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Journal of Clinical Microbiology, April 1999, p. 1062-1068, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Analysis with a Combination of Macrorestriction Endonucleases
Reveals a High Degree of Polymorphism among Bordetella
pertussis Isolates in Eastern France
G.
Prevost,1
F. I. S.
Freitas,1
P.
Stoessel,1
O.
Meunier,2
M.
Haubensack,1
H.
Monteil,1 and
J.
M.
Scheftel1,*
Institut de Bactériologie de la
Faculté de Médecine de Strasbourg1
and Institut d'Hygiène de la Faculté de
Médecine de Strasbourg,2 Université
Louis Pasteur-Hôpitaux Universitaires de Strasbourg, F-67000
Strasbourg, France
Received 23 March 1998/Returned for modification 4 April
1998/Accepted 29 December 1998
 |
ABSTRACT |
From 1990 to 1996, routine screening for whooping cough identified
399 patients with a calmodulin-dependent adenylate
cyclase-positive test result and yielded 69 Bordetella
pertussis isolates. None of the patients were fully vaccinated,
and most were less than 6 months old. Analysis of total DNA by
pulsed-field gel electrophoresis (PFGE) after XbaI,
SpeI, or DraI macrorestriction yielded 19, 15, and 5 different patterns, respectively, whereas ribotyping failed to
demonstrate any strain polymorphism. Discrimination among the isolates
was improved by combining the PFGE profiles. Some patterns were more
frequent, but the corresponding patients were not clearly
epidemiologically related. The patterns for two strains obtained during
a 3-month period from patients who were neighbors differed by the
length of a single DNA fragment. These data strongly suggest that one
type of isolate is widely spread throughout the world and is carried by
individuals other than patients who develop a true illness.
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INTRODUCTION |
Since the early 1990s, the incidence
of whooping cough has increased notably in Europe including France
(2, 17, 21) and had increased in the United States
before that (3, 9), even though vaccination is still
strongly recommended. Bacterial diagnosis of whooping cough is
hindered by the fragility of Bordetella pertussis and the
interference of the normal nasopharyngeal flora. Direct
fluorescent-antibody testing and serological methods lack sensitivity
and specificity, respectively (11). One rapid screening method is based on the detection of calmodulin-dependent
adenylate cyclase (AC)-hemolysin by an assay with an alginate
swab specimen from the nasopharynx (7). Multitarget
PCR-based assays (13, 26) are sensitive and more efficient
than culture, but nasopharyngeal sampling with Dacron swabs is
required. No comparison of the results of the AC test and PCR-based
diagnosis has been reported to date.
To distinguish among the clinical isolates responsible for pertussis
outbreaks, existing epidemiological tools necessitate previous
bacterial isolation (1, 14). Pulsed-field gel
electrophoresis (PFGE) can reveal polymorphisms among clinical
isolates, but isolates from a defined locality (1, 9, 17)
had similar DNA profiles. A recent comparison of PFGE, random
arbitrarily primed detection, and enterobacterial repetitive intergenic
consensus-PCR showed that PFGE was the most discriminatory. PCR-based
detection of B. pertussis repeat DNA sequences failed
to reveal strain polymorphism (17).
The incidence of whooping cough diagnosed by both biochemical testing
and bacterial isolation has increased in eastern France in the past 7 years. We used DNA fingerprinting to distinguish between endemic and
epidemic isolates.
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MATERIALS AND METHODS |
Patients and samples.
Both hospitalized and ambulatory
patients were included in this epidemiological study. They were
eligible for participating in the study if they had signs of pertussis
with intense, dry, and emetic coughing for at least 10 days. Apnea
and cyanosis were frequent in infants younger than 3 months of age and
sometimes necessitated intensive care in Strasbourg University
Hospital. Samples were obtained by a hospital microbiologist or in
an ambulatory consultation to limit the processing time.
