Clinical Utility of PCR for Common Viruses in Acute
Respiratory Illness
WHAT’S KNOWN ON THIS SUBJECT: Quantitative real-time
polymerase chain reaction allows sensitive detection of respiratory
viruses. The clinical significance of detection of specific viruses is
not fully understood, however, and several viruses have been
detected in the respiratory tract of asymptomatic children.
WHAT THIS STUDY ADDS: Our results indicate that quantitative
real-time polymerase chain reaction is limited at distinguishing
acute infection from detection in asymptomatic children for
rhinovirus, bocavirus, adenovirus, enterovirus, and coronavirus.
abstract
BACKGROUND: Acute respiratory illness (ARI) accounts for a large proportion of all visits to pediatric health facilities. Quantitative real-time
polymerase chain reaction (qPCR) analyses allow sensitive detection of
viral nucleic acids, but it is not clear to what extent specific viruses
contribute to disease because many viruses have been detected in
asymptomatic children. Better understanding of how to interpret viral
findings is important to reduce unnecessary use of antibiotics.
OBJECTIVE: To compare viral qPCR findings from children with ARI versus asymptomatic control subjects.
METHODS: Nasopharyngeal aspirates were collected from children
aged #5 years with ARI and from individually matched, asymptomatic,
population-based control subjects during a noninfluenza season. Samples were analyzed by using qPCR for 16 viruses.
RESULTS: Respiratory viruses were detected in 72.3% of the case
patients (n 5 151) and 35.4% of the control subjects (n 5 74)
(P 5 .001). Rhinovirus was the most common finding in both case
patients and control subjects (47.9% and 21.5%, respectively), with
a population-attributable proportion of 0.39 (95% confidence interval:
0.01 to 0.62). Metapneumovirus, parainfluenza viruses, and respiratory syncytial virus were highly overrepresented in case patients.
Bocavirus was associated with ARI even after adjustment for coinfections with other viruses and was associated with severe disease.
Enterovirus and coronavirus were equally common in case patients
and control subjects.
CONCLUSIONS: qPCR detection of respiratory syncytial virus, metapneumovirus, or parainfluenza viruses in children with ARI is likely
to be causative of disease; detection of several other respiratory
viruses must be interpreted with caution due to high detection rates
in asymptomatic children. Pediatrics 2014;133:e538–e545
e538
RHEDIN et al
AUTHORS: Samuel Rhedin, MB BB,a Ann Lindstrand, MD,
MPH,b Maria Rotzén-Östlund, MD-PhD,c,d Thomas
Tolfvenstam, MD-PhD,a,e Lars Öhrmalm, MD-PhD,a Malin
Ryd Rinder, MD-PhD,f Benita Zweygberg-Wirgart, MD-PhD,c,g
Ake Ortqvist, MD-PhD,f,h Birgitta Henriques-Normark,
MD-PhD,b,c,d Kristina Broliden, MD-PhD,a and Pontus Naucler,
MD-PhDa,e
aDepartment of Medicine Solna, Unit of Infectious Diseases,
Center for Molecular Medicine, Karolinska Institutet, Karolinska
University Hospital, Stockholm, Sweden; bSwedish Institute for
Communicable Disease Control, Stockholm, Sweden; cDepartment
of Clinical Microbiology, eDepartment of Infectious Diseases,
hDepartment of Medicine Solna, Infectious Diseases Unit,
fDepartment of Communicable Disease Control and Prevention,
Stockholm Karolinska University Hospital, Stockholm, Sweden;
gDepartment of Clinical Science and Education, South General
Hospital, Karolinska Institutet, Sachs’ Children and Youth
Hospital, Stockholm, Sweden; dDepartment of Microbiology
Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
KEY WORDS
children, case-control study, etiology, respiratory illness, viral
infections
ABBREVIATIONS
ARI—acute respiratory illness
CI—confidence interval
HAdV—human adenovirus
HBoV—human bocavirus
HCoV—human coronavirus
HEV—human enterovirus
hMPV—human metapneumovirus
HRV—human rhinovirus
OR—odds ratio
PAP—population-attributable proportion
PIV—parainfluenza virus
qPCR—quantitative real-time polymerase chain reaction
RSV—respiratory syncytial virus
URTI—upper respiratory tract infection
The findings and conclusions in this article are those of the
authors and do not necessarily represent the views of the
funding agency.
