Accepted Manuscript Is viral co-infection a predictor of severity in children with viral respiratory infections? Sandra A. Asner, MD Msc Winsley Rose, MD Astrid Petrich, PhD Susan Richardson, MD Dat Tran, MD MSc PII:
S1198-743X(14)00062-7
DOI:
10.1016/j.cmi.2014.08.024
Reference:
CMI 61
To appear in:
Clinical Microbiology and Infection
Received Date: 17 April 2014 Revised Date:
21 July 2014
Accepted Date: 4 August 2014
Please cite this article as: Asner SA, Rose W, Petrich A, Richardson S, Tran D, Is viral co-infection a predictor of severity in children with viral respiratory infections?, Clinical Microbiology and Infection (2014), doi: 10.1016/j.cmi.2014.08.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Is viral co-infection a predictor of severity in children with viral respiratory infections?
2
Intended category: Original article
3
Sandra A. Asner1,3,4, MD, Msc; Winsley Rose¹, MD; Astrid Petrich2, PhD; Susan Richardson2,
4
MD; Dat Tran¹, MD, MSc
RI PT
1
5
1
8 9
Division of Infectious Diseases, Department of Paediatrics, The Hospital for Sick
SC
7
Affiliations
Children, University of Toronto, Toronto, Ontario, Canada 2
Division of Microbiology, Department of Paediatric Laboratory Medicine and
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10
Department of Pathobiology and Laboratory Medicine, The Hospital for Sick Children,
11
University of Toronto, Toronto, Ontario, Canada 3
13
15 16
Lausanne, Lausanne, Switzerland 4
Division of Infectious Diseases, Department of Medicine, University Hospital Lausanne, Switzerland
EP
14
Paediatric Infectious Diseases unit, Department of Paediatrics, University Hospital
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12
Address correspondence to: Dat Tran; Division of Infectious Diseases; Department of
18
Paediatrics; The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada
19
M5G 1X8, Tel: 416-813-7654 ext 203530, Fax: 416-813-8404 Email: [email protected]
20
Running title : Respiratory viral co-infection in children
21
Keywords: viral respiratory infections, viral co-infection, clinical disease severity, molecular
22
diagnosis, rhinovirus/enterovirus (HRV/ENT), respiratory syncytial virus (RSV)
23
Word count: Abstract: 240; Text: 2’647; Tables: 4; Figures: 0; References: 20
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Authorship/Contribution
25 Sandra A. Asner: Dr. Asner helped conceptualizing, designing the study. She was involved in the
27
data collection (patient’s chart review) and built a database with all clinical information. She
28
carried out all the analyses, drafted the initial manuscript and approved the final manuscript as
29
submitted.
RI PT
26
SC
30
Winsley Rose: Dr Rose was involved in the data collection (patient’s chart review) and
32
preliminary analyses. He also critically reviewed the manuscript and approved the final
33
manuscript as submitted.
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34
Astrid Petrich: Dr Petrich helped conceptualizing the study. She critically reviewed and
36
approved the final manuscript as submitted.
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Susan E. Richarsdon: Dr. Richardson helped coordinating the data collection and helped
39
conceptualizing and designing the study. She critically reviewed and approved the final
40
manuscript as submitted.
42
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Dat Tran: Dr. Tran conceptualized and designed the study, critically reviewed, revised the
44
manuscript and the analyses. Dr Tran approved the final manuscript as submitted.
45 46
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Abstract
48
Background: Molecular assays have resulted in increased detection of viral respiratory
49
infections, including viral co-infection, from children with acute respiratory infections (ARIs).
50
Yet, the clinical severity of viral co-infection compared to single viral infection remains
51
uncertain.
52
Methods: Retrospective study of children presenting with ARIs comparing clinical severity of
53
single respiratory viral infection to viral co-infection, detected on mid-turbinate swabs by
54
molecular assays. Patient characteristics and measures of clinical severity were abstracted from
55
health records.