Nasopharyngeal swab specimens from both nostrils were obtained with
calcium alginate-tipped applicators (Puritan Hardwood Products Co.,
Guilford, Maine). The swabs were immediately immersed in Stainer and
Scholte medium and were agitated for several seconds (21,
22). Calmodulin-activated AC activity was assayed as described
previously (7, 21). Samples were also inoculated onto fresh
(
7 days) Bordet-Gengou agar plates (Bacto Bordet-Gengou agar base;
Difco Laboratories, Detroit, Mich.) supplemented with 15% (vol/vol)
defibrinated sheep blood and 40 µg of cephalexin (Sigma) per ml. The
plates were incubated at 37°C for at least 7 days in a humid
atmosphere and were observed daily for the presence of "mercury
droplet" colonies surrounded by a zone of hemolysis. B. pertussis was identified by classical methods, and the reference
strain B. pertussis ATCC 18323 was used as a control.
All isolates that were collected were stored at 
80°C in a 9
(wt/vol) NaCl solution containing 20% (vol/vol) glycerol.
DNA preparations for PFGE analysis.
The isolates were grown
at 37°C on Bordet-Gengou agar plates for at least 72 h, until
colonies 1 mm in diameter appeared. The bacteria were harvested with a
Pasteur pipette and were resuspended in 10 ml of 10 mM Tris-HCl-5 mM
EDTA-1 M NaCl (pH 8.0) and then pelleted by centrifugation at 4°C
and 5,000 × g, for 10 min and processed as described
elsewhere (18). DNA fingerprinting was carried out after
restriction with XbaI, SpeI, or DraI.
The gels were run at 12°C with a Geneline Transverse Alternating
Field Electrophoresis (TAFE) system (Beckman) at 140 mA in 0.6× TAFE running buffer (20× TAFE running buffer is 200 mM Tris, 0.5 mM free
acidic EDTA, and 87 mM acetic acid [pH 8.2]). Separation of the
restricted DNA fragments started with 4-s pulses for 2 h, followed
by 12-s pulses for 8 h, 8-s pulses for 6 h, and 6-s pulses
for 2 h. The gels were stained for 30 min with 2 µg of ethidium
bromide per ml and were washed in water before being photographed under
UV light at 300 nm. Pulsotypes were compared and classified in
dendrograms by using the Dice coefficient and the unweighted pair group
method with arithmetic mean clustering methods provided by Molecular
Analyst (version 1.5) and Fingerprinting (version 1.12) software
(Bio-Rad).
Ribotyping.
Ribotyping was performed as described previously
(19) by using Immobilon P membranes (Millipore). Intact DNA
embedded in agarose for PFGE analysis was restricted with
AccI, PvuII, or SalI endonuclease,
according to the manufacturer's recommendations (New England Biolabs,
Beverly, Mass.). Southern blots were made by using
32P-nick-translated DNA fragments (20)
corresponding to the Pfu DNA polymerase-amplified whole 16S
and 23S Escherichia coli rRNA genes (rDNAs) (15).
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RESULTS |
Disease epidemiology.
From 1990 to 1996, 867 patients were
included in this study. The number of the patients sampled was stable
until 1994, but the number increased during the last 2 years (almost
300 patients in 1995 and 1996). The AC test was positive for 399 patients (45.9%) and progressed as described above. Sixty-nine
B. pertussis isolates (representing 17.3% of the
positive AC tests) were obtained. The sex ratio for patients infected
with B. pertussis isolates was 59% males and 41%
females, and that for patients with AC-positive test results was 54%
males and 46% females.
As shown in Fig. 1, 67 and 59% of AC
test-positive and culture-positive patients, respectively, were less
than 6 months old. The frequency of AC test positivity and culture
positivity did not differ between children aged 6 months to 18 months
(9 and 6%, respectively) and those aged 18 months to 6 years (13 and 12%, respectively). Older patients with a diagnosis of pertussis after sampling comprised 11% of the AC test-positive patients and 23%
of the culture-positive patients. Figure 1 indicates the vaccination
status of the patients. None of those with a positive biochemical test
result or from whom bacteria were isolated had been fully vaccinated
when pertussis occurred. The majority of patients were
unvaccinated patients, but infants who were less than 3 months
old accounted for 38% of the AC test-positive patients and 38% of the
culture-positive patients. Only two of these patients had received a
first vaccine injection. The patients in the 3- to 6-month and the
18-month to 5-year age groups with a diagnosis of pertussis were not
completely vaccinated, and the patients in these groups combined
corresponded to 19 and 26% of the subjects with positive AC test
results and B. pertussis culture positivity, respectively. Overall, 65% of the patients had not been vaccinated at
all, but 35% were less than 3 months old. Among the
patients in the 6- to 10-year and 
10-year age groups, no
patient received a full vaccination because the last vaccination dose
at age 6 years, at least, was omitted. The patients in these
groups combined accounted for 11 and 23% of the subjects with positive
AC test results and culture positivity, respectively.
