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Diagnostic Microbiology and Infectious Disease xxx (2013) xxx–xxx
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Diagnostic Microbiology and Infectious Disease
journal homepage: www.elsevier.com/locate/diagmicrobio
Whooping cough in South-East Romania: a 1-year study
Sorin Dinu a,⁎, Sophie Guillot b, c, Cristiana Cerasella Dragomirescu d, e, Delphine Brun b, c, Ştefan Lazăr f,
Geta Vancea f, Biatrice Mariana Ionescu g, Mariana Felicia Gherman g, Andreea-Florina-Dana Bjerkestrand g,
Vasilica Ungureanu d, Nicole Guiso b, c, Maria Damian a
a
Molecular Epidemiology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independenţei, 050096 Bucharest, Romania
Institut Pasteur, Molecular Prevention and Therapy of Human Diseases, 25-28 rue du Dr Roux, 75015 Paris, France
URA-CNRS3012, 25-28 rue du Dr Roux, 75015 Paris, France
d
Respiratory Bacterial Infections Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independenţei,
050096 Bucharest, Romania
e
“Carol Davila” University of Medicine and Pharmacy, Department of Microbiology, 103 Splaiul Independenţei, 050096 Bucharest, Romania
f
“Dr Victor Babeş” Clinical Hospital for Infectious and Tropical Diseases, 281 Şos. Mihai Bravu, 030303 Bucharest, Romania
g
General practitioner, 15 Blv. Timişoara, 061303 Bucharest, Romania
b
c
a r t i c l e
i n f o
Article history:
Received 3 June 2013
Received in revised form 29 August 2013
Accepted 1 September 2013
Available online xxxx
Keywords:
Whooping cough
B. holmesii
Pertactin negative isolate
a b s t r a c t
The incidence of whooping cough in Romania is substantially underestimated, and, as noted by the health
authorities, this is mostly due to the lack of both awareness and biological diagnosis. We conducted a 1-year
study in Bucharest in order to assess the circulation of Bordetella pertussis, the main etiological agent of
whooping cough. Fifty-one subjects suspected of whooping cough were enrolled. Culture, real-time PCR, and
enzyme-linked immunosorbent assay were used for laboratory diagnosis. Whooping cough patients (63%)
were distributed among all age groups, and most were unvaccinated, incompletely vaccinated, or had been
vaccinated more than 5 years previously. Bordetella holmesii DNA was detected in 22% of the bordetellosis
cases; these patients included adults; teenagers; and, surprisingly, young children. B. pertussis isolates were
similar to the clinical isolates currently circulating elsewhere in Europe. One isolate does not express
pertactin, an antigen included in some acellular pertussis vaccines.
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
Bordetella pertussis and Bordetella parapertussis are the etiological
agents of whooping cough, a highly contagious human respiratory
disease. After the introduction of mass immunization in infants in the
1950s, with whole-cell pertussis vaccine (wP), the incidence of
whooping cough decreased substantially in the child population
(Edwards and Decker, 2008; Mattoo and Cherry, 2005; Zepp et al.,
2011).
Mass immunization against pertussis was introduced in Romania
in 1961 using a local diphtheria-tetanus–whole cell pertussis vaccine
(DTwP). The primary immunization schedule included 3 vaccine
doses (at 2, 4, and 6 months of age) followed by 2 boosters, at 12 and
30–35 months of age (Lutsar et al., 2009). In 2008, DTwP was replaced
by acellular vaccines and 2 other changes in the vaccination program
followed in 2010 and 2012. According to the current national program
(http://www.ms.gov.ro/?pag=133), the children are vaccinated with
acellular vaccine when 2, 4, 6, and 12 months old and 1 booster is
administered at 6 years of age. Official reports indicate that the
⁎ Corresponding author. Tel.: +40-21-3069276; fax: +40-21-3069307.
E-mail addresses: [email protected], [email protected] (S. Dinu).
vaccination coverage is under the target of 95% with a significant
decrease (88.2%) registered for the cohort of July 2011 (Popovici,
2013a). The data collected by the Romanian Ministry of Health for the
last 14 years describe a median incidence of whooping cough of
around 0.4 per 100,000 inhabitants. Peak values in pertussis incidence
were recorded in 2000, 2004, and 2008. In 2012, the year we
conducted this study, the estimated incidence of whooping cough was
the same as the median value for the last 14 years (Popovici, 2013b).
