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Validation of carbapenemase and extended-spectrum b-lactamase
multiplex endpoint PCR assays according to ISO 15189
P. Bogaerts1*, R. Rezende de Castro1, R. de Mendonc¸a2, T-D. Huang1, O. Denis2and Y. Glupczynski1
1
Laboratory of Bacteriology, CHU UCL Mont-Godinne-Dinant, Yvoir, Belgium;
2
Laboratory of Bacteriology, ULB– Erasme Hospital,
Brussels, Belgium
*Corresponding author. Tel: +32-81-42-32-41; Fax: +32-81-42-32-04; E-mail: pierre.bogaerts@uclouvain.be
Received 2 January 2013; returned 26 January 2013; revised 29 January 2013; accepted 31 January 2013
Objectives: To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important
carbapenemase and minor extended-spectrum b-lactamase (ESBL) resistance genes, according to the inter-
national ISO 15189 particular requirements for the quality and competence of medical laboratories.
Methods: Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed
internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and
accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced iso-
lates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to
PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system,
Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by
the Belgian organization for accreditation (BELAC).
Results: Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three
EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by
BELAC, and the PCR protocols were accepted as accredited according to ISO 15189.
Conclusions: Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A
ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards.
This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.
Keywords: accreditation, minor ESBLs, molecular detection
Introduction
The worldwide spread of genes conferring resistance to broad
spectrum b-lactams including carbapenems in Gram-negative
bacteria is a source of global concern.
1–3
Class A extended-
spectrum b-lactamase (ESBL)-encoding genes such as bla
TEM
,
bla
SHV
and especially bla
CTX-M
have largely disseminated world-
wide among Enterobacteriaceae. Other bla genes encoding
minor ESBLs (bla
BEL,
bla
VEB,
bla
GES and
bla
PER
) are more rarely
reported, although they have also been observed worldwide
especially among Gram-negative non-fermenters.
4
Even more worrying is the recent emergence and spread of
genes encoding carbapenemases.
3
Although class D carbape-
nem-hydrolysing b-lactamases of group OXA-23, OXA-24,
OXA-58 or OXA-143 type are almost exclusively reported in Aci-
netobacter baumannii,
5
OXA-48 (and related types) seems to
be exclusively and widely expressed in Enterobacteriaceae.
6
Besides OXA-48, the spread of carbapenemases of class A (KPC
and some GES variants such as GES-2 and GES-5) and of class
B (VIM, IMP and NDM) have been reported, albeit at differing fre-
quencies, in Enterobacteriaceae, Pseudomonas aeruginosa and
Acinetobacter spp.
1,7,8
Rapid and reliable detection methods are important for the
early implementation of infection control measures and for
preventing the subsequent dissemination of ESBLs and of carba-
penemases. Although a promising, rapid and easy detection
method based on the antibiotic hydrolytic properties of the
expressed b-lactamases has been recently published,
9,10
con-
firmation and identification of the precise types of b-lactamase
genes involved still need the use of molecular PCR-based
methods.
There is a plethora of PCR methods described in the literature
that are often validated only internally.
11 –15
Nevertheless,
quality standards requirements for medical laboratories are
#The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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J Antimicrob Chemother 2013; 68: 1576–1582
doi:10.1093/jac/dkt065 Advance Access publication 18 March 2013
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now evolving as a general rule, and compliance with the inter-
national ISO 15189 requirements for the quality and compe-
tence of medical laboratories is becoming mandatory.
16
The
accreditation process according to ISO 15189 represents a
highly challenging task for medical laboratories. One particular
difficulty is related to the fact that these requirements have to
be fulfilled, but there are no universal ways or hints about how
to reach them.
We present here the validation scheme for three multiplex
endpoint PCR assays targeting some of the most epidemiologi-
cally relevant carbapenemases (PCR CARBA targeting bla
VIM,
bla
IMP,
bla
NDM,
bla
KPC
and bla
OXA-48
and PCR OXACARBA targeting
bla
OXA-23,
bla
OXA-24
and bla
OXA-58
) and the minor ESBLs (PCR
MINESBL targeting bla
BEL,
bla
GES,
bla
PER
and bla
VEB
). The latter
two tests were particularly important for our activity of reference
centre as no commercial assay is currently available for these
targets. These PCR assays were validated and accredited accord-
ing to the international ISO 15189 standards after an external
audit performed by the Belgian accreditation body BELAC. This
validation scheme could be useful for implementing accredited
home-made PCRs in the routine laboratory.
