TECHNICAL REPORT

External quality assurance scheme

on PCR for Bordetella pertussis, 2012
On behalf of EUpert-labnet network

www.ecdc.europa.eu
ECDC TECHNICAL REPORT

External quality assurance scheme on PCR

for Bordetella pertussis, 2012
On behalf of the EUpert-labnet network
This report was commissioned by the European Centre for Disease Prevention and Control (ECDC), coordinated by
Dr Adoracion Navarro Torné and produced by Dr Norman Fry, Health Protection Agency (London, UK), Dr Tine
Dalby, Statens Serum Institut (Copenhagen, Denmark) and Dr Qiushui He, National Institute for Health and
Welfare (Turku, Finland), on behalf of the EUpert-labnet consortium as part of the coordination of activities for
laboratory surveillance of whooping cough in Member States/EEA countries (referring to Specific Contract
ECDC/2011/013).

Acknowledgements
We acknowledge the expert technical assistance of Lalita Vaghji and John Duncan (HPA, London) in the
preparation, testing and dispatch of this EQA panel.

Suggested citation: European Centre for Disease Prevention and Control. External quality assurance scheme on
PCR for Bordetella pertussis, 2012. On behalf of the EUpert-labnet network Stockholm: ECDC; 2012.

Stockholm, September 2012

ISBN 978-92-9193-383-9
doi 10.2900/62246

© European Centre for Disease Prevention and Control, 2012

Reproduction is authorised, provided the source is acknowledged
TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis 2012

Contents
List of tables ......................................................................................................................................................................... iii
Abbreviations ........................................................................................................................................................................iv
Executive summary................................................................................................................................................................ 1
1. Background ....................................................................................................................................................................... 2
2. Introduction ...................................................................................................................................................................... 4
3. Materials and methods ....................................................................................................................................................... 5
3.1. Organisation ............................................................................................................................................................. 5
3.2. Selection of panel ..................................................................................................................................................... 5
3.3. Carriage of panels ..................................................................................................................................................... 5
3.4. Testing ..................................................................................................................................................................... 5
3.5. Data analysis ............................................................................................................................................................ 5
Qualitative EQA data scoring system ............................................................................................................................ 6
4. Results .............................................................................................................................................................................. 7
Bordetella pertussis dilution series .................................................................................................................................... 7
Bordetella holmesii........................................................................................................................................................... 7
Bordetella parapertussis and Bordetella bronchiseptica....................................................................................................... 7
Negative controls ............................................................................................................................................................. 7
Haemophilus influenzae ................................................................................................................................................... 7
Crossing thresholds .......................................................................................................................................................... 7
Controls .......................................................................................................................................................................... 7
Questionnaire comments on use of Bordetella PCR by European laboratories .................................................................... 10
5. Discussion ....................................................................................................................................................................... 12
Sensitivity, specificity and reproducibility ......................................................................................................................... 12
Differentiation between B. pertussis and other Bordetella species associated with respiratory clinical infections, namely B.
parapertussis, B. holmesii and B. bronchiseptica .............................................................................................................. 12
B. pertussis targets ................................................................................................................................................... 12
B. parapertussis targets ............................................................................................................................................. 12
Other Bordetella species targeted .............................................................................................................................. 12
Evidence of good laboratory practice of the PCR technique by checking for evidence of contamination ............................... 12
Controls ................................................................................................................................................................... 12
Training needs .......................................................................................................................................................... 12
Establishment of best practice in current assays, interpretation and reporting ............................................................... 13
Limitations .................................................................................................................................................................... 13
6. Recommendations ........................................................................................................................................................... 14
References .......................................................................................................................................................................... 15
Annex 1. Invitation letter to participants for the Bordetella pertussis PCR external quality assurance programme ..................... 16
Annex 2. List of participants ................................................................................................................................................. 17
Annex 3. The EUpert-labnet Bordetella pertussis PCR EQA questionnaire ................................................................................ 18
Annex 4. The EUpert-labnet Bordetella pertussis PCR EQA result submission form ................................................................... 20

List of tables
Table 1. Characteristics of the strains used to prepare genomic DNA for inclusion in the first EUpert-labnet B. pertussis PCR EQA
(February 2012) .................................................................................................................................................................... 5
Table 2. Intended results for the first EUpert-labnet Bordetella pertussis PCR EQA (February 2012) ........................................... 6
Table 3 Number of correct qualitative results per panel member and technology type................................................................ 8
Table 4. Summary of responses to questionnaire ..................................................................................................................... 9
Table 5. Crossing thresholds for samples containing Bordetella DNA reported by submitting laboratories .................................. 11

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Abbreviations
CFU Colony forming unit(s)
Cq Crossing threshold
DSN Dedicated surveillance network
ECDC European Centre for Disease Prevention and Control
EEA European Economic Area
EQA External quality assurance
EU European Union
EUpert-labnet Consortium of European pertussis experts funded by ECDC for this current programme
EUVAC.NET A former European surveillance network for selected vaccine-preventable diseases hosted at the Statens
Serum Institut, Denmark
HPA Health Protection Agency (UK)
IS Insertion sequence element
NIBSC National Institute for Biological Standards and Control (UK)
PCR Polymerase chain reaction
QCMD Quality Control for Molecular Diagnostics (Scotland)
qPCR Quantitative (real-time) PCR
RSIL Respiratory and Systemic Infection Laboratory (of the HPA, London)
THL Terveyden ja Hyvinvoinnin Laitos / National Institute for Health and Welfare (Finland)