56
Results: 472 virus-infected children were included: 391 with a single viral infection and 81 with
57
viral co-infection. Viral status did not affect admission to hospital (OR=0.8; 95 % C.I. 0.5-1.4;
58
p=0.491) or clinical disease severity among inpatients (OR=0.8; 95% C.I. 0.5-1.5; p=0.515) after
59
adjusting for age and underlying comorbidities. However, children infected with
60
rhinovirus/enterovirus (HRV/ENT) alone were more likely to be admitted to the hospital
61
compared to those co-infected with HRV/ENT and at least another virus, albeit not significant in
62
multivariable analyses (OR 0.47; 95% C.I. 0.22-1.0; p=0.051). In multivariable analyses,
63
children co-infected with RSV and other viruses were significantly more likely to present with
64
radiologically-confirmed pneumonia compared to those with an isolated RSV infection (OR
65
3.16, 95%C.I. 1.07-9.34, p=0.037).
66
Conclusions: Equivalent clinical severity was observed between children with single and viral
67
co-infection although children co-infected with RSV and other viruses presented more frequently
68
with pneumonia compared to those with single RSV infection. Increased disease severity
69
observed among children with single HRV/ENT infection requires further investigation.
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Introduction The recent use of molecular assays for viral detection has resulted in the identification of respiratory viruses in almost 70% of children admitted with lower respiratory tract infection
74
(LRI), commonly defined as any patient with cough, tachypnea and/or any respiratory distress or
75
wheezing (1). Simultaneous detection of multiple viral pathogens has been reported in 30% of
76
such cases (1-7). While respiratory syncytial virus (RSV) and influenza A (FLU-A) have been
77
mainly identified among children with single viral infection, other viruses including human
78
bocavirus (HBoV) have been mainly reported in children with co-infection (1, 8, 9). To date, the
79
relationship between the clinical severity and infection status with single versus multiple
80
respiratory viruses remains uncertain.
SC
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81
RI PT
73
The objective of this study was to compare the clinical characteristics and the severity of illness in children with multiple simultaneous respiratory viral infections to those with single
83
viral infection while mitigating the shortcomings encountered in previous studies by 1) using a
84
large dataset which includes outpatients in addition to inpatients, 2) considering a composite end-
85
point to assess the severity in patients admitted to hospital, and 3) conducting multivariable
86
analysis to adjust for potential confounding variables.
89 90 91
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Materials and Methods
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Participants and definitions
96
Single-center retrospective study of children presenting to The Hospital for Sick Children, Toronto with an acute respiratory illness and at least one viral infection documented by
98
molecular assays from mid-turbinate swabs. Specimens were collected from November 2007 to
99
April 2008 and January to March 2009, the time periods during which multiplex PCR testing was
RI PT
97
utilized in randomly selected patients under 18 years of age presenting with any respiratory
101
symptom. Multiple simultaneous viral infections (herein referred to as co-infection) were those
102
in which two or more viral pathogens were detected from the same respiratory sample. Viral
103
infection status referred to viral co-infection versus single respiratory viral infection. Information
104
on patient demographics, relevant baseline characteristics and outcomes were extracted from
105
health records. Underlying co-morbidities were grouped into 3 mutually exclusive categories:
106
cardio-respiratory condition, prematurity and any immunosuppressive/metabolic condition. In
107
cases of multiple comorbidities, patients were assigned to the group considered as the highest-
108
risk comorbidity, where an underlying immunocompromised/metabolic condition was
109
considered as the highest-risk comorbidity, followed by a cardio-respiratory condition and then
110
prematurity.
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Bacterial co-infection was defined as the presence of any bacterial pathogen, identified
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112
by culture from blood or respiratory samples upon initial consultation with respiratory
113
symptoms or within 30 days of their initial consultation in association with a documented viral
114
infection. Staphylococcus epidermidis was considered a pathogen if isolated from more than one
115
peripheral blood culture, from one central line blood culture, or from one peripheral blood
116
culture in high risk patients such as those with underlying immunosuppressive conditions,
5
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117
prosthetic devices or newborns according to recent guidelines. Positive bacterial urinary or stool
118
cultures or bacterial pathogens identified from skin or wound swabs were not considered as
119
bacterial co-infection (10). Severity of illness was measured by two primary outcomes: 1) hospital admission for the
RI PT
120
entire cohort; and 2) a composite end-point of intensive care (ICU) admission, hospitalization >
122
5 days, oxygen requirements or death in hospitalized patients. Secondary outcomes examined
123
were radiologically-confirmed pneumonia, ICU admission and mortality. Ethics approval was
124
obtained from the Research Ethics Board at The Hospital for Sick Children.