As Romanian health authorities state, pertussis incidence values have
been greatly underestimated. The relevant epidemiological analyses
were biased due to under-reporting of cases, assessing only the
clinical diagnosis, disregarding adults as potential whooping cough
patients, and the use of laboratory diagnosis methods with poor
sensitivity and specificity.
To improve the description of the circulation of B. pertussis in
Romania, we performed a 1-year pilot surveillance study involving
the “Dr Victor Babeş” Clinical Hospital for Infectious and Tropical
Diseases in Bucharest, 3 general practitioners, the “Cantacuzino”
National Institute of Research-Development for Microbiology and
Immunology (National Reference Centre for Pertussis, and Molecular
Epidemiology Laboratory), and Institut Pasteur in Paris. The project
also aimed to improve biological diagnosis performed by the
Romanian National Reference Centre.
0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.diagmicrobio.2013.09.017
Please cite this article as: Dinu S, et al, Whooping cough in South-East Romania: a 1-year study, Diagn Microbiol Infect Dis (2013), http://
dx.doi.org/10.1016/j.diagmicrobio.2013.09.017
2
S. Dinu et al. / Diagnostic Microbiology and Infectious Disease xxx (2013) xxx–xxx
2. Materials and methods
2.1. Patients and samples
Suspected patients were addressing to “Dr Victor Babeş” Clinical
Hospital for Infectious and Tropical Diseases or to a general
practitioner facility both in Bucharest.
The criteria for enrolment were cough lasting for more than 1
week and at least 1 of the following symptoms: paroxysmal cough,
nocturnal cough, post-tussive emesis, fever, apnea, or facial cyanosis.
A single asymptomatic individual was enrolled because she was a
contact of a possible whooping cough case. Clinical data were
collected from the questionnaire that accompanied the specimens.
Clinical variables recorded included gender and age, the clinical
symptoms listed above, contact with a laboratory-confirmed case,
administration of antibiotics prior to sampling, and immunization
history. Nasopharyngeal swabs (NPS) and sera were collected from
suspected whooping cough patients. Physicians were trained to
perform NPS sampling correctly (http://youtu.be/d6d-y7SX_dY),
and recommended collectors were used (Copan, 482CE; Brescia,
Italy).
The patients/legal guardians were informed about the study; they
signed a consent form, and the study was carried out in an anonymous
way.
2.2. Culture
NPSs were plated on Bordet Gengou agar (Difco, Le Pont-de-Claix,
France) supplemented with 1% glycerol (Calbiochem, Darmstadt,
Germany) and 15% sheep blood and incubated at 37 °C. All the
samples were tested in duplicate on media containing and not
containing 1% cephalexin. The plates were visually inspected every
day for 1 week.
identified using primers and probes targeting the ptxA-Pr as
previously described (Njamkepo et al., 2011). Bordetella holmesii
DNA was detected using primers and probes targeting the H-IS1001–
like insertion sequence (Tatti et al., 2011). The limits of detection by
real-time PCR in our conditions were 30 CFU for the in-house ptxA-Pr–
based PCR and 1 CFU for the in-house H-IS1001–based PCR (Njamkepo
et al., 2011).
2.5. Serology: enzyme-linked immunosorbent assay (ELISA)
Anti-pertussis toxin IgG antibodies were detected and quantified
using the in-house reference ELISA method (Simondon et al., 1998).
The purified PT was kindly provided by Sanofi Pasteur, and the
reference serum was purchased from NIBSC as recommended (Guiso
et al., 2011). The criteria used to confirm the disease were those
proposed previously (Riffelmann et al., 2010). Briefly, a value of antiPT IgG below 40 IU/mL indicates no recent contact with B. pertussis
strains. A second serum sample collected after an interval of 3 weeks
is required if the value is between 40 and 100 IU/mL. A value greater
than 100 IU/mL confirms a recent infection, unless the individual has
been vaccinated during the previous year.