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

Executive summary
Twenty-one laboratories from 21 EU/EEA countries participated in the first external quality assurance scheme for
Bordetella pertussis PCR by ECDC on behalf of the EUpert-labnet network. The panel included dilutions of purified
genomic DNA from B. pertussis at three concentrations designated ‘high’, ‘medium’ and ‘low’. Duplicate samples of
the B. pertussis ‘medium’ dilution were included to test the reproducibility of the PCR assays. Genomic DNA from
other Bordetella species — B. parapertussis, B. holmesii, B. bronchiseptica — were included in the panel, together
with Haemophilus influenzae. Two ‘blank’ samples, i.e. with no added DNA, were also included to check for
potential contamination.
Of 22 datasets (one laboratory submitted two datasets) all (100%) reported the ‘high’ concentration as positive for
B. pertussis and 21/22 also reported the ‘medium’ concentration duplicate samples as B. pertussis positive.
Only 15 out of 22 reported the ‘low’ concentration as positive for B. pertussis or Bordetella spp.
Real-time Bordetella PCR assays (both in-house and commercial) demonstrated greater sensitivity than
conventional PCR assays as demonstrated by 85% (11/13) of those using qPCR reporting the ‘low’ concentration
positive for B. pertussis or Bordetella spp. compared with 57% (4/7) of those using conventional PCR.
None of the laboratories reported Bordetella DNA in the two blank samples, demonstrating good molecular
laboratory practice.
The most common targets for B. pertussis and B. parapertussis were IS481 and IS1001, respectively. However,
these targets are not completely specific for these species. Therefore IS481 positive-only results should be
reported as probable B. pertussis (B. pertussis / B. holmesii / B. bronchiseptica) and IS1001 positive-only results
should be reported as probable B. parapertussis (B. parapertussis / B. bronchiseptica).
Only laboratories using specific B. holmesii assays (e.g. targeting recA) could correctly differentiate this species
from other Bordetella species including B. pertussis.
Several commercial kits are available for the detection of B. pertussis and/or B. parapertussis; however, care
should be taken in the interpretation of these results as indicated above.
An internal process control to check for the presence of PCR inhibitors is recommended to avoid false-negative
reporting.
This report presents the results of the first external quality assurance (EQA) scheme for Bordetella pertussis PCR
funded by the European Centre for Disease Prevention and Control (ECDC). The EQA study was conducted
between February and March 2012.

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

1. Background
The European Centre for Disease Prevention and Control (ECDC) is a European Union (EU) agency with a mandate
to operate the dedicated surveillance networks (DSNs) and to identify, assess, and communicate current and
emerging threats to human health from communicable diseases. Within its mission, ECDC shall ‘foster the
development of sufficient capacity within the Community for the diagnosis, detection, identification and
characterisation of infectious agents which may threaten public health. The Centre shall maintain and extend such
cooperation and support the implementation of quality assurance schemes.’ 1
External quality assurance (EQA) is part of quality management systems and evaluates performance of laboratories,
by an outside agency on material that is supplied specially for the purpose. ECDC’s disease-specific networks
organise a series of EQA for EU/EEA countries. In some specific networks non-EU/EEA countries are also involved
in the EQA activities organised by ECDC. The aim of the EQA is to identify areas for improvement in laboratory
diagnostic capacities relevant to surveillance of the diseases listed in Decision No. 2119/98/EC 2 and to ensure
comparability of results between laboratories from all EU/EEA countries. The main purposes of external quality
assurance schemes include:
• assessment of the general standard of performance;
• assessment of the effects of analytical procedures (method principle, instruments, reagents, calibration);
• evaluation of individual laboratory performance;
• identification of problem areas;
• providing continuing education;
• identification of training needs.
Pertussis (whooping cough) is an acute bacterial infection usually caused by Bordetella pertussis, which can affect
people of all ages. A similar illness is caused by Bordetella parapertussis, but this is not affected by current
pertussis vaccines. Infants are the most vulnerable group with the highest rates of morbidity and mortality, whilst
older children and adults usually display milder symptoms. Increases in both awareness of pertussis infections and
reported numbers of cases in many countries have highlighted the need for good laboratory tests for the detection,
identification and characterisation of clinical infections caused by B. pertussis and other Bordetella species.
The utility of PCR in the laboratory confirmation of B. pertussis infection is now well established and can provide
improved sensitivity over culture [1,2]. In addition, improvements in both nucleic acid extraction and real-time PCR
(qPCR) [3,4] technologies can now provide more rapid turnaround times [2,5].
The absence of existing EQA programmes for B. pertussis PCR in European diagnostic laboratories was reported in
2005 by Muyldermans, et al [2]. These authors described results obtained with two proficiency panels, sent to six
and nine European Laboratories, respectively, containing: (i) a series of dilutions of three previously characterised
B. pertussis clinical isolates and two negative controls; and (ii) a series of dilutions of reference strains of B.
pertussis, B. holmesii, B. hinzii, and B. bronchiseptica, as well as negative controls. Results from the first panel
revealed no false positives by six laboratories (seven datasets) and limits of detection of the three B. pertussis
strains varied from 4 to 4 000, 9 to 9 000, and 3 to 30 000 CFU/ml. Results from nine laboratories for the second
panel were as follows: one laboratory reported a positive result for one of the negative controls and also found a B.
parapertussis-positive sample to be positive for B. pertussis. Eight laboratories using IS481-based assays, reported
positive results for the samples containing B. holmesii and B. bronchiseptica. One laboratory using an assay
targeting the pertactin gene designed to be specific for B. pertussis detected B. pertussis with 100% specificity
illustrating the critical nature of the choice of the target gene for specific identification in B. pertussis PCR assays.
Since the above study, two commercial EQA programmes for B. pertussis PCR became available from Quality
Control for Molecular Diagnostics (QCMD), Glasgow, Scotland and INSTAND e.V. Düsseldorf, Germany.
An ECDC-funded EQA distribution for the ‘Evaluation and standardisation of real-time PCR for detection of B.
pertussis’ was first distributed by the Statens Serum Institut (Copenhagen, Denmark), under the auspices of the
EUVAC.NET (ECDC Grant ECD.2042 Work area 4) in January 2011. A panel of eight samples comprising one
negative, and seven containing Bordetella DNA (five with B. pertussis DNA in varying concentrations, one with B.
parapertussis DNA and one with B. holmesii DNA). Twenty-four laboratories from 19 European countries
participated in this EQA. Bordetella parapertussis was misidentified by three laboratories and B. holmesii was
misidentified by 15 laboratories as either B. pertussis or B. parapertussis. Real-time PCR demonstrated greater

1
Regulation (EC) No 851/2004 of the European Parliament and of the Council of 21 April 2004 establishing a European centre for
disease prevention and control. OJ L 142, 30.4.2004, p. 1. Article 5(3).
2
2002/253/EC: Commission Decision of 19 March 2002 laying down case definitions for reporting communicable diseases to the
Community network under Decision No 2119/98/EC of the European Parliament and of the Council (notified under document
number C(2002) 1043).

