What to do and what not to do in serological diagnosis of pertussis: recommendations from EU reference laboratories.

Abstract

Bordetella pertussis-specific antibodies can be detected by enzyme-linked immunosorbent assays (ELISAs) or multiplex immunoassays. Assays use purified or mixed antigens, and only pertussis toxin (PT) is specific for B. pertussis. The interpretation of results can be based on dual-sample or single-sample serology using one or two cut-offs. The EU Pertstrain group recommends that: (i) ELISAs and multiplex immunoassays should use purified non-detoxified PT as an antigen, that they should have a broad linear range and that they should express results quantitatively in International Units per millilitre (IU/ml); (ii) a single or dual diagnostic cut-off for single-serum serology using IgG-anti-PT between 50 and 120 IU/ml should be used, and diagnostic serology cannot be validly interpreted for one year after vaccination with acellular pertussis (aP) vaccines; (iii) IgA-anti-PT should only be used with indeterminate IgG-anti-PT levels or when a second sample cannot be obtained. This group discourages using: (i) other antigens in routine diagnostics, as they are not specific; (ii) micro-agglutination, due to its lack of sensitivity; (iii) immunoblots for pertussis serodiagnosis, as results cannot be quantified; (iv) other methods, such as complement fixation or indirect immunofluorescence, due to their low sensitivity and/or specificity.

Full text

Page 1

REVIEW
What to do and what not to do in serological diagnosis
of pertussis: recommendations from EU reference laboratories
N. Guiso & G. Berbers & N. K. Fry & Q. He &
M. Riffelmann & C. H. Wirsing von König &
EU Pertstrain group
Received: 26 August 2010 /Accepted: 18 October 2010 /Published online: 11 November 2010
# The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract Bordetella pertussis-specific antibodies can be
detected by enzyme-linked immunosorbent assays (ELISAs)
or multiplex immunoassays. Assays use purified or
mixed antigens, and only pertussis toxin (PT) is specific
for B. pertussis. The interpretation of results can be based
on dual-sample or single-sample serology using one or
two cut-offs. The EU Pertstrain group recommends that:
(i) ELISAs and multiplex immunoassays should use
purified non-detoxified PT as an antigen, that they should
have a broad linear range and that they should express
results quantitatively in International Units per millilitre
(IU/ml); (ii) a single or dual diagnostic cut-off for single-
serum serology using IgG-anti-PT between 50 and 120 IU/ml
should be used, and diagnostic serology cannot be validly
interpreted for one year after vaccination with acellular
pertussis (aP) vaccines; (iii) IgA-anti-PT should only be used
with indeterminate IgG-anti-PT levels or when a second
sample cannot be obtained. This group discourages using: (i)
other antigens in routine diagnostics, as they are not specific;
(ii) micro-agglutination, due to its lack of sensitivity; (iii)
immunoblots for pertussis serodiagnosis, as results cannot be
quantified;(iv)othermethods,suchascomplementfixationor
indirect immunofluorescence, due to their low sensitivity and/
or specificity.
Indications for pertussis diagnostics
The diagnosis ofpertussis shouldonlybe attemptedinpatients
with symptoms compatible with pertussis, such as prolonged
coughing with paroxysms and/or whooping or choking. In
infants, older vaccinated children, adolescents and adults, the
clinical course may not be typical, and prolonged coughing
may be the only symptom. In these cases, the diagnosis of
pertussis requires laboratory methods for confirmation.
Direct and indirect tests are available. Direct tests are
real-time polymerase chain reaction (PCR) and culture,
whereas indirect tests measure specific antibodies in oral
fluid or sera. Here, we focus on the detection of antibodies
against Bordetella pertussis antigens.
Development of the recommendations
The EU Pertstrain group consists of representatives of
the Bordetella reference laboratories in their respective
EU countries. In a face-to-face meeting at the Istituto
Superiore di Sanità (ISS), Rome, Italy, in 2009, the outline
of a manuscript was discussed and agreed upon. After a
search of relevant databases, a first draft was written (NG)
and intensively reviewed by members of the group. In a
second face-to-face meeting, at the Rijksinstituut voor
Members of the EU Pertstrain group are listed in the appendix.
N. Guiso
Institut Pasteur,
Paris, France
G. Berbers
National Institute for Public Health and the Environment,
Bilthoven, The Netherlands
N. K. Fry
Health Protection Agency Centre for Infections,
London, UK
Q. He
National Institute for Health and Welfare,
Turku, Finland
M. Riffelmann:C. H. Wirsing von König (*)
Institut für Hygiene und Labormedizin, HELIOS Klinikum
Krefeld,
Lutherplatz 40,
47805, Krefeld, Germany
e-mail: carlheinz.wirsingvonkoenig@helios-kliniken.de
Eur J Clin Microbiol Infect Dis (2011) 30:307–312
DOI 10.1007/s10096-010-1104-y