(Continued on last page)
ARTICLE
Although acute respiratory illness (ARI)
in childrenaccountsfora large part ofall
visits to pediatric health facilities and is
a great economic burden on society,1,2
our tools to diagnose the etiologic
agents have until recently been limited.3
Treatment with antibiotics induces development of antibiotic resistance in
bacteria4 and has a negligible effect on
most ARIs, which generally are of viral
origin.5,6 Nevertheless, antibiotics are
frequently prescribed due to lack of
clinically valid diagnostic tests verifying
a viral etiology.7 Sensitive methods, such
as quantitative real-time polymerase
chain reaction (qPCR) analyses on nasopharyngeal samples, for a number of
viruses have been introduced in the
clinic as a sensitive diagnostic tool
among children with respiratory tract
infection.8 However, whereas detection
of some viruses, such as influenza virus
and respiratory syncytial virus (RSV),
clearly is predictive for respiratory
disease,9–11 the clinical significance
upon detection of several other viruses
needs further investigation. The interpretation of a viral detection is
complicated by the fact that infections
with multiple viruses are common in
children with ARI and that many viruses
have lately been reported to be found
also in asymptomatic children.12–17
Numerous case series investigating respiratory viruses in hospitalized children with ARI have been published.18–20
However, data are available from only
a few case-control studies that examined to what extent specific respiratory
viruses cause disease.12,14,15,21 Furthermore, these studies had limitations in
that they used hospital-based control
subjects, which might have biased
the results. To address these issues,
we conducted a matched case-control
study outside the influenza season to
compare viral qPCR findings in nasopharyngeal aspirates from children with
ARI versus population-based asymptomatic control subjects matched on
age and calendar time.
PEDIATRICS Volume 133, Number 3, March 2014
Study Population
collected from the parents of all included children before sampling.
A matched case-control study was
conducted with case patients consecutively enrolled at the pediatric emergency department at Sachs’ Children
and Youth Hospital (Stockholm, Sweden)
between September 1, 2011, and
January 30, 2012. Case patients were
children aged #5 years with $1 of the
following symptoms: coryza (rhinorrhea or nasal congestion), sore throat,
earache, cough, sputum production, or
dyspnea. Children were only included
once in the study. One matched control
subject for each case subject according to calendar time (614 days) and
age (66 months) was selected from an
ongoing population-based study of
pneumococcal carriage.22 In that study,
children were enrolled at the time of
their routine visits to child welfare
centers in Stockholm for vaccination
within the childhood immunization
program (covering ∼99.6% of the
population in Sweden). If no control
subject was found, intervals were expanded to calendar time (630 days)
and age (612 months). Children who
were reported to have had episodes of
respiratory disease within the last 7
days were not eligible as control subjects. Written informed consent was
Information from both case patients and
control subjects regarding chronic diseases, vaccination, and sociodemographic status was collected by using
a standardized questionnaire. Chronic
diseases were categorized into 2 groups:
(1) “asthma” (including both allergic and nonallergic asthma); and (2)
“other” (Table 1). Clinical parameters of
case patients were registered at admission. Elevated breathing frequency
was defined as $50 breaths per minute
in children aged ,1 year and $40
breaths per minute in children aged 1 to
5 years. Tachycardia was defined as
a pulse $160 beats per minute in children aged ,1 year and $120 beats per
minute in children aged 1 to 5 years.
Fever was defined as a body temperature $38.0°C. Information about treatment, outcome, and routine biochemical,
microbiologic, and radiologic analyses
were collected from medical records.
Children with reported regular (.3 times
a month) inhalation of b2-adrenergic
agents and/or glucocorticoids but
without reported chronic disease were
designated as having asthma.23 Discharge diagnoses were categorized
according to International Classification of Diseases, 10th Revision, codes
METHODS
TABLE 1 Characteristics of Study Subjects
Characteristic
Age, mo, median (IQR)
Male gender
No. of children in household, median (IQR)
Attending day care
Breastfeeding
$1 parent smoking
Chronic diseases
Asthma
Othera
Ongoing antibiotic treatment
Antibiotic treatment last year
Influenza vaccination last year
Visit abroad last 6 mo
University degree of $1 parent
Case Patients (n = 209) Control Subjects (n = 209)
10 (5–21)
124 (59.3)
2 (1–2)
115 (55.0)
79 (38.7)
31 (15.0)
21 (10.0)
16 (7.7)
5 (1.9)
11 (5.3)
64 (31.1)
6 (3.1)
35 (16.8)
140 (67.6)
7 (4–13)
106 (50.7)
2 (1–2)
94 (45.0)
102 (48.8)
21 (10.0)
4 (1.9)
2 (1.0)
2 (1.0)
1 (0.5)
25 (12.0)
1 (0.5)
40 (19.1)
161 (77.0)
P
.06
.10
.20
.03
.01
.24
,.001
.001
.45
.006
,.001
.13
.60
.04
P values calculated with McNemar’s test and paired t test. Data are presented as absolute number and percentage if not
otherwise specified. IQR, interquartile range.
a Case patients: Down syndrome, n = 1; bronchopulmonary dysplasia, n = 1; heart murmur (suspected bronchopulmonary
dysplasia), n = 1; laryngomalacia, n = 1; hydronephrosis, n = 1. Control subjects: mild epilepsy, n = 1; organic heart disease, n = 1.
e539
(Fig 1). The study was approved by
the regional ethical review board in
Stockholm.