SC
121
126 127
M AN U
125 Virologic studies
From November 2007 to April 2008 and January 2009 to March 2009, 750 mid-turbinate flocked swabs (COPAN Diagnostics, Murrieta, CA, USA) collected in 3 ml of Universal
129
Transport Medium (UTM-RT, COPAN Diagnostics, Murrieta, CA, USA) were randomly
130
selected among all specimens collected from hospitalized children with symptoms of acute
131
respiratory tract infection (ARI). Specimens were submitted to the clinical laboratory for routine
132
testing for respiratory viruses, which comprised direct fluorescent antigen assay (DFA) and
133
culture. All specimens received in the laboratory were set up for routine examination, and the
134
remaining material was immediately aliquoted and frozen at -80°C until further testing. The first
135
25 specimens received each week for a 24-week period were selected for a total of 600
136
specimens from 2007 to 2008. The same method was used in 2009 to obtain a further 150
137
specimens. In preparation for PCR, a single aliquot of each specimen was thawed and extracted
138
on the biorobot M48 workstation using the MagAttract Virus Mini M48 kit (Qiagen,
139
Mississauga, ON, Canada) and eluted in 100 µL of elution buffer.. The DNA obtained was
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divided into six aliquots and immediately frozen at -80°C until further testing. One aliquot from
141
each specimen was subsequently tested by each of four different nucleic acid amplification-based
142
assays: ResPlex II v2.0 (Qiagen, Mississauga, ON, Canada); Seeplex RV15 kit (Seegene Inc.,
143
Seoul, Korea); xTAG- RVP and xTAG-RVP Fast, (Luminex, Austin TX). These assays,
144
together, detect up to18 respiratory viruses RSV [A, B], OC43, 229E, NL63, HKU1,
145
rhinovirus/enterovirus (HRV/ENT), coxsackie/echovirus, parainfluenza virus (PIV) [ 1, 2, 3, 4],
146
FLU-A, FLU-B, HBoV , adenovirus (ADV) [A,B,C,D,F] and human metapneumovirus (hMPV).
147
ResPlex II v2.0 and Seeplex RV15 assays distinguished HRV from ENT whereas xTAG-RVP
148
and xTAG-RVP fast assays reported a combined result of HRV/ENT as reported in our study.
149
All specimens were also examined by DFA for 8 respiratory viruses RSV, FLU A/FLU B, PIV
150
1-3, ADV (SimulFluor®, Millipore, Temecula, CA) and hMPV (Diagnostic HYBRIDS, Athens,
151
OH)) and/or viral culture. We defined a positive viral result as a true positive if the sample tested
152
positive by viral culture regardless of other tests, or by DFA and at least one molecular test, or by
153
two different molecular tests. For viruses that are only detectable by molecular assays
154
(HRV/ENT, coronaviruses, HBoV and PIV 4), a true positive result was defined as two or more
155
positive test results from the four molecular assays (11).
156
158
Statistical analyses
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Standard descriptive and comparative statistics were used on data categorized by viral
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infection status (viral co-infection and single respiratory viral infection). For all statistical
160
testing, only the first mid-turbinate swab was included in cases with multiple swabs within the
161
same child since these samples cannot be considered independent. For skewed data (age), we
162
derived medians and used the Mann-Whitney method for comparisons. The
2
test or Fisher’s
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exact test was used to compare categorical variables between groups as appropriate.
164
Multivariable logistic regression was used to assess the clinical correlates of disease severity
165
between children infected with single respiratory viruses and those with viral in a model adjusted
166
for age and underlying comorbidities. All available predictors perceived to be important based on
167
the literature regardless of their significance in univariable analysis were included (8).
168
Multivariable regression analyses were performed for each virus (HRV/ENT and RSV co-
169
infection versus single infection) when allowed by the sample size. A two-sided p-value < 0.05
170
was considered to be statistically significant. Data were analyzed by SPSS statistical software
171
(version 20.0, SPSS Inc, Chicago, IL, USA).