Page 2

Volksgezondheid en Milieu (RIVM), Bilthoven, the
Netherlands, in June 2010, the draft recommendations were
broadly discussed and agreed upon. A second version of the
recommendations was drafted (NG, CHWvK). This version
was then reviewed again by all authors.
Serological tests
Blood/serum
Most serological assays are validated to test serum; some
may also be validated to test heparinised plasma or
ethylenediaminetetraacetic acid (EDTA) plasma. Capillary
blood samples may be used if a sufficient volume cannot be
obtained otherwise. Serum or plasma must be separated as
soon as possible after blood sampling (24 h at room
temperature). If acute and convalescent serum samples
taken at least three weeks apart from one individual are
available, they should be tested together in one run. All
serum samples may be frozen (at −20°C) after the primary
assay and re-analysed later together with a possible second
sample.
ELISA in serum samples
Inpreparationfortheacellularvaccinestudiesinthe1990s,the
enzyme-linked immunosorbent assay (ELISA) methodology,
the type of antigens, as well as the reference sera have been
standardised, and they have been used in all acellular vaccine
trials, in sero-epidemiological studies in many countries and
for diagnostic purposes in various laboratories [1–5].
Antigens
ELISA is normally done with highly purified antigens. The
antigens most frequently used are pertussis toxin (PT) and
filamentous haemagglutinin (FHA), and to a lesser extent,
pertactin (PRN) and fimbriae (FIM). Sometimes, adenylate
cyclase-haemolysin toxin (ACT) is also used. These
antigens are used in their active, i.e. non-detoxified, form.
The storage conditions of the antigens as well as the
duration of storage can vary significantly according to the
manufacturers who provide the purified antigens. Anti-PT
antibodies are specific for B. pertussis, whereas anti-FHA,
anti-PRN, anti-FIM and anti-ACT are less specific due to
cross-reactivity with other microbial antigens (e.g. other
Bordetella species, Haemophilus species, Mycoplasma
pneumoniae, Escherichia coli). For this reason, in routine
diagnosis, only the measurement of anti-PT antibodies is
recommended.However,the measurementofotherantibodies
may be used for transmission, immunogenicity or vaccine
non-inferiority studies. Some ELISAs, especially commercial
kits, use a mixture of antigens or supernatants or sonicates of
whole B. pertussis cells. The use of kits with mixed antigens
is not recommended [6].
Reference sera
World Health Organization (WHO) references are available
from the National Institute for Biological Standards and
Control (NIBSC, Potters Bar, UK) (“WHO International
Standard (06/140)” and “WHO Reference Reagent (06/
142)”) for the measurement of human antibodies to B.
pertussis antigens, and, thus, quantitative results should be
reported in IU/ml [7].
Plates
For in-house ELISAs, differences between plates were
observed, and Thermo Scientific Nunc MaxiSorp (Nunc AS,
Copenhagen, Denmark) plates and Greiner MICROLON
High Binding (Greiner Bio-One, Frickenhausen, Germany)
plates seemed to work better than others [8].
Flow cytometry
Bead-based multiplex assays that simultaneously measure
antibodies to various antigens have also been applied to B.
pertussis antigens. The assays use fluorescent micropar-
ticles to which purified antigens are covalently coupled and
are based on a proprietary method patented in 1983 in
Germany [9] or on xMAP Technology from Luminex™
[10]. These assays seem to produce results comparable to
conventional ELISA systems.
Immunoblots, line and dot blots
Many commercial kits detect anti-B. pertussis and/or anti-PT,
anti-FHA, anti-PRN and also ACTantibodies in serums using
blotting techniques. Kits using whole-cell suspensions as
antigens are not specific for the detection/diagnosis of
pertussis infections due to the great cross-reactivity with
other bacteria (e.g. other Bordetella species, Haemophilus
species, Mycoplasma pneumoniae). Antigens used in these
kits have to be validated concerning their purity, as
contamination with other antigens or antigen aggregates can
also produce false-positive results. A quantitative interpreta-
tion of blot results is not possible, but a semi-quantitative
reporting may be possible. Although various CE-marked
commercial kits are available, a recent comparison showed
significant differences between kits and an insufficient
correlation to the values of the 1st International Reference
Preparation (Kennerkuecht et al. submitted). Consequently,
the use of immunoblots for pertussis serology is not
recommended by the EU Pertstrain group.
308Eur J Clin Microbiol Infect Dis (2011) 30:307–312

