruses and act as an RNA stabilization agent (Daum et al. 2011(Daum et al. , 2014Zar et al. 2016). Once the suspension dried out, the viral particles were eluted using the following two methods. ...
Article
Full-text available
Environmental air sampling of the SARS-CoV-2 virus in occupational and community settings is pertinent to reduce and monitor the spread of the COVID pandemic. However, there is a general lack of standardized procedures for airborne virus sampling and limited knowledge of how sampling and storage stress impact the recovery of captured airborne viruses. Since filtration is one of the commonly used methods to capture airborne viruses, this study analyzed the effect of sampling and storage stress on SARS-CoV-2 surrogate virus (human coronavirus OC43, or HCoV-OC43) captured by filters. HCoV-OC43, a simulant of the SARS-CoV-2, was aerosolized and captured by PTFE-laminated filters. The impact of sampling stress was evaluated by comparing the RNA yields recovered when sampled at 3 L/min and 10 L/min and for 10 mins and 60 mins; in one set of experiments, additional stress was added by passing clean air through filters with the virus for 1, 5, and 15 hours. The impact of storage stress was designed to examine RNA recovery from filters at room temperature (25 °C) and refrigerated conditions (4 °C) for up to one week of storage. To our knowledge, this is the first report on using HCoV-OC43 aerosol in air sampling experiments, and the mode diameter of the virus aerosolized from the growth medium was 40-60 nm as determined by SMPS + CPC system (TSI Inc.) and MiniWRAS (Grimm Inc.) measurements. No significant difference was found in virus recovery between the two sampling flow rates and different sampling times (p > 0.05). However, storage at room temperature (25 °C) yielded ∼2x less RNA than immediate processing and storage at refrigerated conditions (4 °C). Therefore, it is recommended to store filter samples with viruses at 4 °C up to one week if the immediate analysis is not feasible. Although the laminated PTFE filter used in this work purposefully does not include a non-PTFE backing, the general recommendations for handling and storing filter samples with viral particles are likely to apply to other filter types.
... The viral RNA remains detectable and does not degrade for up to 7 days or longer in VTM (Rogers et al., 2020). In fact, stability studies of the influenza virus A (H1N1) in a similar storage medium (PrimeStore MTM) indicate that viral RNA can be preserved and stabilized for up to 30 days under these conditions (Daum et al., 2011). Since the Coronavirus is an enveloped virus, its recovery rate from water samples is substantially lower than that of nonenveloped viruses (Rusiñol et al., 2020). ...
Article
Full-text available
Airborne spread of COVID‐19 by infectious aerosol is all but certain. However, easily implemented approaches to assess the actual environmental threat are currently unavailable. We present a simple approach with the potential to rapidly provide information about the prevalence of SARS‐CoV‐2 in the atmosphere at any location. We used a portable dehumidifier as a readily available and affordable tool to collect airborne virus in the condensate. The dehumidifiers were deployed in selected locations of a hospital ward with patients reporting flu‐like symptoms which could possibly be due to COVID‐19 over three separate periods of one week. Samples were analyzed frequently for both virus envelope protein and SARS‐CoV‐2 RNA. In several samples across separate deployments, condensate from dehumidifiers tested positive for the presence of SARS‐CoV‐2 antigens as confirmed using two independent assays. RNA was detected, but not attributable to SARS‐CoV‐2. We verified the ability of the dehumidifier to rapidly collect aerosolized sodium chloride. Our results point to a facile pool testing method to sample air in any location in the world and assess the presence and concentration of an infectious agent in order to obtain quantitative risk assessment of exposure, designate zones as ‘hot spots’ and minimize the need for individual testing which may often be time consuming, expensive and laborious. This article is protected by copyright. All rights reserved.
... After throat swab samples were collected aseptically, transportation was carried out using Amies transport medium [22] to Bahir Dar university Microbiology Resaerch Laboratory Center by maintaining cold chain, or cold box with dry ice [23]. The culture media was prepared aseptically by autoclaving and the sterlity was checked by incubating 5% of the batch prepared media overnight. ...