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179 180 181 182 183
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184 185
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Results
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Patient characteristics A total of 750 mid-turbinate swabs from 742 children suspected to have a respiratory
189
illness were tested for respiratory viruses by DFA, cell culture and four molecular assays. Of
190
these 742 children, 391 (82.8%) tested positive for a single respiratory virus and 81 (17.2%) for
191
more than one respiratory virus. Three hundred and one children (63.8%) were males, with a
192
median age of 1.2 years (interquartile range (IQR) 0.4-3.7). Ninety-two children (43.8%)
193
presented with CXR-confirmed pneumonia and 264 (55.9%) with common cold. An underlying
194
co-morbidity was documented in 156 (33%) patients: 80 (16.9%) had an underlying
195
immunosuppressive condition, 36 (7.6%) had pre-existing cardio-respiratory illness. There was
196
no difference in baseline demographic characteristics between children with single respiratory
197
viral infection and those with viral co-infection (Table 1). Underlying cardio-respiratory
198
conditions were overrepresented among children infected with HRV/ENT alone compared to
199
those infected with HRV/ENT and at least one other virus (32.2 % vs. 8.2 %; p=0.001) but not
200
with any other virus.
203
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Proportion of viruses detected
Altogether, HRV/ENT (167/472; 35.4%), RSV (140/472; 29.7%), FLU (95/472; 20.1%)
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204
and coronavirus (42/472; 8.9%) were the most commonly detected viruses. FLU (88/95, 92.6%),
205
RSV (104/140; 74.3%), HRV/ENT (118/167; 70.7%), hMPV (30/41; 73.2%) and PIV (17/32;
206
53.1%) were more frequently detected as single respiratory viral infections whereas HBoV
207
(17/24; 70.8%), ADV (9/15; 60%) and coronavirus (23/43; 53.5%) were more often identified in
208
co-infection with other respiratory viruses. Among the 81 children with viral co-infection, only 4
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209
had more than 2 viruses detected. In the 77 children with dual viral infections, HRV/ENT-RSV
210
(13/77; 16.9%), Coronavirus-RSV (12/77; 15.6%) and HRV/ENT-HBoV (9/77; 11.7%) were the
211
most common combinations identified. The most common bacterial pathogen identified in blood cultures from patients with any
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viral infection was Staphylococcus epidermidis (9.3%). From the 92 children with pneumonia,
214
23 (25%) presented with bacterial co-infection: 6 (6.5%) with Staphylococcus epidermidis
215
bacteremia, 3 (3.3%) with Pseudomonas aeruginosa bacteremia, 3 (3.3%) with Staphylococcus
216
aureus isolated from BAL samples and 4 (4.3%) with H. influenza cultured from BAL samples.
217
The overall rate of bacterial co-infection documented with a viral infection was low. Similar
218
rates of bacterial co-infection were observed between patients with single viral infection versus
219
those co-infected with at least another virus (12 % vs. 8.6%; p=0.662).
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Clinical outcomes
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In univariable analysis, children with any viral co-infection were significantly more likely to present with pneumonia compared to those with any single respiratory viral infection (29.6%
224
vs. 17.4%; p=0.048), albeit not significant in multivariable analysis (OR 1.7; 95% C.I. 0.9-3.1;
225
p=0.102). However, children co-infected with RSV and at least another virus were significantly
226
more likely to present with pneumonia compared to those with a single RSV infection (OR 3.16,
227
95%C.I. 1.1-9.3; p=0.037) in multivariable analyses. Eight children with single viral infection
228
died. Among these, 5 presented with HRV/ENT infection, one presented with progressive acute
229
respiratory distress syndrome (ARDS) after ADV infection, and died four weeks after Influenza
230
A (H1N1)pdm09 (pH1N1) infection, one presented with HBoV infection and the remaining one
231
with PIV 4 infection. All but one fatal case had underlying cardio-respiratory or
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immunosuppressive/metabolic conditions. Of the five fatalities with HRV/ENT infection, three
233
had underlying immunosuppressive conditions and two had a cardiac condition. Two of these
234
patients died from a sepsis-like picture with respiratory failure and possible underlying
235
pneumonia with no other pathogens identified, thus suggesting that HRV/ENT may have
236
potentially contributed to the mortality. The remaining three patients likely died of their
237
underlying disease. No deaths were observed among children with viral co-infection. (Table 2)
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Predictors of hospitalization
In multivariable analysis, viral status did not affect admission to hospital after adjustment
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for age and the presence of underlying conditions. However, when analyzed by viral pathogen,
242
children infected with HRV/ENT alone were more likely to be admitted to the hospital (73.3%
243
vs. 49%; p
S1198-743X(14)00062-7
DOI:
10.1016/j.cmi.2014.08.024
Reference:
CMI 61
To appear in:
Clinical Microbiology and Infection
Received Date: 17 April 2014 Revised Date:
21 July 2014
Accepted Date: 4 August 2014
Please cite this article as: Asner SA, Rose W, Petrich A, Richardson S, Tran D, Is viral co-infection a predictor of severity in children with viral respiratory infections?, Clinical Microbiology and Infection (2014), doi: 10.1016/j.cmi.2014.08.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Is viral co-infection a predictor of severity in children with viral respiratory infections?