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FIG. 1.
Percentages, as compiled histograms, of the patients
with different vaccination statuses and with a diagnosis of pertussis
with an AC-positive test result (AC+; left columns, n = 331)
and from whom B. pertussis bacteria were isolated (Bp+;
right columns; n = 69), according to age (bottom).
Vaccination statuses were as follows: no vaccination dose
( ), one
vaccination dose ( ), two vaccination doses
( ), three
vaccination doses ( ), three vaccination doses without a booster dose
at 18 months
( ), four
vaccination doses including that at 18 months
( ), and
five vaccination doses ( ). The normal vaccination is three doses
monthly from the 3rd to the 6th months of life and two booster doses at
18 months.
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Fingerprinting by PFGE and ribotyping of B. pertussis DNA.
The PFGE profiles obtained after
XbaI digestion contained from 10 to 15 DNA fragments ranging
from 50 kb to 550 to 600 kb (Fig. 2).
Some DNA preparations were retested six times over a 1-year period and
always gave reproducible profiles after digestion with XbaI
or SpeI, as determined previously with the TAFE system (18). This accounts for the stability of the epidemiological marker and that of genomes of B. pertussis during this
study. The best and most reproducible resolution was for fragments in the range of 100 to about 500 kb, and the pattern generally comprised 8 to 10 DNA fragments. For the pulsotypes obtained after digestion with
XbaI (Fig. 2), smaller fragments were not separated
and were not visible enough, whereas the size of the upper DNA fragment could not be determined precisely. Therefore, differentiation of
the patterns was done with the variable bands, whose sizes ranged from
120 to 530 kb. Nevertheless, when the upper DNA fragment is
considered with the one that is 600 kb in length, the
different lengths of genomes varied from 3,200 to 3,800 kb, which
are comparable to those reported previously (23). From the
profiles obtained after digestion with XbaI, some DNA
fragments appeared to be very well conserved, such as those at 135, 160, and about 200 kb (see Fig. 3). One DNA fragment of about 310 kb was always encountered except in a pulsotype 19 strain (from
patient 6). Pulsotypes like those observed in Fig. 2, lanes 6, 7, and
1, were considered to be very similar on the basis of the separation of
two DNA fragments of 270 to 275 kb in lane 1; these fragments
comigrated in lanes 6 and 7. The corresponding patterns were designated
1 and 1' and will be considered again in this work. The pulsotypes of
the strains in lanes 2 and 5 (Fig. 2) were considered to be identical
and were assigned to pattern (pulsotype) 15. The pulsotypes of the strains in lanes 9, 15, 16, 18, 19, and 21 were distinguished from
those of the strains in lanes 4, 17, and 20 by the addition of a large
DNA fragment of approximately 330 kb. This addition did not seem to
affect significantly the length of any other visible fragment, even
that of about 600 kb. The two groups of patterns were designated
patterns 13 and 12, respectively. Pulsotype 15 (Fig.
3) differed from pattern 13 by the
presence of a DNA fragment of 400 kb and a different distribution of
DNA fragments ranging from 200 to 250 kb. Pulsotype 15 differed
from patterns 6 and 17 (Fig. 3) by the presence of DNA fragments
ranging from 150 to 250 kb and another one of 120 kb for
pulsotype 17 (Fig. 3). Finally, 19 different XbaI
digestion patterns (Fig. 3) were characterized from the series of
69 isolates, and these different patterns accounted for the
pronounced polymorphism of the B. pertussis
strains obtained from within a small geographic area (about 12,000 km2 with 1,700,000 individuals). Pulsotypes 13, 15, and 1 contained 22, 9, and 11 isolates (61% of the total), respectively.
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FIG. 2.
PFGE analysis of XbaI-restricted intact DNAs
from random B. pertussis isolates evidenced
polymorphism and frequent pulsotypes. Lanes: T, polymers of
bacteriophage  DNA (New England Biolabs); 1, patient 43; 2, patient
60; 3, patient 55; 4, patient 52; 5, patient 61; 6, patient 11; 7, patient 23.