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

sensitivity than traditional block-based PCR and two laboratories reported the negative sample as weakly positive
for B. pertussis. These findings were presented by Dalby, et al [6] at the 22nd European Congress of Clinical
Microbiology and Infectious Diseases (ECCMID), 31 March – 3 April 2012.
In September 2011, the responsibilities of the EUVAC.NET were transferred to ECDC and subsequently a
consortium of pertussis experts from nine countries, the EUpert-labnet network, was established and was awarded
the Framework Contract ‘Coordination of activities for laboratory surveillance of whooping cough in Member
States/EEA countries’ (ECDC/2011/013). The EUpert-labnet network is led and coordinated by Dr Qiushui He
(Turku, Finland).
The UK Health Protection Agency’s Respiratory and Systemic Infection Laboratory (RSIL) Colindale, UK, and
National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK, were contracted to deliver
Workpackage 2: EQAs for laboratory diagnostics and molecular typing of pertusiss. The Workpackage 2
(coordinated by Dr Norman Fry, HPA – Colindale, Dr Dorothy Xing, HPA - NIBSC and Dr Kevin Markey, HPA –
NIBSC) comprises EQAs for: PCR, serology, and strain typing. This report details the first of these EQAs.

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

2. Introduction
Pertussis remains endemic worldwide and is an important public health issue. There has been a marked increase in
reported pertussis cases in many countries despite high vaccination coverage. Laboratory diagnosis of pertussis is
important for treatment, prevention and surveillance. It is noteworthy that there is wide variation in the reporting
of laboratory-confirmed pertussis cases globally as well as within Europe and one of the reasons for this may be
the differences in the methodologies used for diagnosis.
External quality assurance is important to ensure accurate diagnosis and good laboratory performance. This EQA
programme builds upon the previous collaborative studies on qPCR organised by the EUVAC.NET and EUpertstrain
consortia.
The current programme is designed to develop and standardise diagnostic techniques for confirmation of pertussis
infection in individuals and in outbreak situations. Through EQA schemes, the performance of the national
reference laboratories from each EU/EEA Member State can be independently assessed. Furthermore, through the
scheme, recommendations for improvements to methodologies and areas for further training can be identified.
The specific aims of this Bordetella PCR EQA were:
• to evaluate sensitivity, specificity and reproducibility of current assays to detect B. pertussis DNA;
• to evaluate the ability of assays to differentiate between B. pertussis and other Bordetella species
associated with respiratory clinical infections, namely B. parapertussis, B. holmesii and B. bronchiseptica;
• to assess good laboratory practice of the PCR technique by checking for evidence of contamination;
• to assess differences in interpretation and reporting of Bordetella PCR results;
• to identify training needs;
• to assist the establishment of ‘best practice’ in current assays, interpretation and reporting.

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

3. Materials and methods

3.1. Organisation
The first EUpert-labnet B. pertussis PCR EQA was organised by the UK’s Health Protection Agency, Colindale
(London) intended for National Reference Laboratories in EU Member States, Iceland, Liechtenstein and Norway.
Invitations were initially sent to ECDC-designated pertussis laboratory experts in November 2011 (Annex 1). In
total, 21 laboratories participated, 20 of which were in EU countries and one from Norway (Annex 2).

3.2. Selection of panel

Clinical isolates of B. pertussis, B. parapertussis, B. holmesii, B. bronchiseptica and Haemophilus influenzae were
selected from the culture collection at the HPA’s Respiratory and Systemic Infection Laboratory (see Table 1).
Purified genomic DNA was prepared from bacterial pellets using the Nucleon BACC2 DNA extraction kit (GE
Healthcare Life Sciences) including an RNase A treatment to prevent confounding of quantification. The
concentration of DNA was determined at A260 using the Nanodrop ND-1000 spectrophotometer (Thermo Scientific).
The panel was designed to include dilutions of purified genomic DNA from B. pertussis at three concentrations
‘high’, 2pg/µl; ‘medium’, 0.2pg/µl and ‘low’, 0.02pg/µl. Duplicate samples of the B. pertussis 0.2pg/µl dilution were
included to test reproducibility. The DNA concentration of all other Bordetella species included in the panel (B.
parapertussis, B. holmesii, B. bronchiseptica and Haemophilus influenzae) was 2pg/µl. Two ‘blank’ samples
containing only 10 mM Tris buffer, pH 8.0, i.e. no added DNA, were included to check for potential contamination.
The panel was tested three times prior to dispatch by the sending laboratory.

3.3. Carriage of panels

The panels were prepared, packed according to local regulations, collected by courier on 8 February 2012 from
HPA - Colindale, London and dispatched to the 21 laboratories (Annex 2). All packages were received in a timely
manner allowing laboratories sufficient time to meet the deadline of Friday 30 March 2012.
Table 1. Characteristics of the strains used to prepare genomic DNA for inclusion in the first EUpert-
labnet B. pertussis PCR EQA (February 2012)
HPA reference no. Organism Isolation date Specimen Clinical details
type
H114260371 Bordetella pertussis Oct 2011 Pernasal swab Whooping cough
serotype 1,3
H114560403 Bordetella parapertussis Nov 2011 Pernasal swab Respiratory infection
H104780607 Bordetella holmesii Nov 2010 Blood culture Pyrexia
H111580382 Bordetella bronchiseptica Apr 2011 Sputum Not provided
H120420371 Haemophilus influenzae Jan 2012 Blood culture Bacteraemia
non-capsulated

3.4. Testing
Participants were instructed to treat the panel as ‘extracted DNA samples’ and test them three times using their
usual B. pertussis PCR assay and report their qualitative and/or quantitative results. A reporting sheet for the
results and a questionnaire to ascertain details of each test were sent to each participant by e-mail as Word
(Microsoft) documents to be returned by the deadline.
The main questions concerned each participant’s use of PCR for the diagnosis of Bordetella infections; the type of
assay used (i.e. ‘in-house’, published or commercial kit); whether real time (qPCR) or traditional block-based
thermocycler; which gene targets were used; the types of controls used including internal controls to check for
inhibition; the types of clinical samples tested and approximate number tested per year; the method of DNA
extraction used; participation in any existing EQA programme for Bordetella PCR. Participants were also asked to
provide any other comments on the use of Bordetella PCR by European laboratories.
The full questionnaire is reproduced in Annex 3.