Page 3

Micro-agglutination
Micro-agglutination has been widely used before ELISA
systems became available [11]. As with other bacterial
agglutination assays, micro-agglutination with B. pertussis
cells mainly measures IgM antibodies to outer surface
antigens such as FIM, PRN and lipooligosaccharide (LOS).
Results are reported in titres starting at 1:20 or 1:40. No
agglutinin titre has been attributed to the WHO reference
preparation so far. Agglutinating antibodies can be used to
measure the exposure of a population to B. pertussis antigens;
on an individual level, the micro-agglutination method is not
useful for confirming the clinical diagnosis of pertussis.
CHO cell neutralisation
This test used the ability of PT to induce characteristic
morphological changes in a culture of Chinese hamster ovary
(CHO) cells.Thesechangescanbe neutralisedbythepresence
of anti-PT antibodies. The titres of CHO cell-neutralising
antibodies correlate well with the IgG-anti-PT ELISA values,
and, thus, CHO cell assays are rarely used for diagnostic
purposes[8, 12]. The interpretation of CHO assays cannot be
standardised, and it is difficult to interpret the morphological
changes and to score the results objectively.
Complement fixation
Complement fixation using whole cells of B. pertussis has
been rarely used and suffers from a lack of sensitivity and
specificity.
Indirect immunofluorescence
IndirectimmunofluorescenceusingwholecellsofB. pertussis
has the same disadvantages as complement fixation
Oral fluid
Oral fluid can be sampled by standardised means, e.g.
Oracol swabs (Malvern Medical Developments) or OraSure
oral specimen collection devices (OraSure Technologies
Inc.). The diagnostic sensitivity of measuring antibodies in
oral fluid samples is lower (∼80%) as compared to serum
samples, but the samples can easily be obtained and offer
an adjunct to diagnostic serology
ELISA in oral fluid
An IgG-capture ELISA capable of detecting anti-PT IgG in
oral fluid has been developed. The assay was evaluated by
comparison to a serum ELISA. The results showed that the
oralfluidassaydetectedseropositivesubjectswithasensitivity
of 79.7% [95% confidence interval (CI) 68.3–88.4] and a
specificityof96.6% (95%CI91.5–99.1)relative totheELISA
[13]. This assay has since been modified to include a
monoclonal capture antibody; however, no commercial
assays for measuring antibodies in oral fluid are available.
Reporting of serological results using ELISA or flow
cytometry
Concentrations of antibodies to B. pertussis antigens should
be quantitatively expressed in IU/ml as reference preparations
are available [7]. The reference preparation defines values for
antibodies of isotypes IgG and IgA to PT, FHA, PRN and to
FIM. Assigned values of the reference preparation are
described in Table 1. The numerical values of IU/ml are
equivalent to the previously used ELISA units/ml (EU/ml)
derived from the human reference preparations lot 3, lot 4
and lot 5 from the Center for Biologics Evaluation and
Research/Food and Drug Administration (CBER/FDA),
Bethesda, MD, USA.
Interpretation of serological results
PTandFHAarecontainedinsubstantialamountsinallacellular
vaccines licensed in Europe (except in Denmark),andPRN and
FIM are also components of licensed acellular pertussis
vaccines. Thus, the immune response against infection or
vaccination cannot be distinguished and pertussis vaccination
may interfere with the interpretation of serological results. Due
to a continuous circulation of Bordetellae in the population,
IgG-anti-PT and other antibodies to Bordetella antigens are
detectable in the majority of all adolescent and adult
populations tested so far [14]. Thus, serological diagnosis of
pertussis must be performed in immunological non-naïve
populations with different kinetics of antibody production.
Additionally, pertussis serology suffers from various other
drawbacks:
–
Commercial ELISAs are of different antigen composition
and quality
Referenceantigenswithgoodpurityarenoteasilyavailable
Population-based cut-offs for single-sample serology may
needverificationafterchangesinthevaccinationschedule
Interpretation of anti-PT concentrations in recently
vaccinated persons is difficult
–
–
–
Dual-sample serology based on ≥100% increase in
antibody concentration or on ≥50% decrease in antibody
concentration is a sensitive and specific method for
serological diagnosis [1, 4]. However, even in paired sera,
no antibody increase may be seen after infection due to the
secondary immune response, and the diagnosis may also be
Eur J Clin Microbiol Infect Dis (2011) 30:307–312 309