2
Intended category: Original article
3
Sandra A. Asner1,3,4, MD, Msc; Winsley Rose¹, MD; Astrid Petrich2, PhD; Susan Richardson2,
4
MD; Dat Tran¹, MD, MSc
RI PT
1
5
1
8 9
Division of Infectious Diseases, Department of Paediatrics, The Hospital for Sick
SC
7
Affiliations
Children, University of Toronto, Toronto, Ontario, Canada 2
Division of Microbiology, Department of Paediatric Laboratory Medicine and
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6
10
Department of Pathobiology and Laboratory Medicine, The Hospital for Sick Children,
11
University of Toronto, Toronto, Ontario, Canada 3
13
15 16
Lausanne, Lausanne, Switzerland 4
Division of Infectious Diseases, Department of Medicine, University Hospital Lausanne, Switzerland
EP
14
Paediatric Infectious Diseases unit, Department of Paediatrics, University Hospital
TE D
12
Address correspondence to: Dat Tran; Division of Infectious Diseases; Department of
18
Paediatrics; The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada
19
M5G 1X8, Tel: 416-813-7654 ext 203530, Fax: 416-813-8404 Email: [email protected]
20
Running title : Respiratory viral co-infection in children
21
Keywords: viral respiratory infections, viral co-infection, clinical disease severity, molecular
22
diagnosis, rhinovirus/enterovirus (HRV/ENT), respiratory syncytial virus (RSV)
23
Word count: Abstract: 240; Text: 2’647; Tables: 4; Figures: 0; References: 20
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1
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24
Authorship/Contribution
25 Sandra A. Asner: Dr. Asner helped conceptualizing, designing the study. She was involved in the
27
data collection (patient’s chart review) and built a database with all clinical information. She
28
carried out all the analyses, drafted the initial manuscript and approved the final manuscript as
29
submitted.
RI PT
26
SC
30
Winsley Rose: Dr Rose was involved in the data collection (patient’s chart review) and
32
preliminary analyses. He also critically reviewed the manuscript and approved the final
33
manuscript as submitted.
M AN U
31
34
Astrid Petrich: Dr Petrich helped conceptualizing the study. She critically reviewed and
36
approved the final manuscript as submitted.
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35
37
Susan E. Richarsdon: Dr. Richardson helped coordinating the data collection and helped
39
conceptualizing and designing the study. She critically reviewed and approved the final
40
manuscript as submitted.
42
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41
EP
38
43
Dat Tran: Dr. Tran conceptualized and designed the study, critically reviewed, revised the
44
manuscript and the analyses. Dr Tran approved the final manuscript as submitted.
45 46
2
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Abstract
48
Background: Molecular assays have resulted in increased detection of viral respiratory
49
infections, including viral co-infection, from children with acute respiratory infections (ARIs).
50
Yet, the clinical severity of viral co-infection compared to single viral infection remains
51
uncertain.
52
Methods: Retrospective study of children presenting with ARIs comparing clinical severity of
53
single respiratory viral infection to viral co-infection, detected on mid-turbinate swabs by
54
molecular assays. Patient characteristics and measures of clinical severity were abstracted from
55
health records.