Article
Full-text available
Background: Streptococcus pyogenes (S. pyogenes) is a Gram positive bacterium which is a leading cause of pharyngitis, skin and soft tissue infection and post streptococcal syndromes. Due to lack of β-lactamase enzyme production, it was considered universally susceptible to penicillin group and later generation of β-lactam antibiotics. As such, empirical treatment was common which might leads to development of antibiotics resistance. Therefore, the aims of this study were to determine the prevalence, antibiotics susceptibility profile; and associated factors of S. pyogenes among pediatric patients with acute pharyngitis in Felege Hiwot Comprehensive Specialized Hospital (FHCSH), Northwest Ethiopia. Methods: Hospital based cross-sectional study was carried out on 154 pediatric patients, whose age ranged from 0 to 18 years old using consecutive convenient sampling technique from 1st February to 19th June 2020 at FHCSH. S. pyogenes were identified by throat swab culture on 5% sheep blood agar with an overnight incubation at 37 °C in candle jar containing 5% CO2. Gram stain, catalase test and bacitracin test were used to identify S. pyogenes. Then,the data were entered into EpiData version 3.1 and analyzed by SPSS version 20 software. Finally, stepwise, bivariable and multivariable logistic regressions were carried out for identifyying factors having significant ssociation (p<0.05) with acute pharyngitis. RESULTS: From the total throat swabs, 14 (9.1%) with (95% CI; 4.5-14.3) were culture positive for S. pyogenes. From these, all isolates were sensitive to penicillin and ampicillin. On the otherhand, 4 (35.7%), 4 (35.5%), 3 (21.4%), 2 (14.3%), 1 (7.1%), 7 (50.0%) and 1 (7.1%) isolates were resistant for ceftriaxone, vancomycin, erythromycin, tetracycline, chloramphenicol, clindamycin and levofloxacin, respectively. The presence of any smoker in home showed significant association with S. pyogenes acute pharyngitis. Furthermore, having tender lymphadenopathy and recurrence were clinical predictors for S. pyogenes acute pharyngitis (P < 0.05). Conclusion: The prevalence of S. pyogenes was guaged at 9.1% which is considered as low prevalence. All S. pyogenes isolats remain sensitive to penicillin. However, resistance was reported to clindamycin 7 (50.0%), ceftriaxone 5 (35.7%) and erythromycin 3 (21.4%). The current practice of giving erythromycin, clindamycin instead of penicillin and ampicillin is againest the microbiology result. Therefore, current empirical treatment of acute pharyngitis shall take in to account the current evidences. Continuous surveillance of antibiotics resistance pattern of S. pyogenes for acute pharyngitis must be strengthen to improve the use of antibiotics in hospitals.
... Movement restrictions placed on buffalo samples from locations with reportable diseases to laboratories may result in inadequate bTB surveillance in these areas 9 . PrimeStore Molecular Transport Medium (MTM), used in combination with PrimeStore swabs, has been shown to effectively inactivate infectious organisms, while stabilising the nucleic acids, through lysis of the cell membranes, destruction of proteins and enzymes, and inactivation of nucleases 10 . PrimeStore MTM therefore provides a safer option for collection and transportation of samples that potentially pose a risk to human or animal health. ...
Article
Full-text available
Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis- unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis- positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis- unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.
... The PrimeSwab and the accompanying PrimeStore MTM lived to our expectations and performed best in the comparison. This system has been evaluated using both bacterial and viral pathogens, including SARS-CoV-2 and is intriguing because of the safe inactivation of pathogens and preservation of nucleic acids [41][42][43][44]. Whether it is worth using this system or its sequels (PrimeStore HCP) instead of simple swab systems, remains the choice of users based on risk assessment, integration into strategies, and financial resources. ...
Article
Full-text available
African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a "fit-for-purpose" approach.
... PrimeStore® Molecular Transport Medium (MTM), used in combination with PrimeStore® swabs, has been shown to effectively inactivate infectious organisms, while stabilising the nucleic acids, through lysis of the cell membranes, destruction of proteins and enzymes, and inactivation of nucleases 10 . PrimeStore® MTM therefore provides a safer option for collection and transportation of samples that potentially pose a risk to human or animal health. ...
Preprint
Full-text available
Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore® Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore® MTM at ambient temperature until Xpert® MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis- unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis- positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis- unexposed animals, showing the potentially high specificity of Ultra with PrimeStore® swabs. PrimeStore® MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.
Article
The efforts of the Global Poliovirus Eradication Initiative (GPEI) have brought about the near elimination of poliovirus worldwide. The World Health Organization has issued guidelines for the safe handling and containment of infectious materials (IM) and potentially infectious materials (PIM) following poliovirus eradication. Inactivation of poliovirus in IM and PIM is needed to prevent inadvertent re-introduction of polioviruses post-eradication. In this study, we investigated the use of guanidine thiocyanate-based nucleic acid extraction buffers from commercially available nucleic acid extraction kits to inactivate poliovirus in cell culture isolates and stool suspensions, two common types of poliovirus IM and PIM, respectively. Incubation with selected nucleic acid extraction buffers or extraction buffers supplemented with ethanol reduced the infectivity of high-titer wild poliovirus type 1 (WPV1), wild poliovirus type 3 (WPV3), Sabin 1 (SL1), and Sabin 3 (SL3) cell culture isolates below the limit of detection in CCID50 assays. Stool suspensions containing WPV1, WPV3, SL1, SL2, or SL3 were also inactivated by the extraction buffers tested. Blind passage of WPV1-spiked stool suspensions confirmed complete inactivation of WPV1 after incubation with extraction buffers. Moreover, treatment with a buffer consisting of 4 M guanidine thiocyanate with 30% ethanol inactivated a high-titer WPV1 culture isolate and a WPV1-spiked stool suspension. Taken together, these results show that guanidine thiocyanate-based nucleic acid extraction buffers are an effective means of inactivating poliovirus IM and PIM, and thus will be instrumental in ensuring containment compliance and preventing potential re-emergence of contained polioviruses.