Materials and methods
Bacterial isolates (Table 1)
A collection of 137 Gram-negative clinical isolates obtained from the
European FP7 TEMPOtest-QC consortium Number 241742 (http://www.
tempotest-qc.eu/newweb/index.php?pageId=12) was used to validate
the three multiplex PCR assays (82 isolates for PCR CARBA, 79 for PCR
MINESBL and 68 for PCR OXACARBA). This collection comprised 56 Enter-
obacteriaceae isolates [Klebsiella pneumoniae(n¼21), Escherichia coli
(n¼8), Citrobacter spp. (n¼8), Enterobacter spp. (n¼6), Klebsiella
oxytoca (n¼3), Serratia marcescens (n¼2), Providencia spp. (n¼2),
Proteus spp.(n¼2), Hafnia alvei (n¼1), Morganella morganii (n¼1), Sal-
monella enterica (n¼1) and Aeromonas hydrophila (n¼1)] and 81 Gram-
negative non-fermenters [Pseudomonas spp. (n¼41), Acinetobacter spp.
(n¼39) and Alcaligenes xylosoxidans (n¼1)] expressing various resist-
ance genes.
Design of primers (Table 2)
For each gene family, all alleles referenced on the Lahey Clinic web site
(http://www.lahey.org/Studies/) were uploaded from GenBank databases
and aligned using the ClustalX software version 2.0. Primers were
designed within the common coding region of the published alleles. An
additional primer pair that targets the chromosomal AmpC of A. bauman-
nii (bla
ADC
) was used as an internal PCR/extraction control. All the primers
apart from the forward primer targeting bla
VEB17
were designed in our
laboratory.
DNA extraction and multiplex PCR assays
A single colony was suspended in 200 mL of distilled water, and 10 mLof
a McFarland 3 turbidity standard of A. baumannii ATCC 19606 was added
to the suspension before extraction as an internal extraction control
(958C for 10 min in a dry bath). The 25 mL amplification mixture con-
tained 2 mL of DNA extract, 12.5 mLof2×master mix multiplex PCR Kit
(Qiagen Benelux, Antwerp, Belgium) and 200 mM of each primer
(except for IMP, for which the concentration of the primers was raised
to 600 mM). PCR was performed on a ABI 2720 thermocycler (Life Tech-
nologies Europe BV, Gent, Belgium) under the following conditions:
15 min at 958C and 30 cycles of 30 s denaturation at 948C; 90 s anneal-
ing at 578C and 90 s elongation at 728C; and a final elongation step at
728C for 10 min.
Each PCR run had to include three PCR controls: a positive resistance
gene control including each of the targeted genes, a DNA extraction posi-
tive control including only the internal extraction control suspended in
water, and a negative control (only water, no DNA). The amplicons
were visualized by capillary electrophoresis on a QIAxcel instrument
(Qiagen Benelux) using the QIAxcel high-resolution kit, QX DNA size
marker 100– 2500 bp and QX alignment markers 15/5000 bp according
to the manufacturer’s recommendations. The whole process, including
extraction and electrophoresis takes ,4 h. A negative result could only
be technically validated when the band corresponding to the internal ex-
traction control was present. In case of a positive result for any of the tar-
geted genes, the presence of the internal control does not need to be
taken into account.
Validation process
The validation process was based on the procedure proposed by Rabenau
et al.
18
for home-made qualitative nucleic acid testing. This process
includes the control of the specificity, sensitivity, reproducibility and ac-
curacy of each PCR multiplex assay. For sensitivity, at least 10 different
isolates positive for the gene to be detected were tested once. In cases
where the minimum number of isolates harbouring the targeted genes
was not available in the library, the available isolates were extracted
twice or more so as to reach 10 different sample preparations. For speci-
ficity, at least 20 isolates known to be negative for the targeted genes,
but possibly expressing other resistance genes representative of the
current b-lactamase epidemiology, were tested once. For reproducibility,
one isolate positive for each resistance gene was tested three times
intra-run and three times inter-run. Finally, accuracy was certified by
testing a panel prepared and sent by an external laboratory [external
quality control (EQC) process] comprising three positive isolates for
each target to be tested and three negative ones. This panel equally
comprised referenced isolates from the TEMPOtest-QC collection.