3.5. Data analysis

The intended results of the submitting laboratory (Table 2) were used as a basis for the scoring.

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Participants were expected to:

• detect B. pertussis at both the ‘high’ and ‘medium’ dilutions;
• differentiate between B. pertussis and other Bordetella species;
• accurately interpret and report results depending on the targets used and results obtained;
• obtain a ‘negative’ result (for Bordetella) with the two blank samples;
• obtain a ‘negative’ result (for Bordetella) with the sample containing H. influenzae.
The ‘low’ dilution of B. pertussis was designed to be challenging.

Qualitative EQA data scoring system

Results were scored with a possible 10 out of 10 (100%). Results were also considered acceptable if Bordetella
species was reported for samples containing Bordetella DNA, but incorrect if the wrong species was reported.
Table 2. Intended results for the first EUpert-labnet Bordetella pertussis PCR EQA (February 2012)
Sample Qualitative Result Sample details
number B.pertussis Bordetella spp. Strain Concn. Vol (µL)
1 Negative Negative Tris buffer (10mM, pH 8.0) 0 200
2 Positive Positive Bordetella pertussis 2pg/µL 200
(H114260371)
3 Positive Positive Bordetella pertussis 0.2pg/µL 200
(H114260371)
4 Negative Positive Bordetella holmesii 2pg/gµL 200
(H104780607)
5 Positive Positive Bordetella pertussis 0.2pg/µL 200
(H114260371)
6 Negative Negative Tris buffer (10mM, pH 8.0) 0 200
7 Negative Positive Bordetella bronchiseptica 2pg/µL 200
(H111580382)
8 Negative Positive Bordetella parapertussis 2pg/µL 200
(H114560403)
9 Negative Negative Haemophilus influenzae 2pg/µL 200
(H120420371)
10 Positive Positive Bordetella pertussis 0.02pg/µL 200
(H114260371)

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

4. Results
The intended results are shown in Table 2. All 21 laboratories submitted results and completed questionnaires by
the deadline. One laboratory submitted two datasets, thus 22 datasets are presented. The overall results for the 22
datasets from 21 EU/EEA laboratories are shown in Table 3. Results are presented by platform, as conventional (i.e.
traditional block-based PCR with amplification products detected by gel electrophoresis, staining with ethidium
bromide and image capture under ultraviolet light), or real time (qPCR) using fluorescent detection of amplification
products. These categories were further subdivided by use of a commercial kit or an ‘in-house’ assay. Finally, two
laboratories presented data using a combination of methods and these are described as ‘other’. These ‘other’
methods comprise:
• conventional PCR using a commercial kit and in-house PCR and an in-house real-time PCR for B. pertussis
plus real-time PCR commercial kit and a conventional in-house PCR for B. parapertussis (one dataset);
• conventional in-house together with direct amplification and DNA sequencing (one dataset).
A summary of answers from the questionnaire is shown in Table 4.

Bordetella pertussis dilution series

All laboratories (22/22 datasets) detected B. pertusis DNA in the ‘high’ dilution (sample number 2), and all but one
(21/22) detected B. pertusis in the ‘medium’ dilution duplicates (sample numbers 3 and 5). However, the one
laboratory that scored no. 3 negative for B. pertussis PCR also used direct amplification and DNA sequencing of the
16S rDNA and reported B. pertussis identified by sequencing of 16S rRNA gene (99.6%). Only 15/22 reported B.
pertussis (or Bordetella spp.) for the ‘low’ dilution of B. pertussis DNA (sample number 10).

Bordetella holmesii
Only 16/22 correctly reported sample number 4 as B. holmesii (or Bordetella spp.) or negative for B. pertussis.

Bordetella parapertussis and Bordetella bronchiseptica

All (22/22) correctly reported sample number 8 as B. parapertussis (or Bordetella spp.), or negative for B. pertussis
but only 13/22 correctly reported sample number 7 as B. bronchiseptica (or Bordetella spp.) or negative for
B. pertussis.

Negative controls
All 22/22 datasets correctly reported negative results (for any Bordetella spp.) with sample numbers 1 and 6.

Haem ophilus influenzae

Twenty of twenty-two datasets reported negative results (for any Bordetella spp.) with sample number 9
containing Haemophilus influenzae DNA.

Crossing thresholds
Nine of the 13 laboratories that performed qPCR reported crossing thresholds (Cqs) [3,4] for B. pertussis and/or
Bordetella spp. (see Table 5). Generally, the reproducibility for the duplicate samples (sample numbers 3 and 5)
within laboratories was very good (mean values less than 2 Cq apart), apart from one laboratory (using ptxA)
where the difference was >3.7 Cq. Where available, reproducibility within laboratories was also good for repeated
runs on the same sample (i.e. <0.5, <1, <2.3 Cq).

Controls
Only 14/22 laboratories used an internal control (to check for inhibition) and 13/22 used an internal
calibrator/positive control.

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 EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Table 3 Number of correct qualitative results per panel member and technology type
Sample Sample Sample Total PCR
number content concn. datasets
(pg/µl) n = 22 Conventional qPCR Other

Commercial In-house Both Commercial In-house Both

n=1 n=4 n=2 n=1 n = 11 n=1 n=2

n % n % n % n % n % n % n % n %

2 B. pertussis 2pg/µl 22 100 1 100 4 100 2 100 1 100 11 100 1 100 2 100

3 B. pertussis 0.2pg/µl 21 95 1 100 4 100 2 100 1 100 11 100 1 100 1 50

5 B. pertussis 0.2pg/µl 22 100 1 100 4 100 2 100 1 100 11 100 1 100 2 100

10 B. pertussis 0.02pg/µl 15 68 0 0 4 100 0 0 0 0 10 91 1 100 0 0

8 B. parapertussis 2pg/µl 22 100 1* 100 4 100 2 100 1 100 11 100 1 100 2 100

4 B. holmesii 2pg/µl 16 73 1* 100 1 25 2 100 0 0 9 82 1 100 2 100

7 B. 2pg/µl 12 55 1* 100 2 50 0 0 0 0 8 73 0 0 1 50
bronchiseptica
9 Haemophilus 2pg/µl 20 91 1 100 3 75 2 100 1 100 10 91 1 100 2 100
influenzae
1 Negative 22 100 1 100 4 100 2 100 1 100 11 100 1 100 2 100

6 Negative 22 100 1 100 4 100 2 100 1 100 11 100 1 100 2 100

The shading indicates duplicate samples.