Article
BACKGROUND The ability to rapidly detect SARS-CoV-2 and influenza virus infection is vital for patient care due to overlap in clinical symptoms. Roche’s cobas® Liat® SARS-CoV-2 & Influenza A/B Nucleic Acid Test used on the cobas® Liat® was granted approval under FDA’s Emergency Use Authorization (EUA) for nasopharyngeal (NP) and nasal swabs collected in viral/universal transport medium (VTM/UTM). However, there is a critical need for media that inactivates the virus, especially when specimens are collected in decentralized settings. This study aimed to investigate the use of PrimeStore Molecular Transport Medium® (PS-MTM®), designed to inactivate/kill and stabilize RNA/DNA for ambient transport and pre-processing of collected samples. METHODS A limit of detection (LOD) using serially diluted SARS-CoV-2 RNA in PS-MTM® and routine UTM was established using standard qPCR. Additionally, a clinical panel of NP and oral swabs collected in PS-MTM® collected during the 2020 coronavirus disease 2019 (COVID-19) pandemic were evaluated on the cobas® Liat® and compared to ‘gold standard’ qPCR on an ABI-7500 instrument. RESULTS SARS-CoV-2 RNA LOD using standard qPCR was equivalent on the cobas® Liat® instrument. cobas® Liat® detection from oral/NP swabs in PS-MTM® media exhibited equivalent positive percent agreement (100%) and negative percent agreement (96.4%). CONCLUSION PS-MTM® and the Roche cobas® Liat® are compatible and complimentary devices for respiratory specimen collection and rapid disease detection, respectively. PS-MTM® is equivalent to standard VTM/UTM with the added benefit of safe, non-infectious sample processing for near-patient testing.
Article
Full-text available
Introduction: Severe acute respiratory syndrome coronavirus-2 (SARS CoV 2) virus, a causative agent of COVID-19 has led to universal pandemic. During this pandemic there has been an acute shortage of good quality Viral Transport Medium (VTM) because of increase in number of infected people worldwide. It is also difficult to maintain the transport and storing conditions in line with the guidelines in pandemics. Aim: To assess the feasibility of Oropharyngeal Swab (OP)/ Nasal swabs in 0.9% normal saline in place of VTM and to analyse the effect of temperature on nucleic acid detection by rRT PCR on saline samples stored at 4ºC, ambient and at higher temperature (37ºC). Materials and Methods: The present study was an observational analytical study which included 94 positive and 5 negative samples. Patients' nasal or OP samples were collected as dry swabs and in VTM. Normal saline was added once the samples were received in the laboratory. PCR was done with saline and VTM samples both on day 1. Samples were aliquotted in 3 sets and one set was kept at 4º-8º C and other two at 25ºC and 37ºC, respectively. All positive samples were further tested on day 3, day 4 and day 6. Results were analysed and compared. Results: Samples in normal saline showed very good sensitivity at all temperatures (4º-8ºC, 25ºC and 37ºC) till day 6. Both the swab samples (in saline and in VTM) showed nearly 100% agreement in rRT-PCR results. Ct value variation was also ≤±2. Conclusion: Looking into the cost and logistics issues especially during pandemics, saline is a good and cheaper alternative to VTM and with its use, testing capacity can be expanded.
Article
Full-text available
Background: On April 15 and April 17, 2009, novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in specimens obtained from two epidemiologically unlinked patients in the United States. The same strain of the virus was identified in Mexico, Canada, and elsewhere. We describe 642 confirmed cases of human S-OIV infection identified from the rapidly evolving U.S. outbreak. Methods: Enhanced surveillance was implemented in the United States for human infection with influenza A viruses that could not be subtyped. Specimens were sent to the Centers for Disease Control and Prevention for real-time reverse-transcriptase-polymerase-chain-reaction confirmatory testing for S-OIV. Results: From April 15 through May 5, a total of 642 confirmed cases of S-OIV infection were identified in 41 states. The ages of patients ranged from 3 months to 81 years; 60% of patients were 18 years of age or younger. Of patients with available data, 18% had recently traveled to Mexico, and 16% were identified from school outbreaks of S-OIV infection. The most common presenting symptoms were fever (94% of patients), cough (92%), and sore throat (66%); 25% of patients had diarrhea, and 25% had vomiting. Of the 399 patients for whom hospitalization status was known, 36 (9%) required hospitalization. Of 22 hospitalized patients with available data, 12 had characteristics that conferred an increased risk of severe seasonal influenza, 11 had pneumonia, 8 required admission to an intensive care unit, 4 had respiratory failure, and 2 died. The S-OIV was determined to have a unique genome composition that had not been identified previously. Conclusions: A novel swine-origin influenza A virus was identified as the cause of outbreaks of febrile respiratory infection ranging from self-limited to severe illness. It is likely that the number of confirmed cases underestimates the number of cases that have occurred.