Page 4

based on a decrease of antibodies, which may be too slow
to reach 50% between the acute and convalescent sample.
In clinical practice, diagnosis is mostly based on single-
sample serology using a single or a more continuous cut-
off. For single-sample serology, various cut-off values for
IgG-anti-PT have been proposed (Table 2). In Massachu-
setts, 100 EU/ml was used, and, later, in order to increase
specificity, >∼200 EU/ml IgG-anti-PT (>20 μg/ml) was
employed. In the Netherlands, a cut-off >∼125 EU/ml IgG-
anti-PT with higher specificity or 62 EU/ml with slightly
lower specificity has been used [15, 16]. In Germany, a cut-
off of ∼40 EU/ml IgG-anti-PT was suggested, together with
∼50 EU/ml IgA-anti-FHA. A sero-epidemiological survey
was recently performed in various countries in Western
Europe using a cut-off of 125 EU/ml for recent infection,
and it can be used as a population-based reference [14].
Another recently completed sero-surveillance in US sera
found that three populations could be separated according
to their levels of IgG-anti-PT: one cut-off was estimated at
94 EU/ml and a lower cut-off was estimated at 48 EU/ml
[17]. Using the lower 50 EU/ml cut-off may be especially
useful in outbreak situations, and, overall, this cut-off of 50
EU/ml seems to be more appropriate for diagnosis rather
than a higher cut-off with lower sensitivity [18].
Overall, it is astonishing that, irrespective of various
vaccination strategies, cut-offs for single-sample serology
are comparable throughout the countries in which they were
evaluated.
A comparison of receiver operating characteristic (ROC)
curve analyses with data from Denmark, the Netherlands
and the UK showed that, for all three countries, the single
cut-off with optimal sensitivity and specificity may be in
the range between 60 and 75 IU/ml.
It may be sensible to use a dual cut-off between 62 and
125 IU/ml according to countries as a proof of a recent
infection with B. pertussis, provided that the patient was not
vaccinated during the last 12 months (Table 2). If diagnosis
cannot be established with certainty from a single serum, but
is deemed to be necessary according to the clinical symptoms,
antibodies should be measured in a second (convalescent)
serum sample at two to four weeks interval. In case of non-
availability of a second serum sample, measurement of IgA
anti-PT antibodies can be an alternative, but no broadly
accepted cut-off is available for this antibody subclass.
Considering its relatively high specificity and low sensitivity,
a cut-off near the minimal level of quantification, which may
be between 10 and 20 IU/ml, may seem reasonable [19].
Recommendations for serological testing with suspected
pertussis
In neonates and young infants, PCR and/or culture should
be performed on nasopharyngeal samples, nasopharyngeal
swabs (NPSs) or nasopharyngeal aspirates (NPAs) as soon
as possible post-onset of symptoms. The measurement of
Table 1 Reference preparations from the World Health Organization
(WHO) and the Center for Biologics Evaluation and Research/Food
and Drug Administration (CBER/FDA). Values are given in IU/ml for
the WHO preparations and in ELISA units (EU)/ml for the CBER/
FDA preparations; IU/ml and EU/ml are numerically identical [7]
IgG-anti-PT IgA-anti-PTIgG-anti-FHA IgA-anti-FHA IgG-anti-PRN IgA-anti-PRN
WHO international standard (06/140)
WHO reference reagent (06/142)
CBER/FDA #3
CBER/FDA #5
CBER/FDA #4
335 IU/ml
106 IU/ml
200 EU/ml
ND
ND
65 IU/ml
18 IU/ml
15 EU/ml
140 EU/ml
ND
130 IU/ml
122 IU/ml
200 EU/ml
ND
ND
65 IU/ml
86 IU/ml
100 EU/ml
280 EU/ml
ND
65 IU/ml
39 IU/ml
ND
ND
90 EU/ml
42 IU/ml
38 IU/ml
ND
90 EU/ml
25 EU/ml
ND, not declared
CountryType of study Cut-off SensitivitySpecificityReference
MA, USA
MA, USA
NL
Population study
Population study
Population study
∼100 IU/ml
∼200 IU/ml
125 IU/ml
62 IU/ml
40 IU/ml
125 IU/ml
94 IU/ml
46 IU/ml
50 IU/ml
78%
67%
70%
80%
80%
n.a.
n.a.
n.a.
Better than 100 IU/ml
98%
99.9%
99%
95%
95%
n.a.
n.a.
[20]
[21]
[15]
D
EU
USA
Population study
Epidemiological survey
Epidemiological survey, model
[22]
[14]
[17]
AUS Clinical validation[18]
Table 2 Suggested cut-off
values for IgG-anti-PT for ado-
lescents and adults. Adapted from
WHO “The Immunological Basis
for Immunization Series”
(available online at: http://
whqlibdoc.who.int/publications/
2010/9789241599337_eng.pdf)
MA, Massachusetts
n.a., not applicable
310Eur J Clin Microbiol Infect Dis (2011) 30:307–312