* Kit used, Pneumobacter ACE detection kit (Seegene), claims to detect Bordetella pertussis and Haemophilus influenzae; gave
negative results for B. holmesii / B. bronchiseptica / B. parapertussis / H. influenzae (numbers 4, 7, 8, 9) in panel, but scored as
‘correct’ here as ‘not B. pertussis’.

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

Table 4. Summary of responses to questionnaire

No. %
Laboratories
1. B. pertussis PCR used
a. routinely 16 76
b. outbreak/ special investigations only 4 19
2. PCR assay
a. in-house 19 90
b .commercial 8 38
c. both 6 29
3. Assay methodology and platform
a. Published method? For methods, see [7-21] 16 76
b. Real-time (qPCR) 13 62
c. Platform
TaqMan (ABI) 3 14
LightCycler (Roche) 10 48
LC 1.0 2
LC 2.0 7
LC 480 1
RotorGene 2 10
d. Conventional PCR
iCycler (BioRad) 1
Veriti (ABI) 1
9700 (ABI) 1
MasterCycler (Eppendorf) 4
Thermal Cycler Gradient PCR (TaKaRa) 1
Mx300SP (Stratagene) 1
e. Targets used for B. pertussis
Pertussis toxin promoter 11
Pertussis toxin 2
IS481 20
Pertactin 0
Other
f. Target(s) used for B. parapertussis
IS1001 15
g. Other Bordetella spp. targeted?
B.holmesii 8
B.bronchiseptica 3
B.petrii 1
pan-Bordetella 1
Other targets used?
Outer membrane porin 1
recA 7
h. Internal control (to check for inhibition) 14
i. Internal calibrator (positive control /standard curve) 13
4. Commercial kit 8
PneumoBacter (Seegene Inc,) 4
B.pertussis/para (TIB-MOLBIOL) 1
Bordetella pertussis R-gene (Argene) 1

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Bordetella parapertussis R-gene (Argene) 1

Bordetella R-gene (Argene) 1
Dia-Bor-020 (Diagenode) 1
5. Type of clinical specimens analysed
Respiratory 14
Pernasal swabs 10
Nasopharyngeal aspirates 14
Other
Naspharyngeal swabs 2
Bronchial alveolar lavage 2
Post-mortem samples (e.g. lung) 1
6. Approximate no. specimens processed per annum
of 18 laboratories providing figures Range 5 to 9000
(average, 866; median,
225)
7. Method of DNA extraction
Automated 7
Manual 15
Both 1
8. Participation in EQA scheme 14
QCMD 7
INSTAND 6 (+1 used to)
EUVAC.NET B.pertussis EQA (2011) 13
UK NEQAS (every 2 years) 1

Comments (see below) 6

Questionnaire comments on use of Bordetella PCR by

European laboratories
• To help establish DNA contamination procedures
• Specificity problems of IS481
• Other commercial kits also validated (see [22])
• Currently using IS481 as a single target for detection of B. pertussis. Well aware of the issue of false
positive reactions with B. holmesii; in the process of validating another target, the insertion element IS1001,
in order to distinguish between B. holmesii (which is positive for both insertion elements IS1001 and IS481)
from B. pertussis which is positive for IS481 but negative for IS1001
• Taxonomic genes 16S rDNA, and IS481 used for differentiation
• Just starting Bordetella PCR
• Participation in the EQA will be very useful for our laboratory

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

Table 5. Crossing thresholds for samples containing Bordetella DNA reported by submitting
laboratories
Sample Bordetella Laboratory number
number pertussis

6 7 8 13 15 (ptxA) 16 18 20 21
2 B. pertussis 21.96-23.30 24.26 18.4 20.52 27.34 15.21 22.92 22.17 20.36
±0.38 ±0.26 ±0.17
3 B. pertussis 32.58-34.87 34.1 28.1 30.54 38.00 24.33 33.27 31.73 29.82
5 B. pertussis 32.59-34.65 33.8 (ptxAP) 28.3 30.40 34.13 24.25 33.61 31.58 29.35
10 B. pertussis 40.63-43.12 35.2 >35 0 30.77 40.38 35.41
±0.39
8 B. parapertussis 29.44
4 B. holmesii 26.04 0 22.41
±0.32 ±0.13
7 B. bronchiseptica 30.12
Sample Bordetella spp. Laboratory number
number

6 7 8 13 15 (IS481) 16 18 20 21
2 B. pertussis 34.66-35.15 18.90 22.2
±0.08
3 B. pertussis 28.6±0.14 32.21
5 B. pertussis 28.6±0.13
10 B. pertussis 36.6±1.21 40.24 (IS481) 34.74
(IS481) (IS481)
8 B. parapertussis 23.7±0.15 24.43 0 (IS481) 20.73 29.99 25.26
(IS1001)
4 B. holmesii 27.79-28.86 25.2 (IS481) 25.97 28.03 30.56
29.1 (recA)
35.9 ±0.21
(IS1001)
7 B. bronchiseptica 25.7 (IS1001) 30.1 0 22.97 32.63 27.8

ptxP: pertussis toxin S1 promoter

ptxA, pertussis toxin S1 gene
recA, gene for protein RecA

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

5. Discussion
Sensitivity, specificity and reproducibility
All but one laboratory (21/22) detected B. pertussis at both the ‘high’ and ‘medium’ dilutions, the exception was a
laboratory using a block-based assay, just preparing a B. pertussis PCR service and participating in a B. pertussis
PCR EQA for the first time.
The real-time assays demonstrated greater sensitivity than the conventional PCR illustrated by the detection of B.
pertussis/Bordetella spp. DNA at the ‘low’ dilution by only three of seven laboratories using conventional PCR
versus 10 of 11 deploying qPCR.
The major issue for specificity was one of reporting. It is known that IS481 is not specific for B. pertussis and can
be found in B. holmesii and some strains of B. bronchiseptica, thus IS481 positive-only results should not be
reported as confirmation of B. pertussis. Similarly, the insertion element IS1001 can be found in both B.
parapertussis and B. bronchiseptica, and whilst clinical infection by B. bronchiseptica is rare, IS1001 positive-only
results should not be reported as confirmation of B. parapertussis. Commercial kits claiming to specifically detect B.
pertussis and B. parapertussis using these targets should also update their interpretation.