Results and discussion
The three multiplex PCR assays were validated with a panel of
137 characterized and referenced Gram-negative clinical isolates
(Table 1) according to the validation protocol presented in
the ‘Materials and methods’ section. This collection includes
30 metallo-b-lactamase-expressing isolates (10 VIM-, 10 IMP-
and 10 NDM-expressing isolates), 10 OXA-48-expressing isolates,
10 KPC-expressing isolates, 31 OXA-23, OXA-24 or OXA-58-
expressing isolates, 41 minor ESBL-expressing isolates (BEL,
VEB, GES and PER), 27 TEM-expressing isolates, 26 SHV-
expressing isolates and 14 CTX-M-expressing isolates. In add-
ition, 33 isolates expressing additional b-lactamases not
targeted by the PCR assays were used for specificity testing.
For each multiplex PCR assay, the presence of an extraction
control ruled out false-negative results due to PCR inhibition or
extraction faults. In the positive resistance gene control, the
presence of each band at the expected size confirms the ability
of the PCR to detect up to five resistance genes in a single PCR
(Figure 1). The positive extraction PCR control has to be per-
formed in a separate well as the corresponding 1059 bp ampli-
con tends to disappear in the presence of another resistance
gene. It highlights that the extraction/inhibition control does
not interfere with the detection of the targeted resistance
genes generating smaller amplicons.
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Table 1. Collection of characterized clinical isolates used for multiplex PCR assay validation
Species Number
bla
VIM,
bla
IMP
or bla
NDM
bla
OXA-48
bla
KPC
bla
OXA-23,
bla
OXA-24
or
bla
OXA-58 groups
bla
BEL,
bla
GES,
bla
PER or
bla
VEB
bla
TEMa
bla
SHVa
bla
CTX-M
of G1,
2 and 9
a
Other
b
-lactamases
b
Klebsiella pneumoniae 21 5 4 10 11 20 3 10
Escherichia coli 82 2 62 3 9
Enterobacter cloacae 42 2 13 4 1
Klebsiella oxytoca 31 1 1
Citrobacter freundii 31 11
Serratia marcescens 21 11
Enterobacter aerogenes 111
Enterobacter asburiae 1
Other Enterobacteriaceae
c
14 4 3 4 3 2
Acinetobacter baumannii 34 2 27 10 1 1
Acinetobacter spp. other than
A. baumannii
51 4 1
Pseudomonas aeruginosa 39 10 26 10
Pseudomonas spp. other than
P. aeruginosa
22
Total 137 30 10 10 31 41 27 26 14 33
a
Including both ESBLs and non-ESBLs.
b
Including plasmidic AmpC, carbenicillinases, oxacillinases and SPM metallo-b-lactamase.
c
Including Proteus vulgaris,Aeromonas hydrophila,Citrobacter spp., Hafnia alvei,Morganella morganii and Providencia spp.