Sampling and Microbiologic
Analyses
Nasopharyngeal aspirates from case
patients and control subjects were
obtained by using an identical aspiration technique and were diluted in 3 mL
of saline. qPCR was performed at the
Clinical Microbiological Laboratory at
Karolinska University Hospital, which is
accredited by the Swedish Board for
Accreditation and Conformity Assessment (ISO 15189:2007). Sixteen different viruses were included in the
panel: influenza A seasonal as well as
H1N1pdm09, influenza B, human adenovirus (HAdV), human bocavirus
(HBoV), human coronavirus serotypes
229E, NL63, OC43, and HKU1 (HCoV),
human enterovirus (HEV), human
metapneumovirus (hMPV), human rhinovirus (HRV), parainfluenza virus (PIV)
1 to 3, and RSV.24 Due to cross-reactivity
between HRV and HEV, samples positive
for both viruses were further analyzed
by using an in-house qPCR for HEV.25
We used the term coinfection to represent detection of at least 2 viruses in the
same individual (although detection by
using qPCR does not necessarily equal
ongoing infection, as emphasized).8
Statistical Analyses
Data were analyzed by using Stata version12.1 (StataCorp, CollegeStation,TX).
Paired t tests, Wilcoxon signed-rank
tests, and McNemar’s test were used
on paired data as appropriate.26 Withingroup analyses of categorical data were
performed by using x2 tests and Fisher’s exact test, and independent continuous data were analyzed by using the
Mann-Whitney U test. Unadjusted and
multivariate odds ratios (ORs) were
calculated with conditional logistic regression to assess the association between viruses and ARI. Because the risk
associated with 1 virus can be confounded by coinfection with other viruses, we adjusted for infection with
other viruses by including virus-specific
data as single variables in a multivariate regression model. Two multivariate
models were constructed in which the
first model included age and viruses,
and the second model included age,
viruses, and clinical and sociodemographic variables. Selection of variables
for the second model was performed in
the following way. First, all variables
that were associated with ARI in univariate analyses (P , .2) were included
in the model. Second, variables with a
P value $.2 in the multivariate model
were removed from the model. Finally,
we removed variables that caused
large drifts of ORs due to small sample
bias.27 To estimate how much specific
viruses attributed to ARI in the population, population-attributable proportion (PAP) for viruses that were
significantly associated with ARI
was calculated as follows: exposure
among case patients 3 (adjusted OR
– 1)/adjusted OR, with 95% confidence
intervals (CIs).28
RESULTS
Characteristics of Study Subjects
During the study period, 229 case
patients with ARI were enrolled. Due to
limited inclusion of control subjects
during the holiday season, control
FIGURE 1
Discharge diagnosis according to viral detections in case patients. The number of case patients positive for each virus is shown above bars. International
Classification of Diseases, 10th Revision, discharge diagnoses stratified into the following 7 groups: acute URTI (n = 118): J06.9 acute URI, B34.9 viral infection
(unspecified). Bronchitis (n = 24): J20.9 acute bronchitis, J21.0 bronchiolitis (RSV), J21.1 bronchiolitis (metapneumovirus). Pneumonia (n = 20): J12.1 RS virus
pneumonia, J15.9 bacterial pneumonia, J18.0 bronchopneumonia, J18.9 pneumonia (unspecified). Otitis (n = 20): H66.0 acute media otitis. Asthma (n = 8):
J45.1 nonallergic asthma, J45.8 mixed asthma, J45.9 asthma (unspecified). Croup (n = 8): J04.2 laryngotracheitis, J05.0 acute obstructive laryngitis. Other (n =
25): A08.4 diarrhea, viral cause, B34.0 adenovirus infection (unspecified), J03.0 streptococcal tonsillitis, J03.9 acute tonsillitis (unspecified), J44.8 other
obstructive pulmonary disease, L50.9 cyst, pilonidal, N10.9 acute tubulointerstitial nephritis, R05.9 cough, R10.4 colic (recurrent), R50.9 fever, Z00.0 general
medical examination, Z04.8 examination medicolegal reason.