We also purchased and used a new commercial kit (cat. no. 123101, SeroPertussis Toxin IgG; Savyon® Diagnostics Ltd., Ashdod, Israel)
designed to detect anti-PT IgG antibodies. The objective was to
compare this kit with the in-house reference ELISA in terms of
specificity, sensitivity, and rapidity; the performance of this kit had
not been independently and rigorously tested. This ELISA was
performed manually according to the instructions given in the
package insert. Three different investigators tested 15 sera in
duplicate (6 positive sera with values between 52 and N400 IU/mL
and 9 negative sera) and 2 batches of the kit. Intra-assay agreement
was good, and the differences never exceeded 17%. Sixty-five sera
from patients confirmed to have B. pertussis infections by culture or
PCR or non-infected were tested.
2.3. Characterization of the isolates
3. Results
Isolates were identified on the basis of colony aspect, morphological characteristics, and biochemical properties. Susceptibility to
macrolides (azitromycin, clarithromycin, erythromycin) and sulfamethoxazole/trimethoprim was tested by a disc diffusion method.
DNA pulsed-field gel electrophoresis (PFGE) fingerprinting was
performed as previously described (Caro et al., 2005).
DNA was extracted from the isolates with DNeasy Blood and Tissue
Kits (Qiagen, Hilden, Germany). The repeated regions I and II of the
prn gene encoding pertactin (PRN), the region encoding the S1
subunit of pertussis toxin (PT), and the ptxA promoter (ptxA-Pr) were
used for genotyping (Hegerle et al., 2012). The PRN open reading
frame was amplified and sequenced using primers described
previously (Hegerle et al., 2012).
The production of each PT, adenylate cyclase-hemolysin (AC-Hly),
the other major toxin produced by B. pertussis, PRN, and filamentous
hemagglutinin (FHA) were assessed by Western blotting with specific
murine polyclonal sera (Weber et al., 2001). Monoclonal antibodies
were used to detect fimbria 2 (FIM2) and fimbria 3 (FIM3) proteins
(Guiso et al., 2001).
2.4. Real-time PCR
DNA was extracted from 300 μL aliquots of Amies transport
medium provided with the NPS collector, after collection of samples,
using High Pure PCR Template Kits (Roche, Mannheim, Germany)
following the instructions supplied with the kit.
The presence of Bordetella strains harboring IS481 and IS1001 was
assessed by real-time PCR using Argene kits (catalog no. 69-0011B
and 71-012; Argene, Verniolle, France) according to the manufacturer’s instructions. B. pertussis DNA was then retrospectively
3.1. Patients and samples
Fifty-one patients were enrolled in the study between February
2012 and January 2013. Forty-six individuals were enrolled by the
hospital, and 5 were recruited by the general practitioners. Most of the
individuals were living in Bucharest (29), and the others lived in
counties in the South-East of Romania: Călăraşi (8), Ilfov (6), Buzău
(5), Constanţa (1), Dâmboviţa (1), and Ialomiţa (1). NPSs were
collected from all the 51 patients enrolled. Blood was obtained from
35 patients at the same time as NPS sampling. Seventeen of the 35
individuals returned after approximately 21 days to provide a second
blood sample.
3.1.1. Diagnosis of B. pertussis infections
B. pertussis infection was confirmed in 32 (63%) of the 51 patients
by culture, specific real-time PCR, and testing for anti-PT antibodies.
Four B. pertussis infections were diagnosed by culture, specific realtime PCR, and IgG anti-PT detection; 6, by specific real-time PCR and
IgG anti-PT detection; 4, by specific real-time PCR only; and 18, by
detection of anti-PT antibodies only (12/18 cases based on singlesample serology).
The age of the patients ranged from 3 months to 75 years old
(Table 1). All the patients, except a contact of a possible whooping
cough case, were symptomatic. Paroxysmal cough was reported for
94% of the patients, nocturnal cough for 81%, post-tussive emesis for
53%, cyanosis for 25%, apnea for 25%, and fever for 12.5%. All the
symptoms, except fever, were less frequent among non-confirmed
cases: paroxysmal cough (84%), nocturnal cough (58%), post-tussive
emesis (42%), apnea (11%), and fever (16%).