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Table 2. Primer sequences and amplicon sizes
PCR name Targeted gene Primer sequence (5′to 3′)
a
Amplicon size
CARBA bla
NDM
Forward ACT TGG CCT TGC TGT CCT T 603 bp
Reverse CAT TAG CCG CTG CAT TGA T
bla
VIM
Forward TGT CCG TGA TGG TGA TGA GT 437 bp
Reverse ATT CAG CCA GAT CGG CAT C
bla
IMP
Forward ACA YGG YTT RGT DGT KCT TG 387 bp
Reverse GGT TTA AYA AAR CAA CCA CC
bla
KPC
Forward TCG CCG TCT AGT TCT GCT GTC TTG 353 bp
Reverse ACA GCT CCG CCA CCG TCA T
bla
OXA-48
Forward ATG CGT GTA TTA GCC TTA TCG 265 bp
Reverse CAT CCT TAA CCA CGC CCA AAT C
OXACARBA bla
OXA-23 group
Forward CCC CGA GTC AGA TTG TTC AAG G 330 bp
Reverse TAC GTC GCG CAA GTT CCT GA
bla
OXA-24/143 group
Forward GCA GAA AGA AGT AAA RCG GGT 271 bp
Reverse CCA ACC WGT CAA CCA ACC TA
bla
OXA-58 group
Forward GGG GCT TGT GCT GAG CAT AGT 688 bp
Reverse CCA CTT GCC CAT CTG CCT TT
MINESBL bla
PER
Forward AGT GTG GGG GCC TGA CGA T 725 bp
Reverse GCA ACC TGC GCA ATR ATA GCT T
bla
GES
Forward CTG GCA GGG ATC GCT CAC TC 600 bp
Reverse TTC CGA TCA GCC ACC TCT CA
bla
BEL
Forward CGA CAA TGC CGC AGC TAA CC 448 bp
Reverse CAG AAG CAA TTA ATA ACG CCC
bla
VEB
Forward CGA CTT CCA TTT CCC GAT GC 376 bp
Reverse TGT TGG GGT TGC CCA ATT TT
Inhibition control bla
ADCb
Forward GTA CCT CAA TTT ATG CGG RCA ATA C 1059 bp
Reverse TGC GYT CTT CAT TTG GAA TAC G
a
For degenerate primers: D ¼A, G or T; R ¼AorG;Y¼CorT;K¼GorT;W¼AorT.
b
ADC, Acinetobacter-derived cephalosporinase used as an inhibition/extraction control in each of the three multiplex PCRs.
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Sensitivity, specificity and reproducibility (including the extrac-
tion step) for each of the targeted genes were 100% (data not
shown). The resistance genes of the isolates included in the
three EQC panels were correctly identified by each PCR assay
(accuracy 100%; Figure 1). Regarding specificity, non-specific
amplifications generating fragments of unexpected sizes were
C+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 61 17 IC C– C+ 18 MW (a)
(b)
(c)
1 2 3 4 5 6 7 8 9 10 11 12 C– IC C +
MW
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C– C+
ICMW
blaVEB
blaOXA–48
blaKPC
blaIMP
blaVIM
blaNDM
blaADC: IC
blaADC: IC
blaOXA-58
blaOXA-23
blaOXA-24
blaADC: IC
blaPER
blaGES
blaBEL
5000.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
100.0
100.0
2000.0
1200.0
800.0
600.0
400.0
5000.0
5000.0
2000.0
1200.0
800.0
600.0
400.0
200.0
15.0
2500.0
1500.0
1000.0
700.0
500.0
300.0
100.0
2000.0
1200.0
800.0
600.0
400.0
200.0
15.0
200.0
15.0
Figure 1. Capillary electrophoresis of amplicons obtained for accuracy testing (EQC ring test) of the three multiplex PCR assays. MW, molecular weight
(bp); IC, internal control, corresponding to bla
ADC
of Acinetobacter baumannii;C+, positive control consisting of a nucleic acid extracted from a mixture
of strains expressing the resistance genes to be targeted; C-, negative control (water only). (a) PCR CARBA targeting bla
VIM,
bla
IMP,
bla
NDM,
bla
KPC
and
bla
OXA-48
: lanes 1, 6 and 7, negative strains (Acinetobacter radioresistens OXA-23, A. baumannii PER-1 and Pseudomonas aeruginosa BEL-1); lanes 2, 8
and 9, KPC-producing strains (Klebsiella pneumoniae KPC-2); lanes 3, 5 and 12, VIM-producing strains (Citrobacter braakii VIM-1, P. aeruginosa VIM-4
and P. aeruginosa VIM-2); lanes 4, 14 and 16, OXA-48-producing strains (Enterobacter cloacae OXA-48, K. pneumoniae OXA-48 and E. coli OXA-48);
lanes 10, 11 and 15, IMP-producing strains (P. aeruginosa IMP-13, P. aeruginosa IMP-13 and P. aeruginosa IMP-7); and lanes 13, 17 and 18,
NDM-producing strains (K. pneumoniae NDM-1). (b) PCR OXACARBA targeting bla
OXA-23,
bla
OXA-24
and bla
OXA-58
: lanes 1, 2 and 6, negative strains
(Citrobacter freundii TEM-1, Proteus mirabilis CTX-M-2 and P. aeruginosa SPM); lanes 3, 7 and 8, OXA-23-producing strains (A. radioresistens OXA-23
and A. baumannii OXA-23); lanes 4, 9 and 11, OXA-24-like producing strains (A. baumannii OXA-72); and lanes 5, 10 and 12, OXA-58-producing
strains (Acinetobacter haemolyticus OXA-58, A. baumannii OXA-58 and Acinetobacter pittii OXA-58). (c) PCR MINESBL targeting bla
BEL,
bla
GES,
bla
PER
and bla
VEB
: lanes 1, 8 and 11, negative strains (C. braakii VIM-1, P. aeruginosa VIM-2 and P. aeruginosa IMP-7); lanes 2, 5 and 6, PER-producing
strains (A. baumannii PER-1); lanes 3, 4 and 13: BEL-producing strains (P. aeruginosa BEL-1); lanes 7, 9 and 10: GES-producing strains
(A. baumannii GES-12, P. aeruginosa GES-1 and P. aeruginosa GES-18); and lanes 12, 14 and 15, VEB-producing strains (P. aeruginosa VEB-1b, 1a, 1).