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FIG. 3.
Computerized comparison according to the dendrogram and
by schematic magnification of the 19 XbaI profiles and the
number of corresponding fingerprints among the 70 B. pertussis isolates analyzed, including ATCC 18323. Nb, number.
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The intact DNAs of the isolates were also analyzed after
restriction with
SpeI, and again, evidence of polymorphism
and frequently
occurring fingerprints was obtained (Fig.
4). The 15 patterns
identified by
restriction with
SpeI contained 10 to 12 DNA fragments
ranging from 50 to 550 kb (Fig.
4 and
5A). The sum of the estimated
lengths of
these fragments also approximately corresponded to
the length of
the chromosome determined as described above, indicating
that
numerous small DNA fragments were not visible in the PFGE
profiles after restriction with either
XbaI or
SpeI. As for the
profiles obtained after restriction
with
XbaI, several DNA fragments
appeared to be
ubiquitous in the 15 different profiles; they were
60, 80, 100, 130, and 170 kb in length. A great variability in
DNA bands of 210 to 450 kb was observed, and these encompassed
four to seven fragments
and allowed the differentiation of PFGE
profiles. Another 480-kb
SpeI DNA fragment obtained after restriction
with
SpeI seemed to be almost constant in the genomes. As shown
in Fig.
4, the patterns of many isolates corresponded to pattern
5 (Fig.
5A). Pattern 5 was distinguished from pattern 6 by a 200-kb
DNA
fragment, and isolates with this pattern were clustered with
those with
pattern 7 in the dendrogram analysis and supported
relationships of
clonality. Other pulsotypes, like those shown
in Fig.
5A, patterns 3 and 14, differed from each other by the
presence of DNA fragments of
275 and 125 kb, respectively. As
for the profiles obtained after
restriction with
XbaI, two pulsotypes
(pulsotypes 5 and 9)
obtained after restriction with
SpeI covered
56 and 10% of
the isolates, respectively.
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FIG. 4.
PFGE analysis of SpeI-restricted DNAs from
B. pertussis isolates also evidenced polymorphism and
frequent pulsotypes. Lanes: T, polymers of bacteriophage DNA (New
England Biolabs); 1, patient 21; 2, patient 7; 3, patient 2; 4, patient
4; 5, patient 28; 6, patient 42; 7, patient 58; 8, patient 32; 9, patient 29; 10, patient 6; 11, patient 35; 12, patient 38.
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FIG. 5.
Computerized comparison according to the dendrograms and
by schematic magnification of the 15 SpeI profiles (A) and
the 5 DraI profiles (B) and the number (Nb) of corresponding
fingerprints among the 70 B. pertussis isolates
analyzed, including ATCC 18323.
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Finally, PFGE analysis with
DraI restriction revealed only
five different pulsotypes (Fig.
5B) which were composed of
approximately
10 DNA fragments also ranging from 50 to 500 kb. Most DNA
fragments
obtained after restriction with
DraI were not
constant within
the five patterns except in the region of 50 to 110 kb.
Pulsotypes
2 and 3 obtained after restriction with
DraI were
the most frequently
encountered (30 and 36% of the isolates,
respectively).
Ribotyping experiments revealed complete homogeneity in the profiles
that were obtained (data not shown), despite the use
of three
restriction
endonucleases.
Setting epidemiological data with combinations of pulsotypes
obtained by PFGE.
The pulsotypes obtained after restriction with
DraI, SpeI, and XbaI were combined to
increase the level of discrimination (Table
1). This led to 35 combinations. The
series of a given XbaI pulsotype were often differentiated
by the SpeI profiles, as was the case for XbaI
restriction patterns 13, 14, 15, 1, 1', and 9. However, the three
isolates harboring pulsotype 4 after restriction with
XbaI were not further distinguished by restriction with
either DraI or SpeI, although they appeared to be
independent. The most frequently occurring pulsotype after
XbaI restriction (pulsotype 13) was further distinguished
only for isolates from four patients (those from patients 3, 47, 49, and 46) by restriction from DraI and SpeI,
respectively. Also, XbaI pulsotypes 18, 14, 15, and 1 were
distinguished by the profiles obtained after restriction with
DraI and SpeI. Combinations of patterns obtained
by PFGE after restriction with
DraI-SpeI-XbaI, patterns 2-5-1, 3-5-13, and 5-9-15, appeared more frequently and represented 10, 26, and 9% of the clinical isolates, respectively (Table 1). Among these frequent combinations, patterns 2-5-1 and 3-5-13 were found for isolates which were obtained over a 5-year period. No epidemiological link (by age, nursery, family, or close circle) was easily evidenced for the other patients, and these isolates could be considered independent at the level of this study. The pulsotype combination 5-9-15 was found for six isolates collected in 1995 and 1996 from patients living in the city of Strasbourg, with two isolates found in
two patients from the same family.