Page 5

IgG-anti-PT is only meaningful for older children/adults,
including parents and other household members. In
vaccinated children, adolescents and adults with less than
two weeks of coughing culture and PCR from nasopharyn-
geal samples should be done. For adolescents and adults
with coughing of less than three weeks, PCR and the
measurement of IgG-anti-PT should be performed [1]. If
coughing lasted at least 2 to 3 weeks, the measurement of
IgG-anti-PT should be sufficient. In outbreak situations,
PCR and culture should be performed from nasopharyngeal
samples and IgG-anti-PT should be measured in serum
samples.
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
Appendix
Table 3 shows the members of the EU Pertstrain group.
References
1. André P, Caro V, Njamkepo E, Wendelboe AM, Van Rie A, Guiso
N (2008) Comparison of serological and real-time PCR assays to
diagnose Bordetella pertussis infection in 2007. J Clin Microbiol
46(5):1672–1677
2. Guiso N (2007) Laboratory manual for the diagnosis of whooping
cough caused by Bordetella pertussis/Bordetella parapertussis.
World Health Organization (WHO). Available online at: http://
www.who.int/vaccines-documents/DocsPDF04/www788.pdf
3. Meade BD, Deforest A, Edwards KM, Romani TA, Lynn F, O’Brien
CH, Swartz CB, Reed GF, Deloria MA (1995) Description and
evaluation of serologic assays used in a multicenter trial of acellular
pertussis vaccines. Pediatrics 96(3 Pt 2):570–575; Erratum in
Pediatrics 1995 Dec;96(6):following 1199
4. Simondon F, Iteman I, Preziosi MP, Yam A, Guiso N (1998)
Evaluation of an immunoglobulin G enzyme-linked immunosorbent
Table 3 List of participants of the EU Pertstrain network
Members
Qiushui He
and Jussi
Mertsola
Carl Heinz
Wirsing von
König and
Marion
Riffelmann
Nicole Guiso and
Sophie Guillot
Hans Hallander
and Abdolreza
Advani
Frits R Mooi and
Guy Berbers
Email address
qiushui.he@utu.fi; Jussi.Mertsola@utu.fi
Name of organisation
National Institute for
Health and Welfare,
University of Turku
HELIOS Klinikum
Krefeld
Town, country
Turku, Finland
carlheinz.wirsingvonkoenig@helios-kliniken.de; marion.
riffelmann@helios-kliniken.de
Krefeld, Germany
nicole.guiso@pasteur.fr; sophie.guillot @pasteur.frInstitut PasteurParis, France
hans.hallander@smi.se; reza.advani@smi.se Swedish Institute for
Infectious Disease
Control
National Institute of
Public Health and
the Environment
National Institute of
Hygiene
Statens Serum Institut
Solna, Sweden
frits.mooi@rivm.nl; guy.berbers@rivm.nl
Bilthoven, the
Netherlands
Anna Lutyńskaagzyl@pzh.gov.pl Warsaw, Poland
Karen Krogfelt
and Tine Dalby
Norman Fry
kak@ssi.dk; tid@ssi.dk Copenhagen, Denmark
norman.fry@hpa.org.ukHealth Protection
Agency
National Institute for
Biological Standards
and Control
Institut Pasteur de Lille Lille, France
London, UK
Dorothy Xingdxing@nibsc.ac.uk
Potters Bar, UK
Camille Locht
and David Hot
Clara Ausiello
camille.locht@pasteur-lille.fr; david.hot@pasteur-lille.fr
ausiello@iss.it Istituto Superiore di
Sanità
Norwegian Institute
of Public Health
Rome, Italy
Per Sandven and
Jann Storsaeter
per.sandven@fhi.no; jann.storsaeter@fhi.noOslo, Norway
Eur J Clin Microbiol Infect Dis (2011) 30:307–312311