e540
RHEDIN et al
ARTICLE
subjects were not found for 20 case
patients in December and January. These
case patients were excluded in all
matched case-control analyses. Characteristics of study subjects are listed in
Table 1. Case patients were significantly
more likely to attend day care (P = .03)
and less likely to be breastfed (P = .01) at
the time of inclusion. Moreover, 10.0% of
the case patients suffered from a chronic
disease, predominantly asthma (n = 16
[7.7%]), compared with 1.9% in control
subjects (P , .001), and case patients
were to a larger extent receiving current
treatment with antibiotics (P = .006) or
had received antibiotic treatment within
the last year (P , .001). Finally, parents
with a university degree were more common among control subjects (P = .04).
The 20 case patients who were excluded
from the matched analyses due to lack
of control subjects were significantly
older and to a larger extent positive for
RSV and HRV compared with the included case patients (P = .04, P = .03,
and P = .02, respectively) (Supplemental Table 4).
In total, 24 case patients (10.5%) were
admitted to an inpatient ward, and 48
patients (21.0%) were treated with
antibiotics (Supplemental Table 5).
Viruses Associated With ARI
HRV was detected most frequently from
September through November and was
found at substantially lower levels in
December and January (Fig 2). This
finding was true for both case patients
and control subjects. hMPV, HBoV, and
RSV also followed an epidemic pattern,
increasing in December and January; all
RSV episodes in case patients were
detected in late December through
January. No influenza viruses or HCoV
serotype 229E was detected during the
study period.
One or more respiratory virus was
detected in 151 (72.3%) of the case
patients and 74 (35.4%) of the control
subjects (Table 2). PIV (compiled), PIV1,
RSV, and hMPV had the highest ORs for
ARI, followed by HBoV and HRV. PIV was
detected in 7.7% of the case patients
compared with 0.5% of the control
subjects, with PIV1 detected in 4.3% of
case patients. PIV2 and PIV3 were only
detected among case patients (2 and 5
case patients, respectively). RSV was
detected in 5.3% of case patients and
0.5% of control subjects, and hMPV was
detected in 4.8% of case patients and
1.0% of control subjects. HBoV was
detected in 15.8% of case patients but
was also detected relatively often
among control subjects (4.3%). HRV, the
most prevalent virus in the study population, was detected in 47.9% of case
patients and 21.5% of control subjects.
In contrast, HCoV and HEV were equally
prevalent in case patients and control
subjects. Nevertheless, HCoV serotype
OC43 was only found in case patients
(n = 4). The ORs were fairly stable for
most viruses when the estimates of
the 2 adjusted models were compared.
Serving as an estimate of the fraction of
children that would have been prevented from having respiratory disease
if the specific virus had not been present, PAP was calculated for the most
frequently detected viruses (based on
ORs from model 2). HRV, HBoV, and PIV
(compiled) accounted for the largest
PAP: 0.39 (95% CI: 0.01 to 0.62), 0.12 (95%
CI: –0.06 to 0.28), and 0.08 (95% CI: –0.13
to 0.24), respectively.
Viral Association With Clinical
Presentation and Diagnosis
In an attempt to clinically distinguish the
different viral infections, clinical parameters, symptoms, and discharge diagnoses were compared between case
patientspositiveforaspecificvirusversus
case patients negative for that specific
virus. hMPV infection was associated with
fever (P = .003), increased respiratory
rate (P = .035), tachycardia (P = .01), and
decreased O2 saturation (P = .05) (Table 3). HAdV was associated with decreased O2 saturation (P = .05); HBoV was
associated with increased respiratory
rate (P = .003), tachycardia (P = .01), and
reported coughing (P = .017); HRV was
associated with coryza (P = .04) and absence of fever (P = .008); and RSV was
associated with increased respiratory
rate (P = .04) and wheezing (P = .03).
Case patients with croup, bronchitis, and
asthma were most likely to test positive
for $1 virus (all, 88%). Case patients
diagnosed as having pneumonia, acute
upper respiratory tract infection (URTI),
FIGURE 2
Viral detections over time in case patients and control subjects between September 1, 2011, and January 30, 2012. Detections of specific viruses shown as
percentages of sampled children for (A) case patients and (B) control subjects.
