J Infect Chemother 27 (2021) 497e502

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Journal of Infection and Chemotherapy

journal homepage: http://www.elsevier.com/locate/jic

Original Article

Bordetella pertussis is a common pathogen in infants hospitalized for

acute lower respiratory tract infection during the winter season
Yuka Mihara a, 1, *, Shuji Yoshino b, Keigo Nakatani a, Toyoki Nishimura a, Hiromi Kan a,
Yoshiko Yamamura a, Etsuko Tanaka a, Shigeki Ishii a, Hidemi Shimonodan a,
Kenji Okada c, Toshihiro Nishiguchi a
a
Department of Pediatrics, Miyazaki Prefectural Miyazaki Hospital, 5-30, Kitatakamatsu-cho, Miyazaki-shi, Miyazaki 880-0017, Japan
b
Miyazaki Prefectural Institute for Public Health and Environment, 2-3-2, Gakuenkibanadainishi, Miyazaki-shi, Miyazaki 889-2155, Japan
c
Division of Basic Nursing, Fukuoka Nursing College, 2-15-1, Tamura, Sawara-ku, Fukuoka 814-0193, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: There is some evidence that Bordetella pertussis (B. pertussis) can co-infect with viral res-
Received 9 July 2020 piratory infections in young infants.
Received in revised form Methods: B. pertussis infection was studied by culture, polymerase chain reaction (PCR), and loop-
26 October 2020
mediated isothermal amplification (LAMP) from nasopharyngeal swabs (NPSs) in 49 infants < 12
Accepted 2 November 2020
Available online 7 December 2020
months of age, who were admitted for lower respiratory tract infections during the winter season. Seven
other possible viral pathogens were documented by antigen detection or PCR in NPSs. The clinical feature
of infants with mixed infection of B. pertussis and respiratory viruses were examined.
Keywords:
Pertussis
Results: Overall, B. pertussis infection was found in 10 (20.4%) cases, nine were less than 6 months of age
Bordetella pertussis and seven were unvaccinated. Viral etiology was found in 41 (84%) cases and pertussis-viral co-infection
Co-infection was present in eight patients, five of whom had mixed infection with respiratory syncytial virus. Only the
Infant presence of staccato coughing, cyanosis, and lymphocytosis were significantly different in B. pertussis-
Lower respiratory tract infection positive cases compared with B. pertussis-negative cases. Of the 10 pertussis cases, only the culture-
positive cases showed the typical symptoms and laboratory findings of pertussis in addition to virus-
associated respiratory symptoms with severe hospital course, whereas cases identified as DNA-
positive lacked the characteristics of pertussis and their clinical severities were the same as
B. pertussis-negative cases.
Conclusion: In the absence of typical paroxysmal cough and lymphocytosis, we should carefully consider
diagnosis of pertussis in young children hospitalized for presumed viral respiratory illness according to
local epidemiological surveillance.
© 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.
Published by Elsevier Ltd. All rights reserved.

1. Introduction treated as acute bronchitis, bronchiolitis, or pneumonia in the early

Pertussis is a vaccine-preventable disease and is usually
observed in young, unvaccinated infants in countries with high
vaccination coverage. Young infants with Bordetella pertussis
(B. pertussis) infection present with atypical symptoms, including
apnea, cyanosis, and wheezing, and the laboratory findings, like
leukocytosis and lymphocytosis, are not always specific, being often
* Corresponding author. Department of Pediatrics, Miyazaki Prefectural Miyazaki
Hospital, 5-30, Kitatakamatsu-cho, Miyazaki-shi, Miyazaki 880-0017, Japan.
E-mail address: yuka.mihara@toyota-kai.or.jp (Y. Mihara).
1
Present address: Department of Pediatrics, Kariya Toyota General Hospital, 5-15
Sumiyoshi-cho, Kariya city, Aichi 448-8505, Japan.
stages [1]. B. pertussis and viral co-infections have been reported in
young, unvaccinated infants hospitalized for lower respiratory tract
infection (LRTI), such as bronchiolitis or acute respiratory failure,
during the winter season [2e9]. Pertussis can be especially difficult
to diagnose in these young children with atypical clinical symp-
toms or overlapping respiratory symptoms accompanied by virus-
associated LRTI [2e9].
Our hospital is responsible for the primary to tertiary care
hospitalization of all infants and young children in the central part
of Miyazaki prefecture, where there are about 4500 children un-
der 1 year old. The number of infants aged <12 months who were
admitted to our pediatric ward due to pertussis increased sud-
denly from August, 2014 compared to the previous year. Among