Differentiation between B. pertussis and other Bordetella

species associated with respiratory clinical infections,
namely B. parapertussis , B. holm esii and B . bronchiseptica
B. pertussis targets
All but two laboratories (20/22) used IS481 as a target for B. pertussis; twelve of these also use a single copy
target in addition, 11 use ptxA promoter region and one the ptxA gene. One laboratory used only ptxA gene (in-
house conventional PCR) but in combination with a commercial (conventional) kit.

B. parapertussis targets
Fifteen out of 22 laboratories used IS1001 as a target, 14 of which also used IS481 for B. pertussis.

Other Bordetella species targeted

Seven laboratories used the recA gene to specifically detect B. holmesii. Two laboratories used the fla gene
(conventional PCR) for detection of B. bronchiseptica.
One laboratory targeted pan-Bordetella, B. holmesii, B. bronchiseptica (unpublished data). However, this was one
of two laboratories that reported a Bordetella spp. result for the sample containing H. influenzae DNA.
Thus, several gene targets are required in order to successfully detect and differentiate between B. pertussis, B.
parapertussis, B. bronchiseptica and B. holmesii.

Evidence of good laboratory practice of the PCR technique

by checking for evidence of contamination
This was demonstrated by all laboratories evidenced by no false-positive reporting of the two ‘negative’ samples.

Controls
There is considerable debate about the use of controls in clinical PCRs [23]; an internal control is recommended to
avoid false-negative reporting. If one is not readily available a replicate sample tube can be ‘spiked’ with a known
low amount of positive control material and the signal compared with the ‘unspiked’ one.

Training needs
Many of the participants in this EQA have been providing PCR for B. pertussis for a number of years. However, a
number of laboratories were considering moving from conventional to real-time and/or introducing a PCR service.
In conjunction with ECDC and the EUpert-labnet training activities seven laboratories participated in a workshop on
pertussis real-time PCR (April 23–27, 2012, Turku, Finland).

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

The availability of a particular qPCR platform can dictate which of the existing assays may be used. However, the
main difference in the qPCR platforms is the use of two dual-labelled probes per target with the LightCycler 1.0 or
2.0 (Roche) compared with the single hybridisation probes per target typically used with the LightCycler 480
(Roche), TaqMan (ABI) and RotorGene (Qiagen).

Establishment of best practice in current assays, interpretation and

reporting
In conjunction with ECDC and the EUpert-labnet activities a document providing guidelines and protocols for the
use of real-time PCR in laboratory diagnosis of human infection with Bordetella pertussis or Bordetella
parapertussis has been produced.

Limitations
The strains included were clinical isolates and the whole genome sequence for these was not available.
Although RNase treatment should have allowed accurate DNA quantification using the Nanodrop at A260, greater
accuracy can be achieved using a Fluorimeter, e.g. Qubit (Qiagen).
The samples included in the panel were purified high molecular size genomic DNA and more realistic ‘simulated’
samples could in addition include, for example, human DNA.

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

6. Recommendations
Results which are PCR positive, ‘IS481 only’, should be considered as probable B. pertussis as this insertion
sequence element can also be found in B. holmesii and some strains of B. bronchiseptica. Reports to date indicate
that B. holmesii may be more likely as a potential confounder for B. pertussis in older age groups [20].
Results which are PCR positive, ‘IS1001 only’, should be considered as probable B. parapertussis as this insertion
sequence element can also be found in some strains of B. bronchiseptica. Although B. bronchiseptica infection is
rare in humans, in certain patient groups such as cystic fibrosis patients, it may be more likely.
An internal process control to check for the presence of PCR inhibitors is recommended to avoid false-negative
reporting.
Commercial kits for the detection of B. pertussis and/or B. parapertussis are available, some of which have been
assessed [22]. However, care should be used in the interpretation of these results, as indicated above.
Overall, the results of the 2012 EQA scheme are quite satisfactory and present an improvement compared to the
former edition. Nonetheless, the EQA exercises perform a crucial role and are extremely useful to participants and
to ECDC in order to improve and validate the data collected from the Member States. The support of the EUpert-
labnet network is valued by the participants as reflected in the high level of participation, provision and receipt of
training, and in the harmonisation of methods and protocols for diagnostics and characterisation of Bordetella spp.

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

References
1. He Q, Mertsola J, Soini H, Skurnik M, Ruuskanen O, Viljanen MK. Comparison of polymerase chain reaction with culture
and enzyme immunoassay for diagnosis of pertussis. J. Clin. Microbiol. 1993; 31: 642–645.
2. Muyldermans G, Soetens O, Antoine M, Bruisten S, Vincart B, Doucet-Populaire F, et al. External Quality Assessment for
Molecular Detection of Bordetella pertussis in European Laboratories. J. Clin. Microbiol. 2005; 43: 30–35.
3. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE Guidelines: Minimum Information for
Publication of Quantitative Real-time PCR Experiments. Clin. Chem. 2009; 55:611-622.
4. Bustin, S. A., Beaulieu J.-F., Huggett, J., Jaggi, R., Kibenge, F. S. B., Olsvik, P. A., Penning, L. C., Toegel S. MIQE précis:
Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments.
BMC Mol. Biol. 2010 Sep 21;11:74.