PEDIATRICS Volume 133, Number 3, March 2014
e541
TABLE 2 Respiratory Viruses Associated With Acute Respiratory Illness
Detected Virus
Case Patients (n = 209)
Control Subjects (n = 209)
Unadjusted OR (CI 95%)
Model 1a OR (CI 95%)
Model 2b OR (CI 95%)
HAdV
HBoV
HCoV (any)
HKU1
NL63
OC43
HEV
hMPV
PIV (any)
PIV 1
PIV 2
PIV 3
RSV
HRV
Positive any virus
Single infection
Coinfection
2 pathogens
3 pathogens
4 pathogens
18 (8.6)
33 (15.8)
11 (5.3)
7 (3.4)
—
4 (1.9)
5 (2.4)
10 (4.8)
16 (7.7)
9 (4.3)
2 (1.0)
5 (2.4)
11 (5.3)
100 (47.9)
151 (72.3)
109 (52.2)
42 (20.1)
32 (15.3)
9 (4.3)
1 (0.5)
10 (4.8)
9 (4.3)
12 (5.7)
11 (5.3)
1 (0.5)
—
6 (2.9)
2 (1.0)
1 (0.5)
1 (0.5)
—
—
1 (0.5)
45 (21.5)
74 (35.4)
63 (30.1)
11 (5.3)
9 (4.3)
2 (1.0)
—
2.3 (0.9 to 6.1)
4.4 (2.0 to 10.1)
0.9 (0.4 to 2.0)
0.6 (0.2 to 1.6)
NA
NA
0.8 (0.3 to 2.7)
5.0 (1.1 to 22.8)
16.0 (2.1 to 120.6)
9.0 (1.1 to 71.0)
NA
NA
11.0 (1.4 to 85.2)
3.5 (2.2 to 5.6)
4.9 (3.0 to 7.9)
4.3 (2.6 to 7.1)
12.3 (5.0 to 30.4)
2.1 (0.6 to 7.4)
4.4 (1.6 to 11.8)
1.0 (0.4 to 2.7)
0.5 (0.1 to 1.9)
NA
NA
1.7 (0.4 to 6.9)
12.0 (2.0 to 71.6)
33.6 (3.8 to 294.4)
23.7 (2.4 to 230.0)
NA
NA
8.9 (0.8 to 96.1)
5.1 (2.9 to 9.1)
2.3 (0.6 to 8.2)
4.7 (1.7 to 13.1)
1.1 (0.3 to 3.4)
0.5 (0.1 to 2.0)
NA
NA
1.6 (0.4 to 6.5)
11.9 (1.9 to 73.1)
57.0 (5.3 to 614.6)
41.8 (3.5 to 501.9)
NA
NA
8.4 (0.7 to 96.8)
5.2 (2.9 to 9.4)
ORs calculated with conditional logistic regression analyses. Data are presented as absolute number and percentage if not otherwise specified. NA, not applicable.
a Parameters included in multivariate model: HAdV, HBoV, HCoV, HEV, hMPV, PIV, RSV, HRV, and age.
b Parameters included in multivariate model: HAdV, HBoV, HCoV, HEV, hMPV, PIV, RSV, HRV, chronic disease, university degree of $1 parent, and age.
and otitis tested positive in 58%, 64%,
and 74%, respectively. Viral findings in
relation to discharge diagnosis are
shown in Fig 1. HCoV was negatively
associated with a diagnosis of acute
URTI (P = .005). Furthermore, patients
diagnosed with croup were more likely
to test positive for PIV (P , .001), and
patients with bronchitis were more
likely to be infected with HBoV (P = .04).
To assess the independent effect of specific viral infections, viruses that were
significantly associated with the aforementionedclinicalfindingswerestratified
into single infections and coinfections. No
significant differences were found between patients with single infections
compared with coinfected patients (data
not shown). However, there was a tendency that increased respiratory rate was
more common in HBoV coinfection compared with HBoV single infection (P = .08),
and fever was reported more commonly
in HRV coinfection compared with HRV
single infection (P = .08).
Clinical Impact of Coinfections
In 42 (20.1%) case patients, .1 virus
was detected. Of these, 32 (15.3%) were
e542
RHEDIN et al
positive for 2 viruses, 9 case patients
(4.3%) were positive for 3 viruses, and 1
case patient (0.5%) was positive for 4
concurrent viruses (Table 2). Coinfections of viruses were more common
in case patients compared with control
subjects (20.1% and 5.3%, respectively)
and were associated with a higher risk
of ARI (OR: 12.3 [95% CI: 5.0 to 30.4])
compared with single infections (OR: 4.3
[95% CI: 2.6 to 7.1]). RSV, HAdV, and HBoV
were the viruses most frequently
detected in coinfection with other viruses among the case patients (in 80%,
74%, and 69%, respectively), and the
most common virus pairs were HBoV/
HRV, HAdV/HRV, and HBoV/RSV (n = 13,
n = 11, and n = 9) (Supplemental Table 6).