https://doi.org/10.1016/j.jiac.2020.11.002
1341-321X/© 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Y. Mihara, S. Yoshino, K. Nakatani et al.
these cases, co-infections with virus-associated bronchiolitis were
also observed. There have been only a few sporadic case reports
documenting B. pertussis and viral co-infections in children hos-
pitalized for acute respiratory symptoms in Japan [10]. Therefore,
we conducted a study to investigate the prevalence of pertussis in
young infants admitted to a regional hospital with LRTI during a
winter season and examine the clinical features of infants with
mixed infections of B. pertussis and respiratory viruses.
2. Materials and methods
2.1. Patients
The prospective study was conducted between November 1,
2014 and March 31, 2015 in pediatric department of Miyazaki
Prefectural Miyazaki Hospital, Miyazaki, Japan. Generally healthy
infants aged younger than 12 months who were hospitalized for
an LRTI such as bronchitis, bronchiolitis, pneumonia, acute dys-
pnea, or apnea were included. Those that were born pre-term or
with congenital heart disease, which might affect the severity of
the disease, were excluded. The Ethics Committee of the Miyazaki
Prefectural Miyazaki Hospital approved this study. Informed
consent was obtained from parents or guardians before
enrollment.
2.2. Detection of viral infection
Within 72 h after admission, nasopharyngeal swabs (NPSs) were
systematically obtained from all hospitalized infants < 12 months
of age with LRTI. One of the NPSs was immediately processed for
viral antigens by detection assays using a rapid immunochroma-
tographic assay for respiratory syncytial virus (RSV), adenovirus,
and influenza virus A/B (Tauns Laboratories, Inc., Shizuoka, Japan),
according to routine practice at the hospital laboratory. Remaining
NPSs were stored at 4
C in culture medium containing either
Eagle's MEM or cyclodextrin solid medium (CSM) plates supple-
mented with 5 mg/mL of cephalexin and were transported for
further analysis to Miyazaki Prefectural Institute for Public Health
and Environment, Miyazaki, Japan. For viral diagnosis, extracted
RNA from the NPS solution was subjected to the following three
multiplex reverse transcription polymerase chain reaction (RT-PCR)
methods. Multiplex 1: human metapneumovirus (hMPV) and hu-
man RSV; Multiplex 2: parainfluenza virus type 1e4 (PIV-1, 2, 3,
and 4); Multiplex 3: human rhinovirus (hRV) and human corona-
virus OC43/229E (Hcov). Each multiplex RT-PCR was a single-step,
combined RT-PCR amplification [11].
2.3. Isolation of B. pertussis and detection of genome
B. pertussis was also examined by culture, PCR, and loop-
mediated isothermal amplification (LAMP) assay, as previously
described [12,13]. NPS solutions were inoculated on CSM plates [14]
and were incubated for 4e7 days at 36
C. B. pertussis-like colonies
were subcultured on CSM plates without antibiotics and then
identified as B. pertussis by an agglutination test. The extracted total
DNA was used for PCR and LAMP testing. Primers targeting the
repeated insertion element IS481 were used in the PCR assay and
primers targeting the promoter region of the gene encoding
pertussis toxin (PT) were used in the LAMP assay, as previously
described [12,13]. A case of pertussis was diagnosed if one or more
of the following was found: B. pertussis was isolated by culture, or
IS481-PCR and/or LAMP results were positive.