Please cite this article as: Dinu S, et al, Whooping cough in South-East Romania: a 1-year study, Diagn Microbiol Infect Dis (2013), http://
dx.doi.org/10.1016/j.diagmicrobio.2013.09.017
S. Dinu et al. / Diagnostic Microbiology and Infectious Disease xxx (2013) xxx–xxx
Table 1
Characteristics of the patients enrolled in this study and their clinical symptoms.
Patients without
Patients with
B. pertussis infection B. pertussis infection
(n = 19)
(n = 32)
Age and
gender
Minimum age
Maximum age
Median age
Mean age
Females
Males
Clinical
Patients coughing
symptoms more than 1 week
Patients with
paroxysmal cough
Patients with
nocturnal cough
Patients with posttussive vomit
Patients with facial
cyanosis
Patients with apnea
Patients with fever
None of the above
symptoms
Antimicrobial Patients receiving
treatment
macrolides
Patients receiving
other antibiotics
Vaccination
Vaccinated children
status
Children
incompletely
vaccinated due to the
age
Children
incompletely
vaccinated due to
other reasons
Unvaccinated
children
Teenagers vaccinated
in childhood
Adults vaccinated in
childhood
Patients with no
vaccination data
3 mo
75 y
6y
13 y, 10 mo
20
12
31
3 mo
43 y
3 y, 6 mo
7 y, 1 mo
9
10
19
30
16
26
11
17
8
8
2
8
4
1
3
3
0
2
0
0
4
4
6
2
7
1
2
8
5
2
0
1
2
9
1
3
and the absence of serum samples prevented species-level
identification.
B. holmesii DNA was detected in 8 NPSs: 1 sample was positive only
for B. holmesii DNA; DNA specific for B. holmesii and B. pertussis was
detected in 6 samples; and 1 sample was positive for B. holmesii, B.
pertussis, and IS1001.
3.1.3. Characterization of the isolates
Clinical isolates were recovered from a child vaccinated on time for
his age (Table 2), a 10-year-old child whose last vaccination was at
30–35 months of age, an adult who was vaccinated in her infancy, and
from a patient with no available vaccination history data. None of
these 4 patients who gave culture-positive samples were administered macrolides. All 4 isolates were sensitive to macrolides and
trimethoprim-sulfamethoxazole. They were all classified into PFGE
group IV, 3 of them belonging to the subgroup IV gamma and 1 to
subgroup IV beta (Hegerle et al., 2012). All 4 isolates carried a type 3
ptxA-Pr, a type 1 ptxS1 and a type 2 prn allele (Table 2). However, the
isolates also expressed FIM2 (n = 2) and FIM3 (n = 2). All isolates
expressed AC-Hly, PT, and FHA, but only 3 expressed PRN: a
nucleotide substitution at position 1273 resulting in a stop codon
was detected in the genome of the fourth isolate and explains the lack
of PRN production. This mutation has also been found in isolates from
the United States (Queenan et al., 2013).
3.2. Comparison of the reference ELISA and a commercial kit to detect
anti-PT IgG
The reference in-house ELISA with purified PT and NIBSC reference
sera was used as previously described to assay anti-pertussis toxin IgG
antibodies (Guiso et al., 2011; Simondon et al., 1998). A new
commercial kit (cat. no. 1231-01, SeroPertussis Toxin IgG; Savyon®
Diagnostics Ltd., Ashdod, Israel), not previously evaluated, had
become available in Europe and was used for comparison of the
results with those of the reference ELISA. The estimated sensitivity
and specificity of this test for IgG antibodies, with reference to the
results of the in-house ELISA, were 88% and 100%, respectively.
4. Discussion
No vaccination history data were available for three cases. Eight
children (between 4 months and 7 years of age) were unvaccinated, 7
were incompletely vaccinated due to their age (6) or other reasons
(1), and 4 were vaccinated but had ended their vaccination schedule
at least 5 years before developing the infection (Table 1). All the
vaccinated children, teenagers, and adults found positive for B.
pertussis infection had received wP vaccination when infants.