Case patients with coinfections had
more severe disease compared with
case patients with single infections, with
the following being more prevalent: increased respiratory rate (P = .009),
tachycardia (P = .05), decreased O2 saturation (P = .04), reported fever (P = .05),
discharge diagnosis of pneumonia (P =
.006), and bronchitis (P = .02), as well as
decreased probability of a diagnosis of
acute URTI (P = .01).
DISCUSSION
A better understanding of the etiologic
role of viruses in respiratory diseases is
needed to develop new targeted therapies and reduce overconsumption
of antibiotics. In this matched casecontrol study, we assessed how specific
respiratory viruses can be attributed
to ARI by investigating viral qPCR data
in children with ARI and in matched
asymptomatic control subjects. The
strength of our study was the unique
population-based asymptomatic control
subjects who were sampled consecutively from different areas of Stockholm,
thus serving as a good estimate of the
background prevalence of the different
viruses in the source population. Proper
control selection is of highest importance for garnering valid information
regarding the clinical interpretation of
respiratory virus detection in children.
Moreover, the study design allowed
adjustments for important confounders
such as age, calendar time, and viral
coinfections.
We report that 1 or more respiratory
virus was detected in 72.3% of the case
ARTICLE
TABLE 3 Clinical Parameters and Symptoms Associated With Viral Infections in Case Patients
Parameter
Symptoms on examination
Increased respiratory ratea
Tachycardiac
O2 saturation #95%
Body temperature $38.0°C
Chest wall retractions
Nasal flare
Grunting
Wheezingd
Affected general status
Admitted to inpatient ward
Reported symptomse
Coryza
Sore throat
Earache
Coughing
Fever
Dyspnea
Trouble feeding/drinking
Gastrointestinal symptoms
All (n = 229)
HAdV (n = 19)
HBoV (n = 39)
HCoV (n = 13)
hMPV (n = 13)
PIV (n = 17)
HRV (n = 104)
RSV (n = 15)
73 (33)
97 (46)
26 (11)
67 (30)
38 (17)
4 (2)
3 (1)
30 (13)
30 (13)
24 (10)
7 (37)
9 (50)
5 (26)*,b
9 (47)
2 (11)
—
—
1 (6)
5 (26)
2 (11)
20 (56)**,b
25 (66)*,b
7 (18)
16 (41)
6 (15)
2 (5)
—
9 (23)
4 (11)
6 (15)
6 (46)
3 (23)
1 (7)
4 (29)
3 (21)
—
—
3 (23)
2 (14)
1 (7)
8 (62)*,b
10 (83)*,b
4 (31)*,b
9 (69)**,b
3 (23)
1 (8)
—
2 (15)
4 (31)
2 (15)
7 (41)
8 (53)
1 (6)
5 (29)
3 (19)
—
—
1 (6)
1 (6)
1 (6)
28 (29)
39 (42)
1 (12)
21 (20)**,b
21 (21)
2 (2)
2 (2)
14 (14)
12 (12)
12 (12)
9 (60)*,b
8 (53)
4 (27)
7 (47)
3 (20)
—
—
5 (33)*,b
3 (20)
4 (27)
32 (82)
10 (26)
6 (15)
39 (100)*,b
30 (77)
23 (59)
18 (46)
17 (44)
11 (79)
4 (29)
3 (21)
14 (100)
9 (64)
10 (71)
3 (21)
10 (71)
7 (54)**,b
3 (23)
3 (23)
13 (100)
13 (100)**,b
6 (46)
3 (23)
5 (39)
12 (71)
4 (24)
1 (6)
17 (100)
13 (77)
11 (65)
4 (24)
9 (53)
93 (91)*,b
24 (24)
14 (14)
94 (92)
56 (55)**,b
60 (59)
32 (31)
50 (48)
194 (85)
63 (28)
31 (14)
204 (90)
146 (65)
126 (56)
83 (37)
122 (53)
18 (95)
7 (37)
3 (16)
16 (84)
16 (84)
11 (58)
10 (53)
11 (58)
15 (100)
4 (27)
3 (20)
15 (100)
12 (80)
12 (80)
9 (60)
7 (47)
* P , .05 **P , .01 ***P , .001.
a $50 breaths per minute for childen aged ,1 year, $40 breaths per minute for children aged 1 to 5 years.
b Significant differences between case patients positive and negative for the specific virus.