498
J Infect Chemother 27 (2021) 497e502
2.4. Detection of anti- PT antibody
Acute and convalescent sera from B. pertussis-positive cases
were further assessed by serum PT-IgG antibody. Those with PT-IgG
antibody levels greater than 100 EU/mL or 4-fold increase when
compared with paired serum samples were considered being
recently infected with pertussis.
2.5. Scoring of clinical severity
Detailed epidemiological, clinical, and laboratory data were
obtained from each child from medical files. A clinical severity score
on hospital admission ranging from 0 to 7 was determined by pa-
rameters including respiratory rate (<45/min ¼ 0, 45e60/min ¼ 1,
>60/min ¼ 2), retractions (none ¼ 0, present ¼ 1), oxygen satura-
tion at room air (>95% ¼ 0, 95-90% ¼ 1, <90% ¼ 2), and ability to
feed (normal ¼ 0, reduced ¼ 1, intravenous fluid replacement ¼ 2)
according to the patients’ medical records [9]. An overall disease
severity was also ascertained based on the following parameters:
duration of supplemental oxygen, length of stay (LOS) in the hos-
pital, and clinical severity score on admission.
2.6. Investigation for familial transmission
Household contacts of proven pertussis cases were investigated
for familial transmission, where available. The source of infection
was defined by the individual in the household with the earliest
date of onset of cough.
2.7. Data analysis
For further analysis, patients were divided into three groups: (1)
infants in whom B. pertussis was isolated by culture, “B. pertussis
culture-positive”, (2) identified only by PCR and/or LAMP analysis,
“B. pertussis DNA-positive”, and (3) no B. pertussis was identified,
“B. pertussis-negative”. Group differences in categorical epidemio-
logic and clinical data were analyzed by Chi-squared tests or Fish-
er's exact tests, as appropriate. In cases of non-normally distributed
data, the Mann-Whitney test was used to evaluate whether two
groups were statistically significantly different. P values < 0.05
were considered to indicate statistical significance.
3. Results
3.1. Clinical feature of pertussis
During the study period, 56 infants <12 months of age with an
LRTI were admitted to our hospital, of whom 49 were included in
the study. The median age on admission was 72 days (14e352
days), and there were 27 males and 22 females. B. pertussis was
identified in 10 cases (20.4%), four were culture-positive (Tables 1
and 2), nine were both PCR and LAMP-positive, and one was only
LAMP-positive. Of the 10 cases with confirmed pertussis, nine were
less than 6 months of age, seven were unvaccinated, and two had
not completed the primary pertussis vaccination series. Respiratory
pathogens were detected in 41 (84%) cases. The two most common
viruses detected were RSV and hMPV, which were found in 57% and
20% of all cases, respectively. B. pertussis and viral co-infection was
observed in eight cases: with RSV in five cases, hRV in two cases,
and with both hRV and hMPV in one case. The most commonly
detected virus in infants with B. pertussis negative was RSV, which
was found in 56% of patients. In all the B. pertussis culture-positive
cases, a 4-fold increase of PT-IgG when compared with paired
serum was observed. Among B. pertussis DNA-positive cases, PT-IgG
antibody was already elevated in the acute phase, and no further
Y. Mihara, S. Yoshino, K. Nakatani et al. J Infect Chemother 27 (2021) 497e502