5. Reischl U, Burggraf S, Leppmeier B, Linde H-J, Lehn N. Rapid and specific detection of Bordetella pertussis in clinical
specimens by LightCycler PCR. In Rapid Cycle Real-Time PCR: Methods and Applications, 2001. pp. 313–321. Edited by S.
Meuer, C., Wittwer & K.-I. Nakagawara. Berlin: Springer.

6. Dalby T, Krogfelt K, Fry N, He Q. Bordetella PCR methods in Europe: a EUVAC.NET / ECDC supported study. 22nd
European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) 31 March–3 April 2012 [poster].
7. Van der Zee A, Agterberg C, Peeters M, Schellekens J, Mooi FR. Polymerase chain reaction assay for pertussis:
simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis. J. Clin Microbiol. 1993; 31:
2134-2140.
8. Immunization, Vaccine and Biologicals. Laboratory manual for the diagnosis of whooping cough caused by Bordetella
pertussis/Bordetella parapertussis. 2004. Geneva: WHO. WHO/IVB/04.14. Available at:
http://whqlibdoc.who.int/hq/2004/WHO_IVB_04.14_eng.pdf
9. André P, Caro V, Njamkepo E, Wendelboe AM, Van Rie A, Guiso N. Comparison of serological and real-time PCR assays to
diagnose Bordetella pertussis infection in 2007. J. Clin. Microbiol. 2008. 46:1672–1677.
10. Dragsted DM, Dohn B, Madsen J, Jensen JS. Comparison of culture and PCR for detection of Bordetella pertussis and
Bordetella parapertussis under routine laboratory conditions. J. Med. Microbiol. 2004; 53:749-754.

11. Herwegh S, Carnoy C, Wallet F, Loïez C, Courcol RJ. Development and use of an internal positive control for detection of
Bordetella pertussis by PCR. J. Clin. Microbiol. 2005;43:2462-2464.
12. Houard S, Hackel C, Herzog A, Bollen A. Specific identification of Bordetella pertussis by the polymerase chain reaction.
Res Microbiol. 1989;140:477-487.
13. Antila M, He Q, de Jong C, Aarts I, Verbakel H, Bruisten S, et al. Bordetella holmesii DNA is not detected in
nasopharyngeal swabs from Finnish and Dutch patients with suspected pertussis. J. Med. Microbiol. 2006;55:1043-1051.
14. Kösters K, Reischl U, Schmetz J, Riffelmann M, Wirsing von König CH. Real-time LightCycler PCR for detection and
discrimination of Bordetella pertussis and Bordetella parapertussis. J Clin Microbiol. 2002;40:1719-1722.
15. Grogan JA, Logan C, O'Leary J, Rush R, O'Sullivan N. Real-time PCR-based detection of Bordetella pertussis and
Bordetella parapertussis in an Irish paediatric population. J Med Microbiol. 2011;60:722-729.
16. Fry NK, Duncan J, Wagner K, Tzivra O, Doshi N, Litt DJ, et al. Role of PCR in the diagnosis of pertussis infection in infants:
5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007. J Med
Microbiol. 2009;58:1023-1029.
17. Fry NK, Tzivra O, Li YT, McNiff A, Doshi N, Maple PA, et al. Laboratory diagnosis of pertussis infections: the role of PCR
and serology. J Med Microbiol. 2004;53:519-525.

18. Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real-time PCR assay for detection and differentiation of Bordetella pertussis
and Bordetella parapertussis. APMIS. 2010;118:685-91.
19. Tatti KM, Wu K-H, Tondella ML, Cassiday PK, Cortese MM, Wilkins PP, Sanden GN. Development and Evaluation of Dual-
target Real-time Polymerase Chain Reaction Assays to Detect Bordetella Spp. Diag. Microbiol. Infect. Dis. 2008; 61: 264–272.
20. Njamkepo E, Bonacorsi S, Debruyne M, Gibaud SA, Guillot S, Guiso N. Significant finding of Bordetella holmesii DNA in
nasopharyngeal samples for French patients with suspected pertussis. J. Clin. Microbiol. 2011; 49: 4347-4348.
21. Qin X, Galanakis E, Martin ET, Englund JA. Multitarget PCR for diagnosis of pertussis and its clinical implications. J. Clin.
Microbiol. 2007;45:506-511.
22. Lanotte P, Plouzeau C, Burucoa C, Grélaud C, Guillot S, Guiso N, Garnier F. Evaluation of four commercial real-time PCR
assays for detection of Bordetella spp. in nasopharyngeal aspirates. J. Clin. Microbiol. 2011; 49: 3943-3946.
23. Hoorfar J, Cook N, Malorny B, Wagner M, De Medici D, Abdulmawjood A, Fach P. Making internal amplification control
mandatory for diagnostic PCR. J. Clin. Microbiol. 2003; 41:5835.

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Annex 1. Invitation letter to participants for

the Bordetella pertussis PCR external quality
assurance programme
Health Protection Agency
Respiratory and Systemic Infection Laboratory
HPA – RSIL, Colindale
61 Colindale Avenue
London NW9 5EQ
UK
Tel: +44 (0) 208 327 7330
Fax: +44 (0)208 205 6528
14 November 2011
Dear Colleagues,
Re: Invitation to participate in a Bordetella pertussis PCR External Quality Assurance program
You will have received this letter because we believe you are the officially designated ECDC laboratory expert for
pertussis in your country. If you are not the designated laboratory expert for pertussis please could you let us and
Zaibun Nisa [at ECDC] know, thank you.
Following the award of the Framework Contract No ECDC/2011/013 to a consortium of European pertussis experts
of nine countries (Denmark, Finland, France, Germany, Italy, Netherlands, Norway, Sweden and UK), we are
responsible for Workpackage 2: EQA scheme for the laboratory diagnostics and molecular typing of pertusiss.
Subject to the finalisation of subcontract arrangements with the coordinator of this project National Institute for
Health and Welfare THL, Finland, we intend to distribute a panel of genomic DNA extracts (ready for PCR) for
testing by PCR with the same procedure you would normally use in your laboratory for testing clinical samples for
Bordetella pertussis. The panel will also included DNA from at least one other Bordetella species and non-
Bordetella species.
We plan to send this B. pertussis PCR EQA panel in February 2012.
If you are interested in participating in this EQA please confirm your willingness to participate by emailing Norman
Fry (by November 30, 2011) and let us know the consignee’s details, full address, telephone and fax number and
e-mail address.
An EQA collaborative study to assess current laboratory performance of pertussis serology will be arranged in mid-
2012. Further details of this program will follow in due course
Should you need further information, do not hesitate to contact us.
Many thanks for your attention
With best regards