Article
Full-text available
BACKGROUNDOn April 15 and April 17, 2009, novel swine-origin influenza A (H1N1) virus (S-OIV) was identified in specimens obtained from two epidemiologically unlinked patients in the United States. The same strain of the virus was identified in Mexico, Canada, and elsewhere. We describe 642 confirmed cases of human S-OIV infection identified from the rapidly evolving U. S. outbreak.METHODSEnhanced surveillance was implemented in the United States for human infection with influenza A viruses that could not be subtyped. Specimens were sent to the Centers for Disease Control and Prevention for real-time reverse-transcriptase-polymerasechain-reaction confirmatory testing for S-OIV.RESULTSFrom April 15 through May 5, a total of 642 confirmed cases of S-OIV infection were identified in 41 states. The ages of patients ranged from 3 months to 81 years; 60% of patients were 18 years of age or younger. Of patients with available data, 18% had recently traveled to Mexico, and 16% were identified from school outbreaks of S-OIV infection. The most common presenting symptoms were fever (94% of patients), cough (92%), and sore throat (66%); 25% of patients had diarrhea, and 25% had vomiting. Of the 399 patients for whom hospitalization status was known, 36 (9%) required hospitalization. Of 22 hospitalized patients with available data, 12 had characteristics that conferred an increased risk of severe seasonal influenza, 11 had pneumonia, 8 required admission to an intensive care unit, 4 had respiratory failure, and 2 died. The S-OIV was determined to have a unique genome composition that had not been identified previously.CONCLUSIONSA novel swine-origin influenza A virus was identified as the cause of outbreaks of febrile respiratory infection ranging from self-limited to severe illness. It is likely that the number of confirmed cases underestimates the number of cases that have occurred.
Article
Full-text available
Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted. 14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance. Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment.
Article
Full-text available
To determine the origin of influenza A virus (H5N1) epizootics in Cambodia, we used maximum-likelihood and Bayesian methods to analyze the genetic sequences of subtype H5N1 strains from Cambodia and neighboring areas. Poultry movements, rather than repeated reintroduction of subtype H5N1 viruses by wild birds, appear to explain virus circulation and perpetuation.
Article
Full-text available
Although field-sampling procedures to capture gDNA from individual schistosome larval stages directly from their natural hosts exist, they do pose some technical and logistical challenges hampering certain epidemiological studies. The aim of this study was to develop, refine and evaluate an alternative methodology, which enables better preservation of large numbers of individual schistosome larval stages and eggs collected in low resource endemic areas, to provide PCR-quality DNA for multi-locus genetic analysis. The techniques reported here present simple and effective short-term field and long-term laboratory preservation and storage systems for individually sampled schistosome eggs and larval stages using a commercially available aqueous stabilisation reagent, RNAlater(R) eliminating the need for more cumbersome resources such as refrigerators, heaters and centrifuge equipment for immediate specimen processing. Adaptations to a general gDNA extraction method are described, that enables the acquisition of a gDNA extract (~50 mul), facilitating multiple molecular analyses of each sampled schistosome. The methodology provided PCR-quality mitochondrial and nuclear DNA from laboratory cercariae, miracidia and eggs that had been stored at up to 37 degrees C for 2 weeks and at 4 degrees C for 6 months and also from field collected samples. This present protocol provides significant epidemiological, ethical and practical advantages over existing sampling methods and has the potential to be transferred to studies on other organisms, especially where specimens are unable to be seen by the naked eye, are difficult to handle and need to be obtained from a field environment.
Article
Full-text available
Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).
Article
Abstract Since its identification in April 2009, an A (H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A (H1N1) virus ...
Article
To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples. Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples. Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples. The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.
Article
Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of 'type,' i.e., influenza virus A and B, and 'subtype,' i.e., H1, H3, and H5, specific, single-step/reaction vessel format, real-time RT-PCR assays using total RNA from archived reference strains, shell-vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. 'Type' and 'subtype' specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field-deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.