Table 1
Demographic and medical history data of culture positive pertussis cases.

Cases Age in days Gender Confirmed Primary Pert Vaccination status Previous antibiotic Length of disease before
ussis cases in families (DTaP) treatment NPS taken (days)

1. 37 Male Sibling none no 6

2. 66 Female Mother none yes 14
3. 67 Female Father none no 9
4. 95 Male none none no 15

DTaP: diphtheria, tetanus, pertussis; NPS: nasopharyngeal swab.

Table 2
Laboratory results and clinical characteristics of B. pertussis culture-positive cases.

Cases White Blood Lymphocyte C-reactive Co-infection With Duration of oxygen Duration of hospital Clinical severity score
Cells (n/mm3) count (n/mm3) protein (mg/dl) other pathogen therapy (days) stay (days)

1. 17,340 11,878 0.01 hRV 3 6 6

2. 51,630 34,076 0.2 hRV 8 13 5
3. 22,020 14,401 0.06 Not detected 7 11 4
4. 34,760 20,196 0.08 hMPV, hRV 3 11 5

hRV: human Rhinovirus; hMPV: human metapneumovirus.

increase was observed in convalescent sera in two imcompletely
vaccinated cases and in one unvaccinated case. PT-IgG did not
become seropositive in paired sera in one imcompletely vaccinated
case and in two unvaccinated cases.
3.2. Comparison between pertussis and non-pertussis groups
The 10 infants with B. pertussis-positive were compared to the
39 infants who were B. pertussis-negative. Similar epidemiological
characteristics were found in all infants, with or without B. pertussis
infection (Table 3-A). Clinical symptoms and signs on admission
were not different between the two groups, except for the presence
of staccato coughing and cyanosis that was observed more
frequently in B. pertussis-positive cases. Seven out of 10 (70%)
B. pertussis-positive cases and 29 out of 39 (74%) B. pertussis-
negative cases were accompanied by bronchiolitis (Table 3-B). With
regard to laboratory characteristics, the median absolute white
blood cell (WBC) counts and lymphocyte counts were higher in
B. pertussis-positive cases (Table 4). Clinical severity scores, the
number of patients receiving supplemental oxygen, duration of
oxygen therapy, and LOS in the hospital were not different between
the two groups (Table 4).
3.3. Comparison among B. pertussis culture-positive, DNA-positive
and B. pertussis-negative groups
Pertussis was more suspected in B. pertussis culture-positive
cases than in B. pertussis DNA-positive cases based on clinical
and laboratory findings on admission. Therefore, subsequent
analyses were performed among the three subgroups;
B. pertussis culture-positive cases and B. pertussis DNA-positive
cases were compared to B. pertussis-negative cases. A mixed
respiratory pathogen was identified in three out of four pertussis
cases confirmed by culture, and in five of six confirmed by PCR/
LAMP (Table 4). The epidemiological characteristics between the
three groups were similar, except that B. pertussis culture-
positive cases had a longer duration of time from the onset of
illness until specimen collection than B. pertussis-negative cases
(Table 3-A). As for clinical findings on admission, the frequency
of staccato coughing was significantly higher in B. pertussis
culture-positive cases than in B. pertussis-negative cases
(Table 3-B). B. pertussis culture-positive cases tended to have
post-tussive emesis and less fever (Table 3-B), and nearly three
times higher leukocyte and lymphocyte counts, longer hospital
499
stay, longer oxygen supply, and higher clinical severity score on
admission than B. pertussis-negative cases (Table 4). In contrast,
there was no significant difference between B. pertussis DNA-
positive cases and B. pertussis-negative cases in clinical and
laboratory findings, clinical severity on admission, and disease
severity during hospitalization, except for the presence of
cyanosis that was observed more frequently in DNA-positive
cases (Table 3-B). Since all four B. pertussis culture-positive pa-
tients were less than 3 months of age, the same analysis was
performed next to eliminate the effect of age distribution,
limiting B. pertussis-negative cases to 3 months or less. There
was a stronger correlation between the two groups than when
compared with patients less than 12 months old (data not
shown).
3.4. Familial transmission
In four B. pertussis culture-positive cases, 14 of 18 contacts had
cough, and six of 15 were confirmed with pertussis by examination.
In six B. pertussis DNA-positive cases, 10 of 26 contacts had cough;
24 underwent microbiological testing but none tested positive.
Only one contact was tested serologically and found to be positive.
All contacts who underwent examinations received macrolide an-
tibiotics as post-exposure prophylaxis.
4. Discussion
There are three main results in the present study. First, this
prospective study identified B. pertussis as a common pathogen in
infants <12 months of age hospitalized for acute LRTI during the
winter season and most of them had co-infection with respiratory
viruses, especially RSV. Second, the presence of staccato coughing,
cyanosis, and lymphocytosis was significantly different in
B. pertussis-positive cases compared with B. pertussis-negative
cases; however, there were no differences in clinical severity scores,
duration of oxygen therapy, and LOS in the hospital between two
groups. Third, only the B. pertussis culture-positive cases showed
the typical symptoms and laboratory findings of pertussis with
severe hospital course. To the best of our knowledge, this is the first
report in Japan prospectively examining the co-infection of
B. pertussis in infantile viral LRTI and evaluating the clinical char-
acteristics of mixed infections of B. pertussis and respiratory viruses.
One of the reasons for the relatively high detection of B. pertussis
in this study may be related to a small outbreak in a nearby junior
Y. Mihara, S. Yoshino, K. Nakatani et al. J Infect Chemother 27 (2021) 497e502

Table 3-A
Comparison of epidemiological characteristics in B. pertussis-positive versus B. pertussis-negative infants, hospitalized with LRTI.