56
Results: 472 virus-infected children were included: 391 with a single viral infection and 81 with
57
viral co-infection. Viral status did not affect admission to hospital (OR=0.8; 95 % C.I. 0.5-1.4;
58
p=0.491) or clinical disease severity among inpatients (OR=0.8; 95% C.I. 0.5-1.5; p=0.515) after
59
adjusting for age and underlying comorbidities. However, children infected with
60
rhinovirus/enterovirus (HRV/ENT) alone were more likely to be admitted to the hospital
61
compared to those co-infected with HRV/ENT and at least another virus, albeit not significant in
62
multivariable analyses (OR 0.47; 95% C.I. 0.22-1.0; p=0.051). In multivariable analyses,
63
children co-infected with RSV and other viruses were significantly more likely to present with
64
radiologically-confirmed pneumonia compared to those with an isolated RSV infection (OR
65
3.16, 95%C.I. 1.07-9.34, p=0.037).
66
Conclusions: Equivalent clinical severity was observed between children with single and viral
67
co-infection although children co-infected with RSV and other viruses presented more frequently
68
with pneumonia compared to those with single RSV infection. Increased disease severity
69
observed among children with single HRV/ENT infection requires further investigation.
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SC
RI PT
47
70 3
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71 72
Introduction The recent use of molecular assays for viral detection has resulted in the identification of respiratory viruses in almost 70% of children admitted with lower respiratory tract infection
74
(LRI), commonly defined as any patient with cough, tachypnea and/or any respiratory distress or
75
wheezing (1). Simultaneous detection of multiple viral pathogens has been reported in 30% of
76
such cases (1-7). While respiratory syncytial virus (RSV) and influenza A (FLU-A) have been
77
mainly identified among children with single viral infection, other viruses including human
78
bocavirus (HBoV) have been mainly reported in children with co-infection (1, 8, 9). To date, the
79
relationship between the clinical severity and infection status with single versus multiple
80
respiratory viruses remains uncertain.
SC
M AN U
81
RI PT
73
The objective of this study was to compare the clinical characteristics and the severity of illness in children with multiple simultaneous respiratory viral infections to those with single
83
viral infection while mitigating the shortcomings encountered in previous studies by 1) using a
84
large dataset which includes outpatients in addition to inpatients, 2) considering a composite end-
85
point to assess the severity in patients admitted to hospital, and 3) conducting multivariable
86
analysis to adjust for potential confounding variables.
89 90 91
EP
88
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87
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82
92 93
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94
Materials and Methods
95
Participants and definitions
96
Single-center retrospective study of children presenting to The Hospital for Sick Children, Toronto with an acute respiratory illness and at least one viral infection documented by
98
molecular assays from mid-turbinate swabs. Specimens were collected from November 2007 to
99
April 2008 and January to March 2009, the time periods during which multiplex PCR testing was
RI PT
97
utilized in randomly selected patients under 18 years of age presenting with any respiratory
101
symptom. Multiple simultaneous viral infections (herein referred to as co-infection) were those
102
in which two or more viral pathogens were detected from the same respiratory sample. Viral
103
infection status referred to viral co-infection versus single respiratory viral infection. Information
104
on patient demographics, relevant baseline characteristics and outcomes were extracted from
105
health records. Underlying co-morbidities were grouped into 3 mutually exclusive categories:
106
cardio-respiratory condition, prematurity and any immunosuppressive/metabolic condition. In
107
cases of multiple comorbidities, patients were assigned to the group considered as the highest-
108
risk comorbidity, where an underlying immunocompromised/metabolic condition was
109
considered as the highest-risk comorbidity, followed by a cardio-respiratory condition and then
110
prematurity.
M AN U
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EP
Bacterial co-infection was defined as the presence of any bacterial pathogen, identified
AC C
111
SC
100
112
by culture from blood or respiratory samples upon initial consultation with respiratory
113
symptoms or within 30 days of their initial consultation in association with a documented viral
114
infection. Staphylococcus epidermidis was considered a pathogen if isolated from more than one
115
peripheral blood culture, from one central line blood culture, or from one peripheral blood
116
culture in high risk patients such as those with underlying immunosuppressive conditions,
5
ACCEPTED MANUSCRIPT
117
prosthetic devices or newborns according to recent guidelines. Positive bacterial urinary or stool
118
cultures or bacterial pathogens identified from skin or wound swabs were not considered as
119
bacterial co-infection (10). Severity of illness was measured by two primary outcomes: 1) hospital admission for the
RI PT
120
entire cohort; and 2) a composite end-point of intensive care (ICU) admission, hospitalization >
122
5 days, oxygen requirements or death in hospitalized patients. Secondary outcomes examined
123
were radiologically-confirmed pneumonia, ICU admission and mortality. Ethics approval was
124
obtained from the Research Ethics Board at The Hospital for Sick Children.