3.1.2. Diagnosis of other bordetellosis cases
Other bordetellosis cases were detected only by real-time PCR.
IS1001 DNA, suggestive of B. parapertussis infection, was detected in 1
NPS collected from an adult and in the NPS collected from her 4month-old child. IS481 DNA was also detected in the NPS of the
mother, but unfortunately, the small amount of DNA in the sample
During the 1-year study, 51 patients were enrolled. B. pertussis
infection was confirmed by culture and/or real-time PCR and/or
detection of anti-PT antibodies in 32 patients (63%). The patients
included 12 infants and toddlers, 14 children and adolescents, and 6
adults. These results confirm that, as in all other regions where infants
and toddlers were intensively vaccinated with wP vaccine, B. pertussis
is still circulating.
All the patients presented with standard pertussis symptoms; the
symptoms were more severe among the confirmed cases.
The biological diagnosis was established in 12.5% of the positive
cases (including both children and adults) by culture and specific realtime PCR. Two patients were already being treated with macrolides
when the NPS was collected, possibly contributing to the negative
culture results. The 4 isolates obtained are very similar to isolates
circulating in France (Hegerle et al., 2012) and other parts of Europe
(Advani et al., 2013). All 4 carry a type 3 ptxA-Pr, a type 1 ptxS1, and a
Table 2
Characteristics of the isolates investigated in this study.
Isolate
no.
Patient
Age
Residence
Sex
Vaccination
Comments
1
2
3
4
10 y
1y
1 y, 6 m
32 y
Bucharest
Bucharest
Bucharest
Bucharest
F
M
F
F
Vaccinated
No data
Unvaccinated
Childhood vaccination
HIV infection
-
PFGE
profile
Genotyping
PRN
ptxPr
ptxS1
Western blot
PT
AC-Hly
PRN
FHA
FIM serotyping
IVγ
IVγ
IVγ
IVβ
2
2
2
2
P3
P3
P3
P3
I
I
I
I
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
FIM2+/FIM3−
FIM2+/FIM3−
FIM2−/FIM3+
FIM2−/FIM3+
Please cite this article as: Dinu S, et al, Whooping cough in South-East Romania: a 1-year study, Diagn Microbiol Infect Dis (2013), http://
dx.doi.org/10.1016/j.diagmicrobio.2013.09.017
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S. Dinu et al. / Diagnostic Microbiology and Infectious Disease xxx (2013) xxx–xxx
type 2 prn allele. Like French isolates and other European isolates, they
belong to PFGE group IV (Advani et al., 2013; Hegerle et al., 2012).
They produce FIM2 or FIM3, AC-Hly, PT, and FHA. However, 1 over the
4 isolates does not express PRN, consistent with findings for some
other European isolates, found in France since 2007 (Hegerle et al.,
2012) and in Finland since 2012 (Barkoff et al., 2012). Such isolates
have also been obtained in Japan (Otsuka et al., 2012) and the United
States (Queenan et al., 2013). These currently circulating isolates that
do not express PRN are as virulent in infants as isolates expressing the
antigen (Bodilis and Guiso, 2013).
In 4 cases, IS481 was detected, but the amount of DNA was not
sufficient to determine whether the species present was B. pertussis or
another Bordetella species (Tizolova et al., 2013).
In 2 cases, IS1001 DNA was detected in the NPS suggesting B.
parapertussis or Bordetella bronchiseptica infections (Tizolova et al.,
2013); the cases were a mother and her 4-month-old child. However,
it was not known whether the family had a pet or was in contact with
animals, which could have transmitted B. bronchiseptica. Furthermore,
the NPS obtained from the mother contained IS481 DNA suggestive of
B. pertussis infection.
In 6 cases of infection, B. holmesii and B. pertussis DNAs were both
specifically detected in NPS, as recently reported in France (Njamkepo
et al., 2011) and the United States (Rodgers et al., 2013). Four of these
patients were older than 12 years of age, consistent with reported
observations (Kamiya et al., 2012; Mooi et al., 2012; Njakempoo et al.,
2011; Rodgers et al., 2013; Yih et al. 2000), although 1 patient was 7
months old and the last 7 years old. These observations confirmed
similar reports from South America (Bottero et al., 2013; Miranda et
al., 2012) that B. holmesii DNA can be detected in the NPS collected
from infants and young children. In 1 unvaccinated toddler (2 years,
11 months of age), only B. holmesii DNA was detected: the first serum
was negative for anti PT antibodies, and a second serum was not
available. Unfortunately, there was no search for another cause,
bacterial or viral, of the cough because this study was retrospective.