B. pertussis positive B. pertussis negative Pa

Total Culture positive Culture negative Total Other pathogen No pathogen

(n ¼ 10) (n ¼ 4) (n ¼ 6) (n ¼ 39) (n ¼ 33) (n ¼ 6)

Age (days) 69.5 66.5 82 72 68 96 0.86

(median, range) (31e352) (37e95) (31e352) (14e343) (14e341) (24e343)
Male/Female 5/5 2/2 3/3 22/17 20/13 2/4 0.49
Daycare attendance 2/10 0/4 2/6 6/39 5/33 1/6 0.49
Cough in other family members 8/10 4/4 4/6 31/38 27/32 4/6 0.73
Vaccination status (DTaP)
0 dose 7 4 3 22 19 3 0.38
1 dose 1 0 1 3 2 1
2 doses 1 0 1 6 6 0
3 doses 1 0 1 8 6 2
Previous antibiotic treatment 3/10 1/4 2/6 16/39 13/33 3/6 0.40
Length of disease before NPS taken (days) 6 11.5b 4 5 5 6 0.29
(median, range) (0e15) (6e15) (0e10) (0e28) (0e28) (2e23)

Culture negative pertussis group consists of B. pertussis PCR/LAMP-positive cases. LRTI: Lower respiratory tract infection; DTaP: diphtheria, tetanus, pertussis; NPS: naso-
pharyngeal swab.
a
B. pertussis-positive group vs. B. pertussis-negative group.
b
B. pertussis culture-positive group vs. B. pertussis-negative group, P < 0.05.

Table 3-B
Comparison of symptom, signs on admission and diagnosis at discharge in B. pertussis-positive versus B. pertussis-negative infants, hospitalized with LRTI.

B. pertussis positive B. pertussis negative Pa

Total Culture positive Culture negative Total Other pathogen No pathogen

(n ¼ 10) (n ¼ 4) (n ¼ 6) (n ¼ 39) (n ¼ 33) (n ¼ 6)

Symptoms
Paroxysmal cough 7/10 (70%) 4/4 (100%) 3/6 (50%) 22/39 (56%) 19/33 (58%) 3/6 (50%) 0.40
Stacatto 4/10 (40%) 4/4 (100%)b 0/6 (0%) 0/39 (0%) 0/33 (0%) 0/6 (0%) 0.001
Whooping cough 1/10 (10%) 1/4 (25%) 0/6 (0%) 0/39 (0%) 0/33 (0%) 0/6 (0%) 0.46
Apnea 3/10 (30%) 0/4 (0%) 3/6 (50%) 7/38 (18%) 6/32 (19%) 1/6 (17%) 0.34
Cyanosis 6/10 (60%) 2/4 (50%) 4/6 (67%)c 7/39 (18%) 6/33 (18%) 1/6 (17%) 0.01
Post-tussive emesis 6/10 (60%) 4/4 (100%) 2/6 (33%) 20/39 (51%) 17/33 (52%) 3/6 (50%) 0.45
Fever 6/10 (60%) 1/4 (25%) 5/6 (83%) 24/39 (62%) 21/33 (64%) 3/6 (50%) 0.68
Sleep disturbance 9/10 (90%) 4/4 (100%) 5/6 (83%) 24/33 (72%) 21/27 (78%) 3/6 (50%) 0.24
Feeding difficulties 9/9 (100%) 4/4 (100%) 5/5 (100%) 37/39 (95%) 32/33 (97%) 5/6 (83%) 0.66
Diagnosis at discharge
Bronchitis 2/10 (20%) 0/4 (0%) 2/6 (33%) 9/39 (23%) 6/33 (18%) 3/6 (50%) 0.53
Bronchiolitis 7/10 (70%) 4/4 (100%) 3/6 (50%) 29/39 (74%) 26/33 (79%) 3/6 (50%) 0.60
Pneumonia 3/10 (30%) 0/4 (0%) 3/6 (50%) 7/39 (18%) 6/33 (18%) 1/6 (17%) 0.32

More than one diagnosis was mentioned in some cases.

Culture negative pertussis group consists of B. pertussis PCR/LAMP-positive cases.
a
B. pertussis-positive group vs. B. pertussis-negative group.
b
B. pertussis culture-positive group vs. B. pertussis-negative group, P < 0.05.
c
B. pertussis-culture negative group vs. B. pertussis-negative group, P < 0.05.