Norman K Fry, PhD HPA – RSIL, Colindale

Dorothy Xing, PhD HPA – NIBSC, Potters Bar
Kevin Markey, PhD HPA – NIBSC, Potters Bar

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

Annex 2. List of participants

Country Contact person Laboratory /Institution
Belgium Denis Pierard, Laboratory of Microbiology,
Oriane Soetens, UZ Brussel Hospital
Fedoua Echahidi
Bulgaria Stefan Panaiotov, Department of Microbiology, National Center of
Nadia Brankova Infectious and Parasitic Diseases
Czech Republic Jana Zavadilova National Reference Laboratory for Pertussis and
Diphtheria, National Institute of Public health
Denmark Jørgen Jensen Department of Microbiological Surveillance and
Tine Dalby, Research, Statens Serum Institut
Marianne Gam
Estonia Laura Kunder Health Board Laboratory for Communicable
Diseases
Finland Qiushui He Department of Infectious Disease Surveillance and
Kirsi Gröndahl-Yli-Hannuksela Control, National Institute for Health and Welfare
France Nicole Guiso Centre National de Reference de la Coqueluche et
Sophie Guillot autres bordetelloses,
Institut Pasteur de Paris
Germany Marion Riffelmann, Labor Medizin Krefeld MVZ, HELIOS Klinikum
Carl Heinz Wirsing von König, Krefeld
Nicole Kennerknecht
Greece Maria Giannaki-Psinaki Serology - Microbiology Department, “Aghia Sophia”
Athens Children’s Hospital
Hungary Ildiko Paluska Legionella - Bordetella Laboratory, Hungarian
National Center for Epidemiology
Ireland Juanita Grogan, Molecular Laboratory, Microbiology Department,
Catriona Logan Our Lady’s Children’s Hospital, Crumlin
Italy Paola Stefanelli Istituto Superiore di Sanità
Latvia Diana Dusacka Infectology Center of Latvia
Lithuania Algirdas Griškevicius Molecular Biology Testing Subdivision, National
Public Health Surveillance Laboratory
Luxembourg Paul Reichert Laboratoire national de sante
Frédéric Decruyenaere
Norway Hege Smith Tunsjø Laboratoriemedisin og Medisinsk Biokjemi (TLMB),
Akershus University Hospital
Poland Katarzyna Piekarska Department of Bacteriology, National Institute of
Public Health
Romania Vasilica Ungureanu, Bacterial Respiratory Infections Laboratory, National
Sorin Dinu, Institute of Research Development for Microbiology
Maria Damian
and Immunology
Spain Carmen Pelaz-Antolin Centro Nacional Microbiología, Instituto de Salud
Carlos III
Slovenia Metka Paragi, Department of Medical Microbiology
Tamara Kastrin
United Kingdom Norman Fry Respiratory and Systemic Infection Laboratory,
Health Protection Agency

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Annex 3. The EUpert-labnet Bordetella

pertussis PCR EQA questionnaire
1. Do you use PCR for the diagnosis of Bordetella infections?
a. Routinely Yes/No
b. Outbreaks/special investigations only Yes/No
2. If yes do you use an ‘in-house’ or a commercial assay or both?
a. In-house: Yes/No
b. Commercial: Yes/No
c. Both Yes/No
3. If you use an in-house assay:
a. Is the method you follow published? Yes/No
(If published please give reference):
b. Real-time? Yes/No
c. If real-time; which platform?
Taqman (TaqMan) Yes/No
Model
LightCycler (Roche) Yes/No
Model
RotorGene (Qiagen) Yes/No
Model
Other (please state):
d. If not real-time, which thermocycler block do you use?
Please state manufacturer and model:
e. Which target(s) do you use for Bordetella pertussis?
Pertussis toxin promoter Yes/No
Pertussis toxin Yes/No
IS481 Yes/No
Pertactin Yes/No
Other (please state):
f. Which target(s) do you use for Bordetella parapertussis?
IS1001 Yes/No
Other (please state):
g. Do you target any other Bordetella spp. apart from B.pertussis and B.parapertussis in your Yes/No
assays?
If so which Bordetella species and which targets do use you?:
h. Do you use Internal Controls to check for inhibition? Yes/No
(if yes please give details):
i. Do you use an internal calibrator (positive control/standard curve)? Yes/No
Please give details:

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TECHNICAL REPORT EQA scheme on PCR for Bordetella pertussis, 2012

4. Do you use a commercial kit Yes/No

If yes please give details of Kit (please state):
Manufacturer (please state):
Platform (please state):
5. What kind of clinical samples do you analyse?
Respiratory samples Yes/No
Pernasal swabs Yes/No
Nasopharyngeal aspirates Yes/No
Other (please state):
6. Approximately how many clinical samples do you process for Bordetella PCR per year?
7. What method of DNA extraction do you use?
Manual or automated?
Which kit?
Which manufacturer?
8. Do you regularly take part in an External Quality Assessment Program for Bordetella PCR? Yes/No
If yes which one?
(please give details):
Any other comments on the use of Bordetella PCR by European laboratories:

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EQA scheme on PCR for Bordetella pertussis, 2012 TECHNICAL REPORT

Annex 4. The EUpert-labnet Bordetella

pertussis PCR EQA result submission form
Qualitative Result Quantitative Result
Crossing Point Calculated Amount
Bordetella (Cq values) (pg or GU) or Comments (e.g. inhibition,
# B. pertussis
* spp. Concentration etc.)
(Pos/Neg/NT )
(Pos/Neg/NT*) B. pertussis Bordetella (pg/µl or GU/ml),
of Bordetella DNA**

10

*
Pos = Positive (detected); Neg = Negative (not detected); NT = Not Tested. Cq = crossing threshold (for real-time PCR)
**
Calculated from standard curve (real-time PCR).

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