c $160 beats per minute for children aged ,1 year, $120 beats per minute for children aged 1 to 5 years.
d Presence of wheezing or rhonchi on pulmonary auscultation.
e Symptoms reported by parents.
patients with ARI and 35.4% of the
control subjects. These findings are in
line with previous studies that have
reported viral findings in 54% to 72% of
children with ARI and 19% to 40% in
asymptomatic children,12,14,15 emphasizing the need for adequate control
subjects when assessing the etiologic
fraction of different respiratory viruses. PIV, hMPV, and RSV were associated with high relative risks for ARI
and were only rarely detected in control subjects, which is concordant with
previous studies.11,17 These viruses all
seem to be rapidly cleared from the
respiratory tract after an infection,
making qPCR a suitable diagnostic
method.8 Interestingly, hMPV was associated with severe disease presentations such as fever, decreased O2
saturation, increased respiratory rate,
and tachycardia. Recent reports support that hMPV is a significant respiratory pathogen capable of causing
severe disease in children,29 yet the
clinical pattern of disease is not fully
understood. In view of our findings that
PEDIATRICS Volume 133, Number 3, March 2014
RSV, PIV, and hMPV were strongly associated with ARI and reports of nosocomial transmission of these viruses,30,31
further studies of control interventions
such as cohort nursing are warranted
for these infections.32
HBoV is reportedly commonly detected
in coinfection with other viruses, and its
pathogenicity in humans has been debated.21,33,34 In our study, HBoV was associated with ARI even after adjustment
for coinfection with other viruses, and
HBoV was associated with tachycardia,
increased respiratory rate, and coughing (which suggest lower respiratory
tract infection). However, both HBoV and
HRV were detected at such a high frequency in control subjects that it might
be hard to interpret the clinical significance of a positive qPCR finding. Our
data indicate that 39% (95% CI: 1 to 62)
of ARI in this population could be attributed to HRV, however.
HEV and HCoV were detected at equal
levels in both case patients and control
subjects. However, HCoV serotype OC43
was only detected in case patients, but
due to limited sample size, we could
perform only grouped analyses of these
viruses; it is likely that different HEV
and serotypes of HCoV have different
disease-causing potential.25,35 Nevertheless, our results indicate that the
current qPCR method needs to be more
specific to have a clinical value for differential diagnostic purposes.
The clinical importance of viral coinfections is not fully understood.19 In our
data, coinfections were associated with
increased risk of ARI compared with
single infections, and they were also
associated with more severe disease.
However, we did not have sufficient
power to fully address how different
respiratory viruses interact in the development of ARI, which is of great importance when assessing the potential
impact of targeted interventions.
We performed sampling of control
subjects from multiple child welfare
centers at the time of routine vaccination to obtain a representative
e543
sample of the background prevalence
of respiratory viruses in the child
population. However, the frequency of
parents having a university degree
was higher among control subjects
compared with the general population
of 20- to 40-year-olds in Stockholm
(68.1% vs 49.7%).36 Although the populations were not perfectly comparable, it might indicate that our control
selection failed to include all socioeconomic groups. Moreover, study
subjects were not followed up with
consecutive sampling. Hence, we
could not assess if viral findings in
control subjects represent early detection of an infection, asymptomatic
carriage, low-virulent infection, or
prolonged shedding. In addition,
samples were not systematically collected on all study subjects to assess
bacterial infections, and some case
patients are likely to have suffered
from bacterial or mixed infection
rather than solely from a viral infection. This possibility is indeed a limitation to the current study that
impairs the translation of the findings
into decision-making policies regarding antibiotic treatment. However, the
diagnostics of bacterial infections in
children with respiratory infections
are difficult because blood culture
results are rarely positive, sputum or
samples from bronchoalveolar lavage
are difficult to obtain,37 and the clinical
significance of bacterial diagnostics
from the nasopharynx in children with
ARI is limited due to high rates of colonization of common bacterial pathogens.38,39 Finally, the qPCR method used
in our study was not appropriate to
adequately assess viral load, which has
been found to be associated with severity of disease.40
ACKNOWLEDGMENTS
We acknowledge Pia Andersson, Kerstin
Jämtberg, Carita Krokstrand, Karolin
Petersson, Jessica Darenberg, Malin
Svensson, Henrik Hurtigh, and the staff
at Sachs’ Children and Youth Hospital,
Karolinska Microbiological Laboratory
and Swedish Institute for Communicable Disease Control.