Page 6

assay for pertussis toxin and filamentous hemagglutinin in diagnosis
of pertussis in Senegal. Clin Diagn Lab Immunol 5(2):130–134
5. Tondella ML, Carlone GM, Messonnier N, Quinn CP, Meade BD,
Burns DL, Cherry JD, Guiso N, Hewlett EL, Edwards KM, Xing
D, Giammanco A, Wirsing von König CH, Han L, Hueston L,
Robbins JB, Powell M, Mink CM, Poolman JT, Hildreth SW,
Lynn F, Morris A (2009) International Bordetella pertussis assay
standardization and harmonization meeting report. Centers for
Disease Control and Prevention, Atlanta, Georgia, United States,
19–20 July 2007. Vaccine 27(6):803-814
6. Riffelmann M, Thiel K, Schmetz J, Wirsing von Koenig CH
(2010) Performace of commercial enzyme-linked immunosorbent
assays for the detection of antibodies to Bordetella pertussis. J
Clin Microbiol, Oct. 13
7. Xing D, Wirsing von König CH, Newland P, Riffelmann M, Meade
BD, Corbel M, Gaines-Das R (2009) Characterization of reference
materials for human antiserum to pertussis antigens by an interna-
tional collaborative study. Clin Vaccine Immunol 16(3):303–311
8. Dalby T, Sorensen C, Pedersen JW, Krogfelt KA (2010) Pertussis
serology: assessment of a quantitative IgG-anti-PT ELISA for
replacement of the CHO cell assay. APMIS (in press)
9. Reder S, Riffelmann M, Becker C, Wirsing von König CH (2008)
Measuring immunoglobulin G antibodies to tetanus toxin,
diphtheria toxin, and pertussis toxin with single-antigen enzyme-
linked immunosorbent assays and a bead-based multiplex assay.
Clin Vaccine Immunol 15(5):744–749
10. van Gageldonk PGM, van Schaijk FG, van der Klis FR, Berbers GAM
(2008)Developmentandvalidationofamultipleximmunoassayforthe
simultaneousdeterminationofserumantibodiestoBordetella pertussis,
diphtheria and tetanus. J Immunol Methods 335(1–2):79–89
11. Blumberg DA, Pineda E, Cherry JD, Caruso A, Scott JV (1992)
The agglutinin response to whole-cell and acellular pertussis
vaccines is Bordetella pertussis-strain dependent. Am J Dis Child
146(10):1148–1150
12. Granström G, Wretlind B, Salenstedt C-R, Granström M (1988)
Evaluation of serologic assays for diagnosis of whooping cough. J
Clin Microbiol 26(9):1818–1823
13. Litt DJ, Samuel D, Duncan J, Harnden A, George RC, Harrison TG
(2006) Detection of anti-pertussis toxin IgG in oral fluids for use in
diagnosis and surveillance of Bordetella pertussis infection in
children and young adults. J Med Microbiol 55(Pt 9):1223–1228
14. Pebody RG, Gay NJ, Giammanco A, Baron S, Schellekens J,