SC
121
126 127
M AN U
125 Virologic studies
From November 2007 to April 2008 and January 2009 to March 2009, 750 mid-turbinate flocked swabs (COPAN Diagnostics, Murrieta, CA, USA) collected in 3 ml of Universal
129
Transport Medium (UTM-RT, COPAN Diagnostics, Murrieta, CA, USA) were randomly
130
selected among all specimens collected from hospitalized children with symptoms of acute
131
respiratory tract infection (ARI). Specimens were submitted to the clinical laboratory for routine
132
testing for respiratory viruses, which comprised direct fluorescent antigen assay (DFA) and
133
culture. All specimens received in the laboratory were set up for routine examination, and the
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remaining material was immediately aliquoted and frozen at -80°C until further testing. The first
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25 specimens received each week for a 24-week period were selected for a total of 600
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specimens from 2007 to 2008. The same method was used in 2009 to obtain a further 150
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specimens. In preparation for PCR, a single aliquot of each specimen was thawed and extracted
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on the biorobot M48 workstation using the MagAttract Virus Mini M48 kit (Qiagen,
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Mississauga, ON, Canada) and eluted in 100 µL of elution buffer.. The DNA obtained was
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divided into six aliquots and immediately frozen at -80°C until further testing. One aliquot from
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each specimen was subsequently tested by each of four different nucleic acid amplification-based
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assays: ResPlex II v2.0 (Qiagen, Mississauga, ON, Canada); Seeplex RV15 kit (Seegene Inc.,
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Seoul, Korea); xTAG- RVP and xTAG-RVP Fast, (Luminex, Austin TX). These assays,
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together, detect up to18 respiratory viruses RSV [A, B], OC43, 229E, NL63, HKU1,
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rhinovirus/enterovirus (HRV/ENT), coxsackie/echovirus, parainfluenza virus (PIV) [ 1, 2, 3, 4],
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FLU-A, FLU-B, HBoV , adenovirus (ADV) [A,B,C,D,F] and human metapneumovirus (hMPV).
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ResPlex II v2.0 and Seeplex RV15 assays distinguished HRV from ENT whereas xTAG-RVP
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and xTAG-RVP fast assays reported a combined result of HRV/ENT as reported in our study.
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All specimens were also examined by DFA for 8 respiratory viruses RSV, FLU A/FLU B, PIV
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1-3, ADV (SimulFluor®, Millipore, Temecula, CA) and hMPV (Diagnostic HYBRIDS, Athens,
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OH)) and/or viral culture. We defined a positive viral result as a true positive if the sample tested
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positive by viral culture regardless of other tests, or by DFA and at least one molecular test, or by
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two different molecular tests. For viruses that are only detectable by molecular assays
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(HRV/ENT, coronaviruses, HBoV and PIV 4), a true positive result was defined as two or more
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positive test results from the four molecular assays (11).
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Statistical analyses
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Standard descriptive and comparative statistics were used on data categorized by viral
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infection status (viral co-infection and single respiratory viral infection). For all statistical
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testing, only the first mid-turbinate swab was included in cases with multiple swabs within the
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same child since these samples cannot be considered independent. For skewed data (age), we
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derived medians and used the Mann-Whitney method for comparisons. The
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test or Fisher’s
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exact test was used to compare categorical variables between groups as appropriate.
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Multivariable logistic regression was used to assess the clinical correlates of disease severity
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between children infected with single respiratory viruses and those with viral in a model adjusted
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for age and underlying comorbidities. All available predictors perceived to be important based on
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the literature regardless of their significance in univariable analysis were included (8).
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Multivariable regression analyses were performed for each virus (HRV/ENT and RSV co-
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infection versus single infection) when allowed by the sample size. A two-sided p-value < 0.05
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was considered to be statistically significant. Data were analyzed by SPSS statistical software
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(version 20.0, SPSS Inc, Chicago, IL, USA).