high school occurring just a few months before the number of
pertussis inpatients began to increase at our hospital. This small
outbreak might have led to a small epidemic in the surrounding
area. In previous reports, the prevalence of pertussis in infants and
children admitted to pediatric ward or pediatric intensive care unit
with various LRTI, mostly co-infected with RSV, varies from less
than 1%e23% [2e8,15e18]. Similar to our data, B. pertussis was
rather a common pathogen among young infants and children
admitted for LRTI including acute bronchiolitis or acute respiratory
failure in 1980s in United States [2] and in 2000e2006 in some
other countries, such as Finland [3,4], France [5], Israel [6,7], and
United Kingdom [8]. However, later studies from US and recent
report from Finland revealed that B. pertussis was no longer a
common pathogen in children hospitalized with bronchiolitis
during the winter season [15e18]. It is speculated that these vari-
ations in prevalence of B. pertussis in children may be related to a
reflection of the country's incidence of pertussis and vaccination
rates against pertussis. Furthermore, pertussis outbreaks occurred
in infants with viral LRTI during the winter season when RSV
bronchiolitis was prevalent in the above reports [2e8] and in our
500
study, which was different from the general epidemic of pertussis
from April to July. Therefore, B. pertussis screening is important,
especially in unvaccinated infants with an LRTI if the prevalence of
pertussis is high according to local epidemiological surveillance, as
shown in our study.
In Japan, with a long history of acellular pertussis vaccine (DTaP)
use with high vaccine coverage, the number of reported pertussis
cases increased in young adolescents and adults in the late 2000s,
and a nationwide epidemic occurred between 2008 and 2010 with
outbreaks in high schools and universities, as well as workplaces
[19]. The two peaks of age-specific incidence of pertussis were
determined as < 12 months old and 7e9 years old among children
[20]. DTaP is administered four times at 3, 4, 5, and 18 months, and
the need for additional preschool booster immunization is being
considered. We should be aware that young children, especially
unvaccinated infants, are still at risk for B. pertussis infection in our
country.
Most previous studies could not find any significant differences
between infants with pertussis alone, viral infection alone, or
mixed pertussis-virus infection, suggesting that pertussis is under
Y. Mihara, S. Yoshino, K. Nakatani et al. J Infect Chemother 27 (2021) 497e502

Table 4
Comparison of laboratory characteristics, viral findings, clinical characteristics during hospitalization in B. pertussis-positive versus B. pertussis-negative infants, hospitalized
with LRTI.

B. pertussis positive B. pertussis negative Pa

Total Culture positive Culture negative Total Other pathogen No pathogen

(n ¼ 10) (n ¼ 4) (n ¼ 6) (n ¼ 39) (n ¼ 33) (n ¼ 6)

White Blood Cells (n/mm3) 14,620 28,390b 9755 9450 9440 11,595 0.01
median (range) (8260e51630) (17,340e51630) (8260e14780) (5640e19580) (5640e19580) (7170e14640)
Lymphocyte Count (n/mm3) 6870 17,298b 5744 5113 4812 5558 0.02
median (range) (3965e34076) (11,878e34076) (3965e7686) (2018e9194) (2018e7909) (2477e9194)
C-reactive Protein (mg/dl) 0.44 0.07 0.45 0.5 0.5 0.63 0.25
median (range) (0.01e0.57) (0.01e0.2) (0.43e0.57) (0.03e13.37) (0.03e13.37) (0.03e1.09)
Identified viruses
RSV (sole) 5 0 5 22 22 e 0.54
hRV (sole) 2 2 0 1 1 e
hMPV (sole) 0 0 0 5 5 e
Parainfluenza 3 (sole) 0 0 0 1 1 e
RSV þ hRV 0 0 0 4 4 e
RSV þ hMPV 0 0 0 0 0 e
hRV þ hMPV 1 1 0 0 0 e
Supplemental oxygen 8 4 4 24 23 1 0.24
Duration of oxygen therapy 3 (0e8) 5d (3e8) 2 (0e7) 2 (0e8) 3 (0e8) 0 (0e2) 0.25
Duration of hospital stay 9 (3e14) 11b (6e13) 7 (3e14) 6 (4e12) 6 (4e10) 5.5 (2e12) 0.08
Clinical severity score 4 (2e6) 5b (4e6) 3 (2e5) 3 (0e6) 3 (0e6) 2 (1e5) 0.38
a
B. pertussis-positive group vs. B. pertussis-negative group.
b
B. pertussis culture-positive group vs. B. pertussis-negative group, P < 0.05.
d
B. pertussis culture-positive group vs. B. pertussis-negative group, P ¼ 0.05. Culture negative pertussis group consists of B. pertussis PCR/LAMP-positive cases. Duration in
days, median (range).