Tischer A, Olander RM, Andrews NJ, Edmunds WJ, Lecoeur H,
Lévy-Bruhl D, Maple PA, de Melker H, Nardone A, Rota MC,
Salmaso S, Conyn-van Spaendonck MA, Swidsinski S, Miller E
(2005) The seroepidemiology of Bordetella pertussis infection in
Western Europe. Epidemiol Infect 133(1):159–171
15. de Melker HE, Versteegh FGA, Conyn-van Spaendonck MAE,
Elvers LH, Berbers GAM, van der Zee A, Schellekens JFP (2000)
Specificity and sensitivity of high levels of immunoglobulin G
antibodies against pertussis toxin in a single serum sample for
diagnosis of infection with Bordetella pertussis. J Clin Microbiol
38(2):800–806
16. Versteegh FG, Mertens PL, de Melker HE, Roord JJ, Schellekens
JF, Teunis PF (2005) Age-specific long-term course of IgG
antibodies to pertussis toxin after symptomatic infection with
Bordetella pertussis. Epidemiol Infect 133(4):737–748
17. Baughman AL, Bisgard KM, Edwards KM, Guris D, Decker MD,
Holland K, Meade BD, Lynn F (2004) Establishment of
diagnostic cutoff points for levels of serum antibodies to pertussis
toxin, filamentous hemagglutinin, and fimbriae in adolescents
and adults in the United States. Clin Diagn Lab Immunol 11(6):
1045–1053
18. Horby P, Macintyre CR, McIntyre PB, Gilbert GL, Staff M,
Hanlon M, Heron LG, Cagney M, Bennett C (2005) A boarding
school outbreak of pertussis in adolescents: value of laboratory
diagnostic methods. Epidemiol Infect 133(2):229–236
19. Ward JI, Cherry JD, Chang SJ, Partridge S, Keitel W, Edwards K,
Lee M, Treanor J, Greenberg DP, Barenkamp S, Bernstein DI,
Edelman R; APERT Study Group (2006) Bordetella pertussis
infections in vaccinated and unvaccinated adolescents and adults,
as assessed in a national prospective randomized Acellular
Pertussis Vaccine Trial (APERT). Clin Infect Dis 43(2):151–157,
Epub 2006 Jun 5
20. Marchant CD, Loughlin AM, Lett SM, Todd CW, Wetterlow LH,
Bicchieri R, Higham S, Etkind P, Silva E, Siber GR (1994) Pertussis
in Massachusetts, 1981–1991: Incidence, serologic diagnosis, and
vaccine effectiveness. J Infect Dis 169(6):1297–1305
21. Yih WK, Lett SM, des Vignes FN, Garrison KM, Sipe PL,
Marchant CD (2000) The increasing incidence of pertussis in
Massachusetts adolescents and adults, 1989–1998. J Infect Dis
182(5):1409–1416
22. Wirsing von König CH, Gounis D, Laukamp S, Bogaerts H,
Schmitt HJ (1999) Evaluation of a single-sample serological
technique for diagnosing pertussis in unvaccinated children. Eur J
Clin Microbiol Infect Dis 18(5):341–345
312 Eur J Clin Microbiol Infect Dis (2011) 30:307–312