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TABLE 1.
Distribution of B. pertussis ATCC 18323 and isolates from patients with whooping cough according to PFGE
profiles obtained after restriction with DraI,
SpeI, and XbaI and the 35 combinations that
were revealed
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PFGE analysis of family-related and geographically related cases of
infection provided evidence that macrorestriction with
only one enzyme
may not be efficient enough to distinguish among
the isolates. Results
for three of these related isolates are
presented in Fig.
6. The isolates were from two patients
with
illnesses in two distinct families (lanes 5 to 12) and two
geographically
related patients (lanes 1 to 4) from a single
village where isolates
were obtained after an interval of 3 months. In
the first series
of family-related cases of illness, no discrimination
was obtained
by macrorestriction with
XbaI and
SpeI (Fig.
6, lanes 5 to 8 and
10 to 12), even though the
isolates were sampled over a period
of 1 month in the second series
(patients 35 and 38). However,
in the two geographically related
series, while the pulsotypes
of the isolates from patients 24 and 21 obtained after restriction
with
XbaI appeared to be almost
identical, with only a slight
difference in the resolution of two
DNA fragments of about 265
kb (Fig.
6, lanes 1 and 2), the
corresponding pulsotypes for the
isolates obtained after
restriction with
SpeI (Fig.
6, lanes 3
and 4) were
different because of the different lengths of one
DNA fragment
(215 and 240 kb). After restriction with
XbaI, pattern
1' for the isolate from patient 24 was finally distinguished from
pulsotype 1 for the isolates from patients 21, 23, and 22, which
also
showed a unique and more intense DNA band at 275 kb. In fact,
it
seemed that after restriction with
SpeI a 240-kb
fragment in
the pulsotype of the isolate from patient 24 was
reduced to a
215-kb DNA fragment in the pulsotype of the isolate
from patient
21. Such a variation was not attributed to DNA separation
since
no variation in the other DNA fragments was observed. It
must
be emphasized that the isolate from patient 21 was obtained 3
months after the isolate from patient 24 was obtained, suggesting
the loss of a small genetic element in the isolate from patient
21 that
was observed in the profile obtained after restriction
with
SpeI. The two corresponding isolates may be assumed to be
related, suggesting evolution within
B. pertussis
isolates.
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FIG. 6.
PFGE analysis of geographically related and
family-related B. pertussis isolates. Lanes: L,
polymers of bacteriophage DNA (New England Biolabs); 1, 2, 3, 4, 9, and 10, XbaI-restricted intact DNAs of isolates from
patients 24, 21, 62a, 62b, 38, and 35, respectively; 3, 4, 7, 8, 11, and 12, SpeI restriction of the same DNAs, respectively.
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DISCUSSION |
Although the sampling procedure and the diagnostic techniques have
been identical during the past 7 years, the numbers of cases of
whooping cough and the rates of isolation of B. pertussis have increased in Eastern France, to a ratio of two
cases of bacterial isolation per 100,000 individuals in 1994, but the
AC tests revealed a sixfold greater incidence of pertussis among
people. This incidence of whooping cough is greater than the evaluation
of 3.5 cases per 100,000 individuals in France (2). As
reported previously (6) and according to the data obtained
in this study, newborns and young infants constitute the population at
major risk, but all the patients included in the study presented with
an incomplete vaccination status. In fact, normally vaccinated children
in France represent 95% of the population less than 6 months old, and
90% of the population from 2 to 7 years of age has been vaccinated, whereas the booster dose given at age 6 years remains facultative (4). It must be noted that for about 10 years (1980 to 1990) the last immunization was rarely administered in eastern France. This lack of receipt of the last immunization may have
favored the asymptomatic or nonsymptomatic carriage of B. pertussis by previously immunized people without the development
of true whooping cough. Noncompliance with immunization due to the
fear of well-publicized neurologic side effects can also play a role in
the upsurge in the incidence of pertussis. While a
diagnosis of whooping cough remained poorly evoked in adults
(3, 10), such persons who are transitory infectious carriers
may be vectors for the spread of a B. pertussis strain
of a given pulsotype (isolates with patterns 13, 14, 15, 1, and
4 after restriction with XbaI were more frequently detected in this study). This would explain the absence of an epidemiological relatedness of the patients included in the study. This
hypothesis is strengthened by the observation that incompletely vaccinated people with pertussis were found in populations positive for
pertussis by both a positive AC test result and bacterial isolation.