7. Byington CL, Castillo H, Gerber K, et al. The
effect of rapid respiratory viral diagnostic
testing on antibiotic use in a children’s
hospital. Arch Pediatr Adolesc Med. 2002;
156(12):1230–1234
8. Jartti T, Söderlund-Venermo M, Hedman K,
Ruuskanen O, Mäkelä MJ. New molecular virus detection methods and their clinical value
in lower respiratory tract infections in children. Paediatr Respir Rev. 2013;14(1):38–45
9. Hall CB, Weinberg GA, Iwane MK, et al. The
burden of respiratory syncytial virus infection in young children. N Engl J Med.
2009;360(6):588–598
10. Peltola V, Ziegler T, Ruuskanen O. Influenza
A and B virus infections in children. Clin
Infect Dis. 2003;36(3):299–305
11. Berkley JA, Munywoki P, Ngama M, et al.
Viral etiology of severe pneumonia among
Kenyan infants and children. JAMA. 2010;
303(20):2051–2057
12. Jansen RR, Wieringa J, Koekkoek SM, et al.
Frequent detection of respiratory viruses
without symptoms: toward defining clinically relevant cutoff values. J Clin Microbiol. 2011;49(7):2631–2636
13. van Benten I, Koopman L, Niesters B, et al.
Predominance of rhinovirus in the nose of
symptomatic and asymptomatic infants.
Pediatr Allergy Immunol. 2003;14(5):363–370
van Gageldonk-Lafeber AB, Heijnen ML,
Bartelds AI, Peters MF, van der Plas SM,
Wilbrink B. A case-control study of acute
respiratory tract infection in general
practice patients in the Netherlands. Clin
Infect Dis. 2005;41(4):490–497
van der Zalm MM, van Ewijk BE, Wilbrink B,
Uiterwaal CS, Wolfs TF, van der Ent CK. Respiratory pathogens in children with and
without respiratory symptoms. J Pediatr.
2009;154(3):396–400, e1
Nokso-Koivisto J, Kinnari TJ, Lindahl P, Hovi
T, Pitkäranta A. Human picornavirus and
coronavirus RNA in nasopharynx of children without concurrent respiratory
symptoms. J Med Virol. 2002;66(3):417–420
Mathisen M, Strand TA, Valentiner-Branth P,
et al. Respiratory viruses in Nepalese children
with and without pneumonia: a case-control
study. Pediatr Infect Dis J. 2010;29(8):731–735
Arden KE, McErlean P, Nissen MD, Sloots TP,
Mackay IM. Frequent detection of human
rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol. 2006;
78(9):1232–1240
CONCLUSIONS
Our study indicates that a qPCR finding of
RSV, hMPV, or PIV is likely to be causative
of disease in children with ARI. In contrast, detection of several other viruses
such as HBoV, HRV, HAdV, HCoV, and HEV
must be interpreted with caution due
to high detection rates among healthy
children. Future studies should focus on
how to improve differential diagnostics
with refined molecular techniques of the
latter viruses to further characterize
their pathogenicity in humans.
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(Continued from first page)
Mr Rhedin coordinated enrollment of case patients at the study site, collected and interpreted data from patient history files of the case patients, performed
statistical analyses, and wrote the initial draft of the manuscript; Dr Naucler designed and conceptualized the study and supervised collection of samples,
analyses, and writing of the manuscript; Dr Broliden designed and supervised the study, interpreted data analyses, and reviewed and revised the manuscript; Drs
Lindstrand, Henriques-Normark, and Ortqvist designed the data collection instruments, managed and supervised data collection of the control subjects,
interpreted sociodemographic and clinical data of the control subjects, and reviewed and revised the manuscript; Drs Öhrmalm, Rotzén-Östlund, Tolfvenstam, and
Zweygberg-Wirgart designed the standardized sampling protocol, conducted, supervised, and interpreted the microbiologic analyses, and reviewed and revised
the manuscript from a clinical microbiologic point of view; and Dr Rinder supervised data collection at one of the study sites, critically reevaluated medical
histories and interpreted clinical signs of infection of the case patients, and reviewed and revised the manuscript from a pediatric point of view. All authors
approved the final manuscript as submitted.
www.pediatrics.org/cgi/doi/10.1542/peds.2013-3042
doi:10.1542/peds.2013-3042
Accepted for publication Dec 18, 2013
Address correspondence to Samuel Rhedin, MD-PhD student, Unit of Infectious Diseases, Center for Molecular Medicine, L8:01, Karolinska Institutet, 171 76
Stockholm, Sweden. E-mail: [email protected]
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright © 2014 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.
FUNDING: This work was supported by grants from the Karolinska Institutet.
POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.
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