diagnosed in infants with respiratory tract infection, including
infants with presumed viral bronchiolitis [2e9]. In the present
study, apnea and cyanosis tended to be more frequently observed
in B. pertussis DNA-positive cases than in the other two groups.
There have been reported that the percentage of apnea and
cyanosis was higher in infants with pertussis than in infants with
RSV bronchiolitis [9], or in those with mixed pertussis-RSV
infection than those with RSV infection alone [5]. Viruses detec-
ted in infants with apnea and/or cyanosis in the present study
were hRV in 1/2 of B. pertussis culture-positive cases, RSV in 3/4 of
B. pertussis DNA-positive cases, and RSV in 8/10 of B. pertussis-
negative cases. The frequency of apnea and cyanosis was higher in
infants with B. pertussis DNA-positive even when compared with
the 26 RSV-positive infants with B. pertussis-negative (apnea; 50%
vs. 27%, cyanosis; 67% vs. 27%, respectively). Therefore, the dif-
ference in detected viruses among cases or co-infection of
B. pertussis and RSV might have affected the frequency of these
symptoms. Among pertussis cases in the present study, only
culture-positive pertussis infants were suspected, on a clinical
basis, as suffering from pertussis in addition to symptoms of virus-
associated bronchiolitis during hospitalization, and had more
severe clinical course than infants with viral infection alone.
However, none of the B. pertussis DNA-positive or B. pertussis-
negative patients were suspected to have pertussis on the bases of
clinical signs and laboratory results. Therefore, the cases diag-
nosed as B. pertussis DNA-positive could not be diagnosed as
pertussis without examination, and may have been a source of
infection to the surroundings. The longer duration of time from
the onset to sample collection in B. pertussis culture-positive cases
compared with that in the other two groups might be because the
samples were collected when the cough became paroxysmal after
catarrhal period, which usually occurs 1e2 weeks after the onset
of illness in classic cases. However, in B. pertussis DNA-positive
cases or B. pertussis-negative cases, staccato coughing or whoop-
ing were not observed even after hospitalization, indicating that
the samples were not collected before paroxysmal stage of
pertussis. Therefore, it was suggested that the difference in the
day of sample collection had no effect on the evaluation of their
symptoms at the time of hospitalization. He et al. suggested that
bacterial number influences clinical severity of respiratory
symptoms in pertussis cases [21]. Thus, the differences in bacterial
numbers between B. pertussis culture-positive cases and
B. pertussis DNA-positive cases might have caused the clinical
differences in each case. We consider that there is no possibility of
false-positive test results. This is because in all cases, a combined
test of both the IS481-based PCR and LAMP assay was performed.
The former is a highly sensitive test, but detects not only
B. pertussis but also Bordetella holmesii, and the latter is a highly
specific test for B. pertussis targeting the PT promoter region [13].
All of the DNA-positive cases showed positive results for both the
PCR and LAMP assay, except for one case. Furthermore, there is
another possibility that using highly sensitive diagnostic tech-
niques for pertussis may indicate infection outside the window of
acute infection. From the results of antibody response to PT,
B. pertussis DNA-positive cases might be asymptomatic carriers.
However, it may be difficult to determine the presence or absence
of infection only by serological evaluation, as there was a possi-
bility that antibodies against PT were not sufficiently elevated due
to the influence of maternal antibody, the effect of vaccination,
and immaturity of immune system in young infants. Our
pertussis-positive group, like the respective groups in many other
studies, was quite small and therefore underpowered to reveal
small, albeit real, differences between the groups. Further studies
with larger numbers of patients are needed to confirm our results.
In conclusion, B. pertussis is a common pathogen in children <12
months of age hospitalized for viral associated LRTI during the
winter season when there are local epidemics of pertussis. In the
absence of typical paroxysmal cough and lymphocytosis, we should
consider a diagnosis of pertussis in young children hospitalized for
presumed viral respiratory illness according to local epidemiolog-
ical surveillance data.
Declaration of competing interest
None declared.

501
Y. Mihara, S. Yoshino, K. Nakatani et al.
Acknowledgments
We thank Dr. Satoshi Honjo for helping with statistical analysis,
and Miho Miura, Asato Yasuda, and Eri Ito for technical assistance.
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