Again, the study with the 69 B. pertussis isolates
confirmed that the results of PFGE analysis provided
epidemiological markers of choice (9, 17). Even though
different data were obtained for Bordetella
bronchiseptica isolates (19), variations in
B. pertussis were not found by three ribotyping
procedures. The use of XbaI and SpeI restriction
endonucleases provided consistent assessments of the polymorphisms of
B. pertussis strains. Restriction with XbaI,
SpeI, and DraI identified 19, 15, and 5 different
pulsotypes, respectively, and accounted for the polymorphisms of the
bacteria collected over a 7-year period in a small geographic
area, although 76% of the isolates were collected during the
last 3 years. Use of combinations of PFGE patterns greatly improved the
ability to distinguish the isolates since 35 patterns were recorded
when combinations were used. Nevertheless, it appeared that each
XbaI, SpeI, and, to a lesser extent,
DraI restriction site may have concerned regions susceptible
to genetic rearrangements. In this study, for isolates from patients
with whooping cough in a small village detected over a 3-month
interval, two clonal XbaI profiles were observed, and
further examination of the SpeI fingerprints improved the
ability to discriminate between the isolates.
The frequent pulsotype combinations did not necessarily account for a
direct epidemiological link between isolates and the corresponding
patients. After comparison, pattern 13 for isolates from patients 31, 33, and 40 obtained after restriction with XbaI (Table 1 and
Fig. 3) seemed to be identical to the pattern most encountered for
isolates involved in an outbreak at Fort Smith, Alberta, Canada
(9), as well as the pattern for isolates involved in another
recent French study of isolates from Paris and its suburbs
(17). This observation suggests that B. pertussis is spread not only by people who have developed a true
illness. Adults and health care workers may be affected by unrecognized
B. pertussis infections (10, 12, 16). This
bacterial carriage may be endemic and may be unknown for most patients.
The efficacy of the common pertussis vaccine, which is estimated to
decrease significantly during the 5th year after immunization (5,
8, 11, 16), strengthened this hypothesis. In a recent
work, 25% of the adult population with a cough for 2 or 3 weeks
had nonsymptomatic pertussis (24), in accordance with the
results of this study. In conclusion, the rapid processing
of samples from patients with whooping cough allowed significant
detection of B. pertussis, although to a lesser extent
than the AC test did. PFGE typing with two or three
endonucleases, e.g., XbaI, SpeI, and
DraI, confirmed the high degree of polymorphism among
B. pertussis strains and strengthened the ability
to distinguish isolates, but the predominant strains suggested the
long-term spread of the bacteria. These routes of spread might be
reduced by more efficient vaccination programs.
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ACKNOWLEDGMENTS |
We are very grateful to the pediatricians and pediatric care
units, especially those at the Strasbourg University Hospital, for
assistance and information concerning the patients. We thank D. Young
for help with the English.
This work was supported by funds from the Institut de
Bactériologie de la Faculté de Médecine de Strasbourg.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut de
Bactériologie de la Faculté de Médecine de
Strasbourg, Université Louis Pasteur-Hôpitaux
Universitaires, 3, rue Koeberlé, F-67000 Strasbourg, France.
Phone: (33) 3 88 21 19 70. Fax: (33) 3 88 25 11 13. E-mail: jmichel.scheftel{at}medecine.u_strasbg.fr.
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Journal of Clinical Microbiology, April 1999, p. 1062-1068, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