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observed in about 2% of the cases, and have to be interpreted as
negative results (Figure 1b, lane 1). These non-specific results
were often related to an Enterobacter asburiae isolate (data
not shown). No explanation could be found to explain this phe-
nomenon, although it was less frequently observed when the
primers were HPLC purified and was not observed when the
PCR was performed in simplex for each individual target. The val-
idation files were controlled by BELAC during an external audit
held in UCL Mont-Godinne on 19 March 2012, and the three
PCR protocols were accepted for accreditation according to ISO
15189 in July 2012 (BELAC Certificate 431-MED).
It is important to point out that, according to ISO 15189, an
efficient separation between the different work areas must be
provided in order to efficiently avoid cross-contamination. All
the processes described here only represent the analytical part
of the accreditation process. Pre-analytical and post-analytical
stages must also follow ISO 15189 standards, but these are
more difficult to export from one laboratory to another. Many ex-
cellent home-made PCRs (endpoint or real-time) have already
been published in the literature (e.g. Dallenne et al.,
11
Huang
et al.,
12
Naas et al.,
13
Poirel et al.,
14
Swayne et al.
15
and Naas
et al.,
19
although many other publications exist). Regarding
other multiplex PCRs for carbapenemases, each method presents
its own characteristics. For example, Huang et al.
12
proposed a
real-time TaqMan multiplex targeting different class D carbape-
nemases from A. baumannii, while Swayne et al.
15
published a
TaqMan PCR targeting five class A and D carbapenemases
encountered in Enterobacteriaceae. The three multiplex PCR
assays presented by Poirel et al.
14
are endpoint PCRs targeting
the largest panel of carbapenemase genes (11 targets). The im-
plementation of PCR in a laboratory will depend on its particular
needs, local epidemiology, technical resources and quality
requirements. Most methods are indeed efficient and have
already been peer-reviewed, but they would most probably not
be accepted as such by external auditors as conforming to ISO
15189. Major non-conformities with ISO 15189 standards are
the lack of an internal control able to rule out false-negative
results and the absence of accuracy testing by participating in
at least one annual EQC.
Testing accuracy is optimally achieved by participation in ex-
ternal quality assurance schemes organized by independent
bodies such as Quality Control for Molecular Diagnostics or the
United Kingdom National External Quality Assessment Service.
Unfortunately, these bodies do not yet organize EQC evaluations
for the detection of resistance genes. In such situations, ISO
15189 accepts that laboratories organize a so-called ring test
(a blind exchange of an EQC proficiency panel) to evaluate
the accuracy of their own methods. This is what was
performed with the strains obtained from the TEMPOtest-QC
consortium.
In summary, we report here the successful accreditation
process of three home-made multiplex PCR assays according
to the ISO 15189 standards. We believe that this validation
scheme should constitute a valuable tool for laboratories in
order to accredit their own protocols. Moreover, as these three
multiplex PCR assays are already accredited, they could easily
be implemented in other diagnostic laboratories through a
lighter verification procedure.
18
Funding
This work was supported by EU grant FP7-HEALTH-2009-SINGLESTAGE
TEMPOtest-QC, project 241742 and by INAMI/RIZIV funding of the
Belgian reference centres.
Transparency declarations
None to declare.
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