Finally, in 1 case, B. pertussis DNA, B. holmesii DNA, and DNA
containing IS1001 were detected.
B. pertussis infection was confirmed by real-time PCR and
detection of anti-PT antibodies in 18.75% of cases and only by realtime PCR in 12.5% of cases. In 18 (56%) cases, B. pertussis infection was
confirmed only by detection of anti-PT antibodies in the serum of the
patients; this illustrates the continuing usefulness of testing for antiPT IgG. For 12 cases, we obtained only 1 serum sample; for all these
cases, the score was between 100 and 400 IU/mL or over 400 IU/mL
although the serum was sampled early, at the onset of the cough.
However, some of these patients have previously been vaccinated.
These observations are coherent with previous findings for the
kinetics of antibody titers after infection: the kinetics differs according
to whether the patients have never been in contact with the
bacterium before the infection and those who have been vaccinated
or previously infected (Simondon et al., 1998). This was further
confirmed in some patients for whom 2 sera were obtained at an
interval of 1 month (3): the antibody titers decreased not increased. In
4 cases, antibodies were not detected, although the B. pertussis DNA
was demonstrated by PCR. Possibly, the serum was sampled too early
after the onset of the cough in these cases.
We also evaluated a new commercial ELISA kit for the detection of
anti-PT antibodies. Intra- and inter-assay agreement was good for
both specificity and sensitivity as compared to the reference
technique. Thus, we found that this kit is comparable to others tested
previously (Riffelmann et al., 2010).
In conclusion, our study confirms that B. pertussis is still
circulating in Romania and supports the view that whooping
cough is underreported: the single hospital in Bucharest included
in this study referred 46 samples to the National Reference
Laboratory, whereas only 251 samples were referred by the whole
country (32 and 70 confirmed cases, respectively) (Popovici, 2013b).
Infections occurred in unvaccinated and partially vaccinated children indicating, as in other regions, that vaccines should be
administered on time according to National and WHO recommendations. Furthermore, our work demonstrates, again, that biological
diagnosis is absolutely required.
This study also confirms that bacterial cultures are required to
monitor both antibiotic resistance (Guillot et al., 2012; Zhang et al.,
2013) and shifts in bacterial populations, such as the loss of
production of vaccine antigens.
Real-time PCR testing for Bordetella IS sequences is the most
sensitive approach and contributes both to preventing transmission
of the disease and to facilitating treatment of contacts of the case.
However, the specificity of such tests needs to be validated by a
national reference laboratory to confirm the diagnosis. In the
present study, the use of real-time PCR targeting IS481 would not
have allowed the discrimination between B. pertussis infection and coinfection with (or co-carriage of) B. holmesii or B. holmesii infection.
Detection of anti-PT IgG antibodies was useful for the diagnosis of
56% of the cases, and in 67% of these cases, a single serum sample
allowed the diagnosis.
Establishment of a national surveillance of whooping cough in
Romania using culture, specific real-time PCR, and serology is clearly
necessary. Greater awareness is also required among both health care
workers and also the public that pertussis is not just a pediatric
disease.
Acknowledgments
This work was funded by the “Cantacuzino” National Institute of
Research-Development for Microbiology and Immunology, Institut
Pasteur Foundation, and CNRS URA 3012. Funds for culture and PCR
reagents were obtained from Sanofi Pasteur.
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Please cite this article as: Dinu S, et al, Whooping cough in South-East Romania: a 1-year study, Diagn Microbiol Infect Dis (2013), http://
dx.doi.org/10.1016/j.diagmicrobio.2013.09.017