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Results
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Patient characteristics A total of 750 mid-turbinate swabs from 742 children suspected to have a respiratory
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illness were tested for respiratory viruses by DFA, cell culture and four molecular assays. Of
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these 742 children, 391 (82.8%) tested positive for a single respiratory virus and 81 (17.2%) for
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more than one respiratory virus. Three hundred and one children (63.8%) were males, with a
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median age of 1.2 years (interquartile range (IQR) 0.4-3.7). Ninety-two children (43.8%)
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presented with CXR-confirmed pneumonia and 264 (55.9%) with common cold. An underlying
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co-morbidity was documented in 156 (33%) patients: 80 (16.9%) had an underlying
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immunosuppressive condition, 36 (7.6%) had pre-existing cardio-respiratory illness. There was
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no difference in baseline demographic characteristics between children with single respiratory
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viral infection and those with viral co-infection (Table 1). Underlying cardio-respiratory
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conditions were overrepresented among children infected with HRV/ENT alone compared to
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those infected with HRV/ENT and at least one other virus (32.2 % vs. 8.2 %; p=0.001) but not
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with any other virus.
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Proportion of viruses detected
Altogether, HRV/ENT (167/472; 35.4%), RSV (140/472; 29.7%), FLU (95/472; 20.1%)
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and coronavirus (42/472; 8.9%) were the most commonly detected viruses. FLU (88/95, 92.6%),
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RSV (104/140; 74.3%), HRV/ENT (118/167; 70.7%), hMPV (30/41; 73.2%) and PIV (17/32;
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53.1%) were more frequently detected as single respiratory viral infections whereas HBoV
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(17/24; 70.8%), ADV (9/15; 60%) and coronavirus (23/43; 53.5%) were more often identified in
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co-infection with other respiratory viruses. Among the 81 children with viral co-infection, only 4
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had more than 2 viruses detected. In the 77 children with dual viral infections, HRV/ENT-RSV
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(13/77; 16.9%), Coronavirus-RSV (12/77; 15.6%) and HRV/ENT-HBoV (9/77; 11.7%) were the
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most common combinations identified. The most common bacterial pathogen identified in blood cultures from patients with any
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viral infection was Staphylococcus epidermidis (9.3%). From the 92 children with pneumonia,
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23 (25%) presented with bacterial co-infection: 6 (6.5%) with Staphylococcus epidermidis
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bacteremia, 3 (3.3%) with Pseudomonas aeruginosa bacteremia, 3 (3.3%) with Staphylococcus
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aureus isolated from BAL samples and 4 (4.3%) with H. influenza cultured from BAL samples.
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The overall rate of bacterial co-infection documented with a viral infection was low. Similar
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rates of bacterial co-infection were observed between patients with single viral infection versus
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those co-infected with at least another virus (12 % vs. 8.6%; p=0.662).
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Clinical outcomes
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In univariable analysis, children with any viral co-infection were significantly more likely to present with pneumonia compared to those with any single respiratory viral infection (29.6%
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vs. 17.4%; p=0.048), albeit not significant in multivariable analysis (OR 1.7; 95% C.I. 0.9-3.1;
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p=0.102). However, children co-infected with RSV and at least another virus were significantly
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more likely to present with pneumonia compared to those with a single RSV infection (OR 3.16,
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95%C.I. 1.1-9.3; p=0.037) in multivariable analyses. Eight children with single viral infection
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died. Among these, 5 presented with HRV/ENT infection, one presented with progressive acute
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respiratory distress syndrome (ARDS) after ADV infection, and died four weeks after Influenza
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A (H1N1)pdm09 (pH1N1) infection, one presented with HBoV infection and the remaining one
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with PIV 4 infection. All but one fatal case had underlying cardio-respiratory or
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immunosuppressive/metabolic conditions. Of the five fatalities with HRV/ENT infection, three
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had underlying immunosuppressive conditions and two had a cardiac condition. Two of these
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patients died from a sepsis-like picture with respiratory failure and possible underlying
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pneumonia with no other pathogens identified, thus suggesting that HRV/ENT may have
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potentially contributed to the mortality. The remaining three patients likely died of their
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underlying disease. No deaths were observed among children with viral co-infection. (Table 2)
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Predictors of hospitalization
In multivariable analysis, viral status did not affect admission to hospital after adjustment
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for age and the presence of underlying conditions. However, when analyzed by viral pathogen,
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children infected with HRV/ENT alone were more likely to be admitted to the hospital (73.3%
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vs